C9orf72-specific phenomena associated with frontotemporal dementia and gastrointestinal symptoms in the absence of TDP-43 aggregation

The most common genetic cause of frontotemporal dementia is the expanded hexanucleotide (GGGGCC) repeat insertion in a non-coding promoter region of the chromosome 9 open reading frame 72 (C9orf72) gene [4,13]. Nearly all patients who carry the C9orf72 expansion show well-developed TAR DNA-binding protein 43 (TDP-43) inclusion pathology at autopsy, and TDP-43 has been considered a key driver of neurodegeneration based on human clinico-pathological correlation approaches [7]. Scattered case reports describing pre-symptomatic C9orf72 expansion carriers suggest, however, that C9orf72-specific phenomena, such as dipeptide repeat (DPR) proteins and RNA foci can be observed in the absence of TDP-43 inclusions [11,15]. One previous report of a patient with behavioral variant FTD (bvFTD) suggested that focal degeneration could occur in brain regions lacking TDP-43 aggregation, but to date no patient with full-blown symptomatic FTD has lacked TDP-43 inclusions altogether [11]. Likewise, although DPRs have been identified in the testis, there has been no evidence of symptomatic DPR aggregation outside the central nervous system (CNS) [2].

Here, we report a 65-year-old woman who developed slowly progressive behavioral changes over at least 1 year. She withdrew from friends, became emotionally labile, and grew disinterested in her hobbies. At times, she struggled to cook for herself. In parallel, she developed hyposmia, compulsive binge-eating, nausea, vomiting, loose stools, bowel incontinence, and a 20-pound weight gain. Classical disinhibition and compulsive behaviors were lacking. She had no known family history of neurodegenerative disorders. Her mother was alive and cognitively normal; but her father, who suffered from a stroke, was described as having a mental illness at the time of his death at age 71 years. Her neurologic exam demonstrated mildly increased tone in her arms, rare myoclonic jerks, and a slightly reduced stride length.

Her medications (see Supplemental Table 1) were without recent changes. Extensive laboratory testing (see Supplemental Table 2) failed to reveal a cause of her gastrointestinal or behavioral symptoms. A brain magnetic resonance image (MRI) without contrast (Fig. 1a, Supplemental Fig. 1) showed moderate, distributed atrophy most prominent in the anterior insula, medial frontal, anterior temporal, and parietal lobes. A routine electroencephalogram showed mild slowing of the posterior dominant rhythm. Esophagogastroduodenoscopy demonstrated moderate esophagitis, a non-obstructive Shatzki ring, and mild non-erosive gastritis, consistent with gastroesophageal reflux disease but considered insufficient to produce her symptoms. Gastric biopsies were negative for H. pylori. Duodenal biopsies were negative for histologic or immunophenotypic evidence of Celiac or Whipple’s disease. A colonoscopy with terminal ileum biopsies was likewise unremarkable.

Fig. 1: A C9orf72 mutation carrier without TDP-43 pathology.

a. Axial slices from the patient’s T1-weighted MRI scan show moderate to severe atrophy, most prominent in the insular, frontal, and parietal brain regions. b-d. Representative images showing the presence of C9orf72-specific phenomena such as RNA foci and dipeptide inclusions in affected brain regions including the pACC and FI. TDP-43 immunoreactivity show normal nuclear staining and a complete absence of inclusions. Scale bar = 5 μm (b), 25 μm (c), and 250 μm (d). Abbreviations, pACC: pregenual anterior mid cingulate cortex, FI: frontal insula, SF: sense RNA foci, ASF: antisense RNA foci.

Over the next 2 years, the patient developed worsening parkinsonism, dysphagia, and intractable vomiting. She could manage only basic household tasks, requiring assistance with driving, shopping, and managing her finances. Neuropsychological testing demonstrated moderate difficulties in executive functioning and processing speed, with relatively preserved visuospatial skills and episodic memory. Because of her progressive behavioral decline, early loss of interest in friends and hobbies (i.e., apathy), changes in eating behavior, executive-predominant neuropsychological profile, and frontal-predominant atrophy on MRI, she met criteria for probable bvFTD [12]. Genetic testing later revealed a large GGGGCC expansion (>1000 repeats) in C9orf72, leading to a diagnosis of definite bvFTD by international consensus criteria [12]. In her final weeks, she could not eat without vomiting. Three years after her first symptoms, she woke up one morning, made oatmeal, and went to the bathroom nauseated. After several rounds of emesis, she collapsed and died of aspiration.

A general autopsy was performed. Gross examination of the gastrointestinal (GI) tract was unremarkable. The fresh brain weighed 1190 grams. Gross neuropathological assessment revealed mild to moderate volume loss of the frontal and temporal lobes bilaterally. Sections stained for hematoxylin and eosin were rated for microvacuolation and astrogliosis (Supplemental Table 3); overall, these changes were absent or mild, with only selected bvFTD-susceptible regions showing evidence of mild neurodegeneration. There were scattered microinfarcts and siderocalcinosis in the globus pallidus, but the substantia nigra showed normal findings. Immunohistochemical studies revealed sparse to frequent diffuse amyloid-beta plaques in the neocortex (Thal Phase 1), tau neurofibrillary pathology involving the entorhinal region, hippocampus, and amygdala (Braak neurofibrillary tangle stage 3), and no Lewy body disease on alpha-synuclein immunostaining. Immunostaining for TATA-binding protein-associated factor 15 (TAF15), one of the most sensitive antibodies for detecting FTLD-FET inclusions, showed no inclusion pathology with normal nuclear TAF15 staining [9]. Given the known C9orf72 expansion, we performed fluorescence in situ hybridization (FISH) for sense and antisense repeat RNA foci, which showed foci of both types in selected areas (Figure 1b). Immunohistochemistry for each of the five DPRs revealed widespread DPR inclusions throughout the brain (Fig. 1c and Supplemental Table 3). Poly-GA was the most abundant DPR while poly-PA and -PR were the least abundant. The distribution of DPRs was consistent with previous studies [3,14]. Immunostaining for p62 showed that most neuronal cytoplasmic inclusions were perinuclear, with a stellate morphology resembling DPR inclusions (Supplemental Fig. 2). Immunostaining for full-length TDP-43 showed normal nuclear staining, without inclusions, in all 45 CNS regions evaluated (see Fig. 1d, Supplemental Table 3, and Supplemental Fig. 3), except for 3–4 scattered neuronal cytoplasmic inclusions in the amygdala, a finding observed in 30–50% of cognitively normal persons of the patient’s age [16].

To determine whether the patient’s GI symptoms might relate to C9orf72-associated pathological features, we performed immunohistochemistry for the five poly-dipeptides on tissue sections from several segments of the GI tract (Fig. 2, Supplemental Table 3). Neuronal cytoplasmic inclusions, chiefly composed of poly-GA and –GP were observed in neurons occupying the myenteric (Auerbach’s) plexus of the gastro-esophageal junction and sigmoid colon (Fig. 2). Meissner’s plexus was free of inclusions. No neurons showed TDP-43 aggregation or depletion. Numerous attempts to perform FISH for repeat RNA foci on enteric samples were unsuccessful due to tissue non-adherence. Hematoxylin and eosin-stained sections throughout the GI tract showed no clear evidence of vacuolation or neuronal loss.

Fig. 2: Dipeptide inclusions are present in the enteric nervous system.

Example images showing the presence of various dipeptide inclusions in the enteric neurons of the myenteric (Auerbach’s) plexus from the gastroesophageal junction or sigmoid colon. In these regions, TDP-43 immunoreactivity shows normal nuclear staining of the enteric neurons. Scale bar = 25 μm.

The present case demonstrates that the C9orf72 expansion can be associated with: (1) probable bvFTD in the absence of FTLD-TDP and (2) severe GI symptoms accompanied by C9orf72-specific phenomena within the enteric nervous system.

Patients with C9orf72-FTD showing limited TDP-43 inclusion pathology have been reported, but to our knowledge the present case is the first to show significant brain atrophy in the near complete absence of TDP-43 inclusions [14]. Prevailing FTLD nomenclature fails to accommodate the patient’s findings, because she does not have FTLD-TDP, and FTLD with ubiquitin proteosomal system inclusions (FTLD-UPS) is intended for use when the protein found within the ubiquitinated inclusions is not known [8]. We therefore offer the following terminology as a temporary way to describe the present case and others like her: C9orf72-associated FTLD without TDP-43 aggregation.

Although some studies have widely surveyed non-CNS tissues for DPR inclusions, these studies have not included the GI tract or patients with prominent GI symptomatology [2, 10]. Patients with bvFTD often have profound alterations in eating behavior, but we are not aware of prior cases in which GI dysregulation was the most disabling feature [1]. Interestingly, GI complications have been observed in transgenic mice with human mutant (A315T) TDP-43 [5]. The mice exhibited dilation of the cecum and terminal ileum, and histopathological analysis revealed vacuolated myenteric plexi with TDP-43 aggregates in associated ganglion cells [5, 6]. Murine models of C9orf72 disease have not, to our knowledge, shown such findings, but the present case adds enteric neurons to the list of nervous tissues vulnerable to C9orf72-specific phenomena.