Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2005 Aug;77(2):280-96.
doi: 10.1086/432518. Epub 2005 Jul 6.

An autosomal dominant cerebellar ataxia linked to chromosome 16q22.1 is associated with a single-nucleotide substitution in the 5' untranslated region of the gene encoding a protein with spectrin repeat and Rho guanine-nucleotide exchange-factor domains

Kinya Ishikawa  1 , Shuta Toru, Taiji Tsunemi, Mingshun Li, Kazuhiro Kobayashi, Takanori Yokota, Takeshi Amino, Kiyoshi Owada, Hiroto Fujigasaki, Masaki Sakamoto, Hiroyuki Tomimitsu, Minoru Takashima, Jiro Kumagai, Yoshihiro Noguchi, Yoshiyuki Kawashima, Norio Ohkoshi, Gen Ishida, Manabu Gomyoda, Mari Yoshida, Yoshio Hashizume, Yuko Saito, Shigeo Murayama, Hiroshi Yamanouchi, Toshio Mizutani, Ikuko Kondo, Tatsushi Toda, Hidehiro Mizusawa
Affiliations

An autosomal dominant cerebellar ataxia linked to chromosome 16q22.1 is associated with a single-nucleotide substitution in the 5' untranslated region of the gene encoding a protein with spectrin repeat and Rho guanine-nucleotide exchange-factor domains

Kinya Ishikawa et al. Am J Hum Genet. 2005 Aug.

