Genomic sequencing analysis of the DYT3 region. a, Physical map of the DYT3 critical region on Xq13.1. Annotated genes in this region that have experimentally verified coding sequences (and their proteins) include NLGN3 (neuroligin 3), GJB1 (gap junction protein, beta-1), ZNF261 (zinc finger protein 261), NONO (non-POU domain–containing octamer-binding protein), ITGB1BP2 (melusin [integrin beta-1 binding protein 2]), TAF1 (TAF-250 [TATA-binding protein-associated factor, 250 kDa]), ING2 (inhibitor of growth 2), OGT (O-linked N-acetylglucosamine transferase), ACRC (acid repeat–containing gene), and CXCR3 (chemokine, CXC motif, receptor 3). The broken arrow indicates MTS. A unique Southern probe was designed for the HindIII-digested fragment containing the SVA retrotransposon insertion. Eight BAC clones comprising a continuous contig map cover 462,651 bp, from 66572462 to 67035112 in NCBI build 30. A total sequence length of 463,567 bp was determined, with 5.7-fold redundancy. Also shown is the distribution of 159 nucleotide variants that were identified by sequencing. None of these variants is located in any exon of these genes, including their alternative splicing forms. A red arrowhead indicates the SVA insertion. b, The SVA insertion in the XDP-related and healthy control populations. Information on the patients with XDP is given in table 1 (patients 1–13). The SVA insertion produces a 6.1-kb fragment, whereas the wild type produces a 3.4-kb fragment. Arrowheads in the gel indicate female individuals. Carriers are defined as mothers or daughters of patients with XDP. Possible carriers are daughters or sons of these carriers. NC = negative control.

Northern analysis. a, Three probes for northern hybridization to TAF1 (detailed information on these probes is given in table 3). b, