Mutated CTSF in adult-onset neuronal ceroid lipofuscinosis and FTD

The Belgian ANCL family.

In this family with unaffected parents, 4 of the 5 sibs developed ANCL (figure 1A). The eldest sister (II-1), born in 1948, is still alive and has no children. She developed generalized myoclonic epileptic seizures at the age of 35 years, followed by extrapyramidal symptoms and cognitive disturbances 7 and 15 years later, respectively. CT scan of the brain at the age of 35 revealed moderate cortical and subcortical atrophy, too pronounced for her age. Brain MRI at the age of 62 showed generalized cortical-subcortical atrophy. In-depth neuropsychological investigations were consistent with a diagnosis of behavioral variant (bv) FTD. The eldest brother (II-2; propositus) was born in 1952 and remained childless. At the age of 26 years, he had developed progressive motor speech problems. MRI of the brain revealed mild generalized cortical-subcortical atrophy. At the age of 54, his condition progressively worsened, with severe motor, cognitive, and behavioral decline, consistent with a diagnosis of bvFTD. Repeat MRI of brain demonstrated generalized cortical-subcortical atrophy. In the final stage, he developed akinetic mutism and died in 2012 at the age of 60. The second daughter (II-3) was born in 1953. At the age of 30 years, she presented with progressive apathy and behavioral, cognitive, and affective disturbances. Brain CT disclosed severe generalized cortical-subcortical atrophy. She died in 2007 at the age of 54, after a long period of progressive behavioral and cognitive disturbances resulting in severe dementia (bvFTD) and akinetic mutism. She was the mother of 2 healthy children. The youngest daughter (II-4), born in 1960, is healthy and the mother of 2 healthy children. The youngest brother (II-5) was born in 1962 and has 2 healthy children. At the age of 50, he developed right-sided tremor, motor speech problems, general slowness, and feelings of anxiety. Neuropsychological testing disclosed a general cognitive decline, with prominent frontal lobe involvement. MRI of the brain showed marked generalized cortical-subcortical atrophy and a cyst of the choroid plexus in the left lateral ventricle. The mother of the 5 sibs was born in 1925 and is still alive at age 90. Their father, born in 1921, died at the age of 89.

Neuropathology and electron microscopy of the ANCL family.

Autopsy was performed on the brain of patient II-2, which weighed 956 g. Macroscopic examination revealed atrophy of the different lobes and dilation of the ventricular system. Microscopic examination of the frontal, parietal, temporal, and occipital neocortices showed generalized signs of neuronal storage of a yellowish pigment, more evident in the large pyramidal cells of the third cortical layer but also in the Betz neurons of the fifth cortical layer at the level of the precentral gyrus. The granular storage was noted in the perikaryon and could be followed in the proximal part of the axon (figure 2, A and C). Sometimes, the axon was enlarged after the axonal hillock. The storage material was autofluorescent (figure 2E), yellowish on cresyl violet and hematoxylin and eosin stains, deep blue on Klüver-Barrera stain, and strongly periodic acid-Schiff positive (figure 2F). AT8 (against hyperphosphorylated tau) elicited a moderate amount of neurofibrillary tangles and sparse neuritic threads in the hippocampus and parahippocampal structures. The presence of these findings had no clinical relevance. TDP-43 immunohistochemistry did not show any abnormalities. Cathepsin D staining showed lipopigmentary granules (figure 2D). Other immunohistochemical stains were negative.

Electron microscopic examination of the brain of patient II-2 confirmed the intraneuronal storage of polymorphic lipofuscin-like inclusions (diameter: 1–2 μm) with granular components displaying distinctive features (figure 3A). At higher magnifications, densely packed short lamellar profiles, coarser granular components (figure 3B), and lipid droplets as well as lamellar complexes (figure 3C) and few fingerprints (figure 3D) were seen. These inclusions were mostly different from the classical lipofuscin (figure 3E). Similar but large coalescing inclusions were present in glial and microglial cells. Numerous polymorphic inclusions revealing coarse and densified granular subunits associated with lipid droplets were observed in Golgi- or basket-like cells. Glial cells and granule cells showed compound lipofuscin-like deposits with granular subunits.

Sporadic granular lipopigments with osmiophilic granules in Schwann cells as well as unmyelinated dystrophic axons were observed in the skin biopsy of patient II-2, while lipofuscin-like inclusions with an uncommon heterogeneous pattern were detected in eccrine sweat glands and blood vessels (figure 3F) of patient II-5.

A more detailed description of the neuropathology and electron microscopic examination can be found in the supplemental data.

Identification of the causal gene defect in the ANCL family.

Analysis of the exomes identified only one rare coding variant that was consistent with a recessive mode of inheritance. The variant predicted a missense mutation p.Ile404Thr in the CTSF gene and was found homozygous in all affected sibs and heterozygous in the unaffected mother and unaffected daughter (figure 1B). Segregation analysis with a panel of flanking STR markers demonstrated that the CTSF p.Ile404Thr mutation was inherited on 2 different haplotypes and, as such, confirmed that the parents were not consanguineous (figure 1B). The presence of the p.Ile404Thr mutation was tested in an extended cohort of 1,177 Belgian control persons. No homozygous carriers were found; however, p.Ile404Thr was identified in the heterozygous state in 2 control individuals, a 76-year-old man and a 67-year-old woman. Inspection of their STR genotypes was suggestive of a common haplotype with the paternal-inherited disease haplotype (figure 1B, yellow haplotype) of the ANCL family.

Genetic screening of CTSF in patients with FTD.

Because the neurocognitive profile of the ANCL family was marked by prominent frontal lobe features evolving to severe FTD with akinetic mutism at the end stage of the disease in 2 sibs, the identified causal CTSF gene was further tested for mutations in 461 unrelated Belgian patients with FTD. We detected 1 heterozygote mutation, p.Arg245His, in 2 patients (0.4%), which was absent in the control cohort (figure 1C).

Mapping of the mutations on the CTSF protein.

The CTSF gene encodes a lysosomal cysteine protease, CTSF, that belongs to the peptidase C1 family and is believed to participate in the intracellular degradation and turnover of proteins. CTSF is a 484 amino acid protein with 2 known functional domains: a peptidase inhibitor I29 domain and a peptidase C1 domain (figure 1C). In line with the majority of the reported CTSF mutations, the p.Ile404Thr missense mutation maps to the peptidase C1 domain near the C-terminal end of the protein. In contrast, the p.Arg245His mutations fall outside the known functional domains, yet flanking to the inhibitor I29 domain. Both the p.Ile404Thr and the p.Arg245His mutations affect conserved residues, although for the latter, in mice, a His-residue is present at the homologous position (figure 1D).

Clinical presentation of the FTD carriers of CTSF p.Arg245His.

One of the FTD patients with the heterozygote p.Arg245His mutation developed signs of bvFTD at the age of 65 years, marked by irritability, socially inappropriate behavior, and verbal disinhibition. He also suffered from severe balance problems causing repetitive falling, nocturnal myoclonus, dysarthria, mild word-finding difficulties, and a mild asymmetrical extrapyramidal syndrome. At age 67, he had developed constructional apraxia, dysphagia, and impaired vertical eye movements. MRI of the brain showed atrophy of the midbrain with a hummingbird sign, compatible with progressive supranuclear palsy (PSP). No clear family history was reported.

The second carrier developed memory problems at the age of 76. She had 1 sister with unspecified dementia, developed at the age of 75. The patient later developed slowness, balance problems with repeated falling, inappropriate and compulsive behavior, incontinence, echolalia, dysarthria, and extrapyramidal signs with a vertical gaze palsy. At the age of 81, she evolved to a state of akinetic mutism with swallowing problems. Her MRI results also showed a hummingbird sign, as in PSP, and a presynaptic dopaminergic degeneration on dopamine transporter imaging.

There was no direct evidence for relatedness between the two p.Arg245His carriers; however, STR genotyping flanking the mutation was indicative of a common disease haplotype, suggesting a common founder.