Abstract

Autosomal dominant cerebellar ataxia (ADCA) is a group of heterogeneous neurodegenerative disorders. By positional cloning, we have identified the gene strongly associated with a form of degenerative ataxia (chromosome 16q22.1-linked ADCA) that clinically shows progressive pure cerebellar ataxia. Detailed examination by use of audiogram suggested that sensorineural hearing impairment may be associated with ataxia in our families. After restricting the candidate region in chromosome 16q22.1 by haplotype analysis, we found that all patients from 52 unrelated Japanese families harbor a heterozygous C-->T single-nucleotide substitution, 16 nt upstream of the putative translation initiation site of the gene for a hypothetical protein DKFZP434I216, which we have called "puratrophin-1" (Purkinje cell atrophy associated protein-1). The full-length puratrophin-1 mRNA had an open reading frame of 3,576 nt, predicted to contain important domains, including the spectrin repeat and the guanine-nucleotide exchange factor (GEF) for Rho GTPases, followed by the Dbl-homologous domain, which indicates the role of puratrophin-1 in intracellular signaling and actin dynamics at the Golgi apparatus. Puratrophin-1--normally expressed in a wide range of cells, including epithelial hair cells in the cochlea--was aggregated in Purkinje cells of the chromosome 16q22.1-linked ADCA brains. Consistent with the protein prediction data of puratrophin-1, the Golgi-apparatus membrane protein and spectrin also formed aggregates in Purkinje cells. The present study highlights the importance of the 5' untranslated region (UTR) in identification of genes of human disease, suggests that a single-nucleotide substitution in the 5' UTR could be associated with protein aggregation, and indicates that the GEF protein is associated with cerebellar degeneration in humans.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Haplotype analysis in the chromosome 16q22.1–linked ADCA critical locus. Ten families (P2, P4, P6, P12, P14, and T1–T5) large enough for haplotype analysis are indicated (yellow boxes at top). A founder haplotype “3-1-4-4-4” was seen in all patients for markers 16cen-D16S421-TA001-GA001-TTTA001-CATG003-16qter, between GATA01 and 17msm. Two alleles are shown (separated by a virgule [/]) for cases in which phase could not be determined. An asterisk (*) indicates that P values show significant linkage disequilibrium by Fisher’s exact probability test. The specific C→T change in the puratrophin-1 gene lies within the founder chromosomal region.
Figure 2
Figure 2
Positional cloning of the chromosome 16q22.1–linked ADCA gene. A, Genetic and physical maps of the interval in chromosome 16q22.1, showing microsatellite markers used to refine the interval. Twenty-one genes and their direction of transcription (Ensembl) are shown. B, Nucleotide sequences of exon 1 flanking the C→T single-nucleotide change in control and patient DNA samples. The patient harbors a heterozygous C→T substitution on the sense strand. C, RFLP by digestion with EcoNI. PCR was performed with primers UK1-E1F1 and UK1-E1R1. Whereas the normal allele produces digested fragments of 209, 92, and 59 bp, the C→T change in the mutant allele disrupts one EcoNI site, producing fragments of 268 and 92 bp.
Figure 3
Figure 3
Genomic and mRNA structure of the puratrophin-1 gene. A, Intron-exon structure of puratrophin-1. Exons are shown as vertical bars, and coding regions are shown in red. The C→T change at nt 4255 (or, 16 nt upstream of the translation initiation codon) is indicated. Nucleotide numbers (indicated by a number sign [#]) are counted from the 5′ end. B, Puratrophin-1 mRNAs cloned from the human cerebellum. Major transcripts are full-length and short-form puratrophin-1 mRNAs; minor transcripts (B1–B8) were also cloned. The CRL-TRIO, spectrin, Rho GEF/DH, and PH domains are indicated. Epitopes for five rabbit anti–puratrophin-1 antibodies (FL01–FL04 and SV01) are also mapped. Antibodies FL02, FL03, and FL04 are designed to specifically detect full-length puratrophin-1, the antibody SV01 specifically detects short-form puratrophin-1, and the antibody FL01 detects both full-length and short-form puratrophin-1.
Figure 4
Figure 4
A, The recombinant, full-length puratrophin-1–HA fusion protein was first immunoprecipitated (IP) with preimmune serum (PIS) (as a negative control), anti-HA antibody (as a positive control), or anti–puratrophin-1 antibodies (FL01, FL02, FL03, and FL04) and was then tested for western blot (WB) detection with the anti-HA antibody. The anti–puratrophin-1 antibodies consistently detected the recombinant fusion protein, with an approximate molecular size of 135 kD. B, Immunoabsorption test. Antiserum against puratrophin-1 (FL01) was completely absorbed with synthetic peptide of 1 mg/liter concentration (FLO1 + peptide). Scale bars = 50 μm.
Figure 5
Figure 5
Southern-blot analysis. The probe of this experiment was ∼10 kb in size, covering the Q9H7K4 coding region. The DNA samples from the control individuals and patients (lane 1 = patient 1; lane 2 = control 1; lane 3 = patient 2; lane 4 = patient 3; lane 5 = control 2) were digested with restriction enzyme EcoRV, were separated on a 0.8% agarose gel, were transferred to nylon membrane, and were hybridized with a radio-labeled probe. In all DNA samples, fragments of ∼9 kb were detected, which suggests that there were no genomic rearrangements.
Figure 6
Figure 6
Puratrophin-1 mRNA expression in various control human tissues. With this nonquantitative RT-PCR analysis, a relatively stronger expression was observed in the testis and pancreas, whereas mild or moderate expression was seen in the spleen, thymus, prostate gland, heart, placenta, lung, liver, and kidney. Puratrophin-1 mRNA expression was low in the ovary, small intestine, colon, peripheral-blood leukocytes, whole brain, and skeletal muscle.
Figure 7
Figure 7
The consequence of a C→T change in the 5′ UTR of puratrophin-1 mRNA. A, The puratrophin-1 mRNA level assessed relative to G3PDH mRNA levels. The puratrophin-1 mRNA levels in the cerebella of individuals with chromosome 16q22.1–linked ADCA appear slightly decreased compared with levels in control individuals. B, Quantitative analysis of puratrophin-1 mRNA, with the use of the TaqMan technique in cerebellar mRNAs of four control individuals (with AD) and of two individuals with chromosome 16q22.1–linked ADCA. Both total puratrophin-1 mRNA (i.e., full-length and short-form mRNAs) and short-form puratrophin-1 mRNAs tended to be reduced in cerebella of individuals with chromosome 16q22.1–linked ADCA, although the significance was not statistically proven (P=.0641 for total mRNA; P=.1649 for short-form mRNA). C, Histogram of in vivo luciferase assay. “F/R” denotes fire-fly luciferase activity versus Renilla activity. The wild-type construct with allele C increases luciferase expression compared with an empty vector (Mock), whereas the mutant construct with allele T demonstrates significantly reduced luciferase activity (P<.001).
Figure 8
Figure 8
Expression of puratrophin-1 in control human and mouse tissues. A and B, Human testis. C and D, Human prostate gland. E and F, Human pancreas. G and H, Human kidney. I and J, Mouse cochlea. A, C, E, G, and I, Immunohistochemical analysis with use of the polyclonal anti–puratrophin-1 antibody SV01. B, D, F, H, and J, Hematoxylin and eosin stain of the section adjacent to that shown in A, C, E, G, and I, respectively. Puratrophin-1 is expressed in many tissues, with varying strength. In particular, Leydig cells in the testis (arrow in A), epithelial cells in the prostate gland (C), and Langerhans islet in the pancreas (E) showed strong immunoreactivity. Immunoreactivity in the kidney was weak (G). Expression in the cochlea was particularly intense at the sensory hairlets (arrow in I). Hair cells of the stria vascularis also showed immunoreactivity (arrowhead in I). All scale bars = 50 μm.
Figure 9
Figure 9
Immunohistochemical analysis of puratrophin-1, Golgi-apparatus membrane protein (G58K), and spectrin in control cells and samples from a patient's cerebellum. A, Puratrophin-1 expression in the cell body of Purkinje cells, visualized with use of rabbit polyclonal anti–puratrophin-1 antibody (FL01). B, With use of same antibody FL01, aggregation of puratrophin-1 (arrow), seen in a Purkinje cell of a brain affected with chromosome 16q22.1–linked ADCA. C, G58K expression, seen diffusely in the cell body of control Purkinje cells. D, G58K aggregation (arrow), morphologically quite similar to the puratrophin-1 aggregate, seen in a patient’s Purkinje cell. E, Spectrin expression, seen in the cell body of a control Purkinje cell. F, Spectrin aggregation in a patient’s Purkinje cell. All scale bars = 50 μm.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

Web Resources

    1. Celera Discovery System, http://www.celeradiscoverysystem.com/index.cfm (for candidate genes)
    1. Ensembl, http://www.ensembl.org/ (for genetic map, genomic sequences, ESTs, and 21 candidate genes, including Q9H7K4 and SLC9A5)
    1. GenBank, http://www.ncbi.nlm.nih.gov/Genbank/ (for microsatellite DNA markers GGAA10 [accession number AB13610], TTCC01 [accession number AB13611], TA001 [accession number AB13612], GA001 [accession number AB197662], and AAT01 [accession number AB13613] and full-length and short-form puratrophin-1 mRNAs [accession numbers AB197663 and AB197664])
    1. NCBI, http://www.ncbi.nlm.nih.gov/ (for DKFZP434I216 [accession numbers BC054486 and AK024475])
    1. Online Mendelian Inheritance in Man (OMIM), http://www.ncbi.nlm.nih.gov/Omim/ (for 16q-linked ADCA type III, or SCA4)

References

    1. Bergeron C, Beric-Maskarel K, Muntasser S, Weyer L, Somerville M, Percy ME (1994) Neurofilament light and polyadenylated mRNA levels are decreased in amyotrophic lateral sclerosis motor neurons. J Neuropathol Exp Neurol 53:221–230 - PubMed
    1. Charron AJ, Bacallao RL, Wandinger-Ness A (2000) ADPKD: a human disease altering Golgi function and basolateral exocytosis in renal epithelia. Traffic 1:675–686 - PubMed
    1. Chen D-H, Brkanac Z, Verlinde CLMJ, Tan X-J, Bylenok L, Nochlin D, Matsushita M, Lipe H, Wolff J, Fernandez M, Cimino PJ, Bird TD, Raskind WH (2003) Missense mutations in the regulatory domain of PKCγ: a new mechanism for dominant nonepisodic cerebellar ataxia. Am J Hum Genet 72:839–849 - PMC - PubMed
    1. Estrach S, Schmidt S, Diriong S, Penna A, Blangy A, Fort P, Debant A (2002) The human Rho-GEF trio and its target GTPase RhoG are involved in the NGF pathway, leading to neurite outgrowth. Curr Biol 12:307–312 - PubMed
    1. Flanigan K, Gardner K, Alderson K, Galster B, Otterud B, Leppert MF, Kaplan C, Ptacek LJ (1996) Autosomal dominant spinocerebellar ataxia with sensory axonal neuropathy (SCA4): clinical description and genetic localization to chromosome 16q22.1. Am J Hum Genet 59:392–399 - PMC - PubMed

Publication types

MeSH terms