Analysis of IFT74 as a candidate gene for chromosome 9p-linked ALS-FTD

Abstract

Background: A new locus for amyotrophic lateral sclerosis--frontotemporal dementia (ALS-FTD) has recently been ascribed to chromosome 9p.

Methods: We identified chromosome 9p segregating haplotypes within two families with ALS-FTD (F476 and F2) and undertook mutational screening of candidate genes within this locus.

Results: Candidate gene sequencing at this locus revealed the presence of a disease segregating stop mutation (Q342X) in the intraflagellar transport 74 (IFT74) gene in family 476 (F476), but no mutation was detected within IFT74 in family 2 (F2). While neither family was sufficiently informative to definitively implicate or exclude IFT74 mutations as a cause of chromosome 9-linked ALS-FTD, the nature of the mutation observed within F476 (predicted to truncate the protein by 258 amino acids) led us to sequence the open reading frame of this gene in a large number of ALS and FTD cases (n = 420). An additional sequence variant (G58D) was found in a case of sporadic semantic dementia. I55L sequence variants were found in three other unrelated affected individuals, but this was also found in a single individual among 800 Human Diversity Gene Panel samples.

Conclusion: Confirmation of the pathogenicity of IFT74 sequence variants will require screening of other chromosome 9p-linked families.

Figure 1

Ideogram of the Chromosome 9p ALS-FTD locus. (A) ideogram of chromosome 9 showing the linked areas defined by Morita et al, Vance et al and Yan et al in 2006. The shared region flanked by D9S1678 and D9S2154 is shaded in grey. (B) Schematic representation of the known genes and predicted transcripts in the area shared by the Dutch, Scandinavian and North American families. In red, IFT74 where sequence variants associated with ALS-FTD, ALS and FTD were identified.

Figure 2

Pedigrees showing affection status, individual IDs, and haplotypes for (a) family 476 and (b) family 2. Gender has been masked to protect the anonymity of the families. Probands are indicated by arrows; open symbol, unaffected; shaded symbol, affected; diagonal line, deceased; current age, or age at death and cause of death, is indicated. AD, Alzheimer Disease. Brackets around an allele indicate an inferred haplotype. Arrows show the limits of the haplotype.

Figure 3

(A) On the left, hemi-brain from individual III-1 of family 476 diagnosed with FTD showing marked frontal and anterior temporal lobe atrophy. On the right, a coronal slice showing severely dilated lateral ventricles with narrowed gyri and widened sulci; (B) ubiquitin-positive, tau-negative inclusions in the superficial laminae of the middle frontal gyrus (arrow, scale bar = 10 nm); (C) high power photomicrograph of a neuronal cytoplasmic inclusion; (D) high power photomicrograph of a dystrophic neurite in the neuropil; (E) high power photomicrograph of a neuronal intranuclear inclusion (C -E, bar = 5 nm); (F) Ubiquitin-positive cytoplasmic inclusions in motor neurons of the lower medulla-spinal cord (arrow, bar = 50 nm); (G) higher power photomicrograph of a neuron containing a cluster of Bunina body-like inclusions; (H) higher power photomicrograph of a loosely aggregated cytoplasmic inclusion (G & H, ubiquitin immunohistocemistry, bar = 5 nm).

Figure 4

Family 476, 95, 549 and 13 originate from North America. WT indicates a mutation-negative subject, Q342X, G58D and I55L indicate subjects carrying a heterozygous sequence variation. Currently unaffected mutation carriers have not been shown; in all instances these individuals have not yet reached the maximum age at onset observed.

Figure 5

Sequence variants in IFT74 are linked to Chromosome 9p ALS-FTD. (A) Schematic of IFT74's structure. (B) Sequence variants I55L, G58D and Q342X are shown relative to affected protein domains and are indicated by black arrowheads. Human IFT74 protein aligned with other orthologs from chimpanzee (XP_520518.1), dog (XP_531964.1), mouse (AAH23760.1) and rat (NP_001007002.1). Amino acid number is based on the human protein. (C) chromatograms showing wild type (top), forward mutant (middle) and reverse mutant (bottom) sequences of I55L (left), G58D (middle) and Q342X (right).

Figure 6

(A) Human brain soluble extracts (40 ug per lane) were separated on 4–20% SDS-PAGE gels and blotted using a goat polyclonal antibody to the C-terminus of IFT74 (Imgenex, anti-CMG1) at a final antibody concentration of 0.5 mg mL^-1. Two major bands at ~90 kDa and ~70 kDa were identified which were blocked by pre-absorption of the antibody with the immunizing peptide (data not shown), indicating specificity. (B) A. Immunofluorescent staining of primary rat cortical neurons with the same antibody showed localization of IFT74 to vesicles in the cell body and along the neuronal processes. B. Secondary antibody alone gave no signal using consistent gain and offset settings.