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Multicenter Study
. 2013 Oct 8;81(15):1332-41.
doi: 10.1212/WNL.0b013e3182a8250c. Epub 2013 Sep 11.

C9ORF72 Repeat Expansions in Cases With Previously Identified Pathogenic Mutations

Free PMC article
Multicenter Study

C9ORF72 Repeat Expansions in Cases With Previously Identified Pathogenic Mutations

Marka van Blitterswijk et al. Neurology. .
Free PMC article


Objective: To identify potential genetic modifiers contributing to the phenotypic variability that is detected in patients with repeat expansions in chromosome 9 open reading frame 72 (C9ORF72), we investigated the frequency of these expansions in a cohort of 334 subjects previously found to carry mutations in genes known to be associated with a spectrum of neurodegenerative diseases.

Methods: A 2-step protocol, with a fluorescent PCR and a repeat-primed PCR, was used to determine the presence of hexanucleotide expansions in C9ORF72. For one double mutant, we performed Southern blots to assess expansion sizes, and immunohistochemistry to characterize neuropathology.

Results: We detected C9ORF72 repeat expansions in 4 of 334 subjects (1.2% [or 1.8% of 217 families]). All these subjects had behavioral phenotypes and also harbored well-known pathogenic mutations in either progranulin (GRN: p.C466LfsX46, p.R493X, p.C31LfsX35) or microtubule-associated protein tau (MAPT: p.P301L). Southern blotting of one double mutant with a p.C466LfsX46 GRN mutation demonstrated a long repeat expansion in brain (>3,000 repeats), and immunohistochemistry showed mixed neuropathology with characteristics of both C9ORF72 expansions and GRN mutations.

Conclusions: Our findings indicate that co-occurrence of 2 evidently pathogenic mutations could contribute to the pleiotropy that is detected in patients with C9ORF72 repeat expansions. These findings suggest that patients with known mutations should not be excluded from further studies, and that genetic counselors should be aware of this phenomenon when advising patients and their family members.


Figure 1
Figure 1. Chromatograms and electropherograms of identified subjects
(A) Chromatograms of subjects included in this study. Shown are GRN mutations p.C466LfsX46 (c.1395_1396insC), p.R493X (c.1477C>T), and p.C31LfsX35 (c.90_91insCTGC), and MAPT mutation p.P301L (c.902C>T). (B) C9ORF72 repeat expansions detected in the subjects with GRN and MAPT mutations. PCR products of repeat-primed PCR reactions separated on an ABI3730 DNA Analyzer and visualized by GeneMapper software, showing 4 double mutation carriers detected in this study with their characteristic stutter amplification. C9ORF72 = chromosome 9 open reading frame 72; GRN = progranulin; MAPT = microtubule-associated protein tau.
Figure 2
Figure 2. Southern blot of patient F
Southern blot demonstrating 3 brain regions of patient F. DIG-labeled DNA Molecular Weight Marker II (Roche) was used with fragments of 2,027; 2,322; 4,361; 6,557; 9,416; and 23,130 base pairs. A positive control that harbors a C9ORF72 expansion, but no additional mutation in GRN or MAPT, is shown in lanes 7, 8, and 9. In lane 10, a negative control without a C9ORF72 expansion is shown; this patient only displays the 2.3-kb wild-type allele. C9ORF72 = chromosome 9 open reading frame 72; DIG = digoxigenin; GRN = progranulin; MAPT = microtubule-associated protein tau.
Figure 3
Figure 3. Neuropathology of patient F
Neuropathology of the GRN/C9ORF72 double mutation in a pathologically confirmed frontotemporal lobar degeneration case. (A) Brain MRI revealed dilated lateral ventricles and flattening of the caudate nucleus (arrow) in a T1-weighted coronal MRI scan acquired 3 years before death. MRI findings were supported (B) at autopsy with marked flattening of the caudate (arrow) and cerebral atrophy most significant in the frontal cortex (arrowhead). Superficial spongiosis in layer II of the cortical ribbon (C) is associated with type A TDP-43 pathology, including neuronal cytoplasmic inclusions (arrow) and neuritic pathology (arrowhead). The inset demonstrates a “lentiform” intranuclear inclusion (arrow) in the dentate gyrus of the hippocampus. (D) p62 and (E) C9RANT immunohistochemistry of the cerebellar granule cell layer shows many neuronal cytoplasmic inclusions, consistent with a mutation in C9ORF72. Bar = 25 µm for C, D, and E, and 10 µm for inset. C9ORF72 = chromosome 9 open reading frame 72; GRN = progranulin; TDP-43 = TAR DNA-binding protein 43.

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