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

Momeni P, Schymick J, Jain S, Cookson MR, Cairns NJ, Greggio E, Greenway MJ, Berger S, Pickering-Brown S, Chiò A, Fung HC, Holtzman DM, Huey ED, Wassermann EM, Adamson J, Hutton ML, Rogaeva E, St George-Hyslop P, Rothstein JD, Hardiman O, Grafman J, Singleton A, Hardy J, Traynor BJ - BMC Neurol (2006)

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

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Neurogenetics, National Institute of Aging, NIH, Bethesda, MD, USA. momeni@mail.nih.gov <momeni@mail.nih.gov>

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.

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Related in: MedlinePlus

(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.
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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.

Mentions: 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 6a). 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 6b).


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

Momeni P, Schymick J, Jain S, Cookson MR, Cairns NJ, Greggio E, Greenway MJ, Berger S, Pickering-Brown S, Chiò A, Fung HC, Holtzman DM, Huey ED, Wassermann EM, Adamson J, Hutton ML, Rogaeva E, St George-Hyslop P, Rothstein JD, Hardiman O, Grafman J, Singleton A, Hardy J, Traynor BJ - BMC Neurol (2006)

(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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1764752&req=5

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.
Mentions: 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 6a). 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 6b).

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

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Neurogenetics, National Institute of Aging, NIH, Bethesda, MD, USA. momeni@mail.nih.gov <momeni@mail.nih.gov>

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.

Show MeSH
Related in: MedlinePlus