Neuropathy target esterase impairments cause Oliver-McFarlane and Laurence-Moon syndromes

Abstract

Background: Oliver-McFarlane syndrome is characterised by trichomegaly, congenital hypopituitarism and retinal degeneration with choroidal atrophy. Laurence-Moon syndrome presents similarly, though with progressive spinocerebellar ataxia and spastic paraplegia and without trichomegaly. Both recessively inherited disorders have no known genetic cause.

Methods: Whole-exome sequencing was performed to identify the genetic causes of these disorders. Mutations were functionally validated in zebrafish pnpla6 morphants. Embryonic expression was evaluated via in situ hybridisation in human embryonic sections. Human neurohistopathology was performed to characterise cerebellar degeneration. Enzymatic activities were measured in patient-derived fibroblast cell lines.

Results: Eight mutations in six families with Oliver-McFarlane or Laurence-Moon syndrome were identified in the PNPLA6 gene, which encodes neuropathy target esterase (NTE). PNPLA6 expression was found in the developing human eye, pituitary and brain. In zebrafish, the pnpla6 curly-tailed morphant phenotype was fully rescued by wild-type human PNPLA6 mRNA and not by mutation-harbouring mRNAs. NTE enzymatic activity was significantly reduced in fibroblast cells derived from individuals with Oliver-McFarlane syndrome. Intriguingly, adult brain histology from a patient with highly overlapping features of Oliver-McFarlane and Laurence-Moon syndromes revealed extensive cerebellar degeneration and atrophy.

Conclusions: Previously, PNPLA6 mutations have been associated with spastic paraplegia type 39, Gordon-Holmes syndrome and Boucher-Neuhäuser syndromes. Discovery of these additional PNPLA6-opathies further elucidates a spectrum of neurodevelopmental and neurodegenerative disorders associated with NTE impairment and suggests a unifying mechanism with diagnostic and prognostic importance.

Keywords: Dermatology; Developmental; Genetics; Neuro endocrinology; Ophthalmology.

Figure 1

Clinical phenotypes of Oliver–McFarlane and Laurence–Moon syndromes. (A–F) Variability in eyelash length and eyebrow and scalp hair quality for patients A:1 (A), A:2 (B), B:1 (C), C:1 (D), F:1 (E) and F:2 (F). (G–J) Chorioretinal atrophy with variable pigment findings including macular and peripapillary sparing and peripheral pigment clumping in patients A:1 (G), C:1 (H), D:1 (I) and F:1 (J). (K–M) Brain MRI findings including small anterior pituitary noted in Oliver–McFarlane syndrome patient A:1 (K) and Laurence–Moon syndrome patient F:1 (M), along with signal abnormalities of the posterior internal capsule and deep white matter bilaterally in patient B:1 (L). (N) Spastic paraplegia findings on hand X-ray of patient F:1. (O–R) Neuropathology findings for patient E:1, including severe depletion of Purkinje and granule cells in cerebellar cortex (O), numerous empty baskets (P), severe astrogliosis (Q) and an increased activated microglial cells (R). (O) H&E stain, (P) α-internexin immunohistochemistry, (Q) glial fibrillary acidic protein immunohistochemistry and (R) IBa1 immunohistochemistry. Scale bar represents 90 μm on (A) and 45 μm on (B–D).

Figure 2

PNPLA6 mutations are associated with Oliver–McFarlane and Laurence–Moon syndromes. (A) An illustration of PNPLA6 gene structure along with the mutations. Exons highlighted corresponding to protein domains. (B) Predicted PNPLA6 protein structure includes three cNMP domains and one patatin-like domain. (C) Alignment of portions of PNPLA6 proteins from various species, showing conservation of the neuropathy target esterase domain residues mutated in patients with Oliver–McFarlane and SPG39 syndromes. (D) A homology model of the catalytic domain of PNPLA6 constructed using SWISS-MODEL, and 10XW.PDB as a template. The location of mutations (red) and the enzymatically active residues (blue) are shown. cNMP, cyclic-nucleotide monophosphate.

Figure 3

Human embryonic expression of PNPLA6. PNPLA6 in situ hybridisation of human embryos as Carnegie stage 19 (E) and stage 23 (A–D and F). (A and D) Horizontal sections with expression in the retina and periventricular zones of the lateral and third ventricles. (B) Eye expression observed in the retina, fovea (arrow), retinal pigment epithelium (arrowhead), choroid, external epithelium and lens (L). (C) Anterior and posterior pituitary expression in the epithelium and parenchyma. (E) PNPLA6 expression in the developing cerebellum (arrow) and hindbrain (arrowhead). (F) PNPLA6 is expressed in the nasal epithelium (arrow), eyelid epithelium (arrowhead), extraocular muscles (EOM) and lens (L). Scale bar represents 400 μm in (A), (D) and (E) 100 μm in (B) and (C), and 200 μm in (F).

Figure 4

Suppression of pnpla6 expression produces developmental defects in zebrafish embryos. (A) Normal embryos class. (B–D) Embryos injected with morpholino (MO) against pnpla6 have developmental defects, curved head (mild), curved head and distal tail (intermediate), and curved, small head with full tail curvature (severe). (E) Percentage of embryos categorised in various phenotypic classes after injection with pnpla6 MO, wild-type mRNA or coinjection of PNPLA6 harbouring mutant alleles (N>100 embyros per condition; error bars=SEM; *p<0.05, **p<0.01, ***p<0.001).

Figure 5

Differential neuropathy target esterase (NTE) hydrolase activity among PNPLA6-associated diseases. (A) Phenol production normalised to wild-type control cells. Statistical significance of analysis of variance post hoc Tukey’s test comparing wild type (blue line), SPG39 patient cells (green line) or family A carrier cells (red line). (B) Values for phenol production (±SEM) for each cell line along with genotype and affected status. Error bars=SEM; *p<0.05, **p<0.01, ***p<0.001.