Previous linkage analysis of F2 using dinucleotide marker data had revealed a single region with a lod score of ~1.5 on chromosome 9p that matched with the published chromosome 9p21.3-p13.3 ALS-FTD locus (figure ). Similarly, marker data showed a haplotype across chromosome 9p that segregated with disease status in F476 (figure ). Based on these data, F476 and F2 were selected for mutational screening of the 14 candidate genes within the previously defined 2.1 Mb region of the chromosome 9p ALS-FTD locus.
In the course of sequencing the intraflagellar transport gene (IFT74
), we identified a C to T sequence variant at nucleotide 1024 in exon 13 in the proband of F476 (III-3, figures and ). This base pair change predicts a premature stop codon at position 342 of the peptide (Q342X) truncating the last 258 residues. This variant segregated with disease within the family as it was present in the two brothers of the proband diagnosed with ALS-FTD (III-1, III-4). The Q342X sequence variant was not present in four unaffected individuals within the kindred (II-3, III-2, IV-2 & IV-4), in 1,000 chromosomes from North American controls or in 900 chromosomes from the Human Genome Diversity Panel although this C to T base pair change is ostensibly in a library clone derived from human thymus (BX436367, clone CS0CAP001YM04, Life Technologies, Inc). One younger unaffected individual carried the Q342X sequence variant. We did not find IFT74
sequence variants in F2 by sequencing or exonic gene dosage methods (data not shown). Neither F476 or F2 had additional mutations in the other 13 genes within the 9p locus defined by the Dutch, Scandinavian and North American families[14
] or in 47 additional genes in the larger haplotype.
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 (more ...)
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 (more ...)
In order to assess the prevalence of IFT74 sequence variants, we sequenced the entire coding region of this gene in a large number of ALS, ALS-FTD and FTD samples (Table ). The Q342X mutation was not found in any of these patients. However, we identified a G58D (nt173 G to A) sequence variant in a Caucasian woman who developed sporadic semantic dementia without motor involvement at the age of 58 (II-1, figures and ). This G58D sequence variant was not found in 900 chromosomes from North American controls or in 1,600 chromosomes from the HGDP. We also identified an I55L sequence variant (nt163 A to T) in three additional affected probands; first, the I55L sequence variant was found in the proband of F549 who had been diagnosed with FTD at the age of 62 (figures and ). His 80-year-old brother (II-7) had been diagnosed with FTD at the age of 75 and carried the I55L sequence variant. A 70-year-old apparently unaffected sister (II-12) also carried the I55L sequence variant; second, the proband of F194, a 59-year-old Caucasian woman diagnosed with ALS-FTD, had the I55L sequence variant. Her sister had died of FTD and her paternal uncle had died of ALS-FTD (DNA samples not available); third, the I55L sequence variant was found in a 67-year-old man with sporadic ALS. He had presented one year before his death with bulbar symptoms including emotional lability, and had become increasingly withdrawn and indifferent to his symptoms during the course of his illness. I55L was not found in 900 chromosomes from North American controls, but it was found in 1 of 1,600 chromosomes from the HGDP in a French sample.
We proceeded to verify that IFT74 is expressed in brain. Refseq (NCBI) provisionally reports the IFT74 gene to contain 14 coding exons based on NM_025103. We sequenced human brain derived cDNA and found a T to A transversion at nt599 of NM_025103 (primers available upon request). This base pair substitution converts a stop codon at position 159 to lysine indicating that IFT74 contains 19 exons encoding a 600 residue protein with a predicted molecular weight of 69.2 kDa. This larger coding region of IFT74 was confirmed by deposited sequences AY040325 and CR617782, which show lysine at codon position 159. Western blotting of human brain lysates with a polyclonal antibody against a peptide from the C-terminus of IFT74 demonstrated two major bands at ~90 kDa and ~70 kDa, of which the lower band is the most prominent (figure ). Both of these bands were blocked by pre-absorption of the antibody with the immunizing peptide (data not shown), indicating specificity. These data show that this gene is expressed throughout the adult human brain and also confirmed that IFT74 contains 19 exons. Staining of primary rat cortical neurons with the same antibody demonstrated that IFT74 is localized to vesicles in the cell body and along the neuronal processes (figure ).
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 (more ...)