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. 2001 Mar;68(3):598-605.
doi: 10.1086/318810. Epub 2001 Jan 22.

Tissue-specific expression of a splicing mutation in the IKBKAP gene causes familial dysautonomia

S A Slaugenhaupt  1 , A Blumenfeld, S P Gill, M Leyne, J Mull, M P Cuajungco, C B Liebert, B Chadwick, M Idelson, L Reznik, C Robbins, I Makalowska, M Brownstein, D Krappmann, C Scheidereit, C Maayan, F B Axelrod, J F Gusella
Affiliations

Tissue-specific expression of a splicing mutation in the IKBKAP gene causes familial dysautonomia

S A Slaugenhaupt et al. Am J Hum Genet. 2001 Mar.

Abstract

Familial dysautonomia (FD; also known as "Riley-Day syndrome"), an Ashkenazi Jewish disorder, is the best known and most frequent of a group of congenital sensory neuropathies and is characterized by widespread sensory and variable autonomic dysfunction. Previously, we had mapped the FD gene, DYS, to a 0.5-cM region on chromosome 9q31 and had shown that the ethnic bias is due to a founder effect, with >99.5% of disease alleles sharing a common ancestral haplotype. To investigate the molecular basis of FD, we sequenced the minimal candidate region and cloned and characterized its five genes. One of these, IKBKAP, harbors two mutations that can cause FD. The major haplotype mutation is located in the donor splice site of intron 20. This mutation can result in skipping of exon 20 in the mRNA of patients with FD, although they continue to express varying levels of wild-type message in a tissue-specific manner. RNA isolated from lymphoblasts of patients is primarily wild-type, whereas only the deleted message is seen in RNA isolated from brain. The mutation associated with the minor haplotype in four patients is a missense (R696P) mutation in exon 19, which is predicted to disrupt a potential phosphorylation site. Our findings indicate that almost all cases of FD are caused by an unusual splice defect that displays tissue-specific expression; and they also provide the basis for rapid carrier screening in the Ashkenazi Jewish population.

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Figures

Figure 1
Figure 1
Genomic structure of IKBKAP, showing orientation and placement of the 37 exons within a 68-kb genomic region of chromosome 9q31. The primers used for analysis of the splice defect are denoted as “18F” (exon 18), “19F” (exon 19), and “23R” (exon 23). The asterisks (*) indicate the locations of the two mutations identified; the mutation associated with the major AJ haplotype is located at base pair 6 of intron 20, whereas the mutation association with the minor AJ haplotype is located at base pair 73 of exon 19. The “4.8” and “5.9” designations at exon 37 indicate the lengths (in kb) of the two IKBKAP messages that differ only in the length of their 3′ UTRs.
Figure 2
Figure 2
Demonstration of mutations in IKBKAP. a, Antisense sequence of the T→C mutation (indicated by arrows adjacent to lanes G and A) at base pair 6 of intron 20, which is associated with the major FD haplotype. Lanes 1 and 2, patients with FD who are homozygous for the major haplotype (homozygous GG); lane 3, patient with FD who is heterozygous for the major haplotype and minor haplotype 2 (heterozygous GA); lane 4, patient with FD who is heterozygous for the major haplotype and minor haplotype 3 (heterozygous GA); lanes 5 and 6, control individuals (homozygous AA). b, Heterozygosity for the G→C mutation (indicated by arrows adjacent to lanes G and C) at base pair 73 of exon 19. Lane 1, patient with FD who is homozygous for the major haplotype (homozygous GG); lanes 2–4, patients with FD who are heterozygous for the major haplotype and minor haplotype 2 (heterozygous GC); lane 5, patient with FD who is heterozygous for the major haplotype and minor haplotype 3 (homozygous GG); lane 6, control individual (homozygous GG). c, Sequence of cDNA generated from the RT-PCR of a patient with FD who is heterozygous for the major and minor 2 haplotypes. The arrow points to the heterozygous G→C mutation in exon 19. The boundary of exons 19 and 20 is also indicated, illustrating that this patient expresses wild-type message that includes exon 20, despite the presence of the major mutation on one allele.
Figure 3
Figure 3
Northern blot analysis of IKBKAP. a, Human multiple-tissue northern blot hybridized with IKBKAP exon 2, showing the presence of two messages, of 4.8 and 5.9 kb (northern blots hybridized with other IKBKAP probes yielded similar patterns). b, Northern blot generated by use of mRNA isolated from lymphoblast cell lines: lanes 1, 2, and 5, patients with FD who are homozygous for the major haplotype; lane 3, individual carrying two definitively non-FD chromosomes; lane 4, patient with FD who is heterozygous for the major haplotype and minor haplotype 2; lane 6, control brain RNA (Clontech). The level of expression of IKBKAP mRNA relative to β-actin mRNA is quite variable in lymphoblasts. No consistent increase or decrease in mRNA levels was observed between patients with FD who are homozygous for the major haplotype, patients with FD who are heterozygous for the major haplotype and minor haplotype 2, and control individuals.
Figure 4
Figure 4
RT-PCR analysis of the exon 20 region of IKBKAP, showing expression of the wild-type message and protein in patients. a, Results of use of primers 18F (exon 18) and 23R (exon 23). Lanes 1 and 2, patients with FD who are homozygous for the major haplotype; lane 3, patient with FD who is heterozygous for the major haplotype and minor haplotype 2; lanes 4 and 5, control individuals; lane 6, water control. b, Results of western blot generated by use of cytoplasmic protein isolated from lymphoblast cell lines of patients with FD and detected with a carboxyl-terminal antibody. Lane 1, patient with FD who is heterozygous for the major and minor haplotype 3; lanes 2, 4, 6, and 8, patients with FD who are homozygous for the major haplotype; lanes 3, 5, 7, and 9, controls individuals; lane 10, patient with FD who is heterozygous for the major and minor haplotype 2; lane 11, Hela cell–line sample.
Figure 5
Figure 5
RT-PCR analysis of the exon 20 region of IKBKAP, showing variable expression of the mutant message in patients with FD patients. The analysis was done by use of primers 19F (exon 19) and 23F (exon 23). Lanes 1 and 2, control fibroblasts; lanes 3–5, fibroblasts of patients with FD who are homozygous for the major mutation; lanes 6 and 7, lymphoblasts of patients with FD who are homozygous for the major mutation; lanes 8 and 9, control lymphoblasts; lane 10, brain stem of patient with FD; lane 11, temporal lobe of patient with FD (showing a faint 319-bp band and no 393-bp band); lane 12, water control. RT-PCR of control brain RNA (Clontech) showed only the 393-bp band (data not shown).
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References

Electronic-Database Information

    1. BLAST, http://www.ncbi.nlm.nih.gov/BLAST/
    1. FlyBase, http://flybase.bio.indiana.edu/
    1. GenBank, http://www.ncbi.nlm.nih.gov/Genbank (for published IKBKAP sequence [accession number AF044195] and 5.9-kb IKBKAP cDNA sequence [accession number AF153419])
    1. GENSCAN Web Server, http://genes.mit.edu/GENSCAN.html
    1. Online Mendelian Inheritance in Man (OMIM), http://www.ncbi.nlm.nih.gov/Omim/ (for FD (MIM 223900])

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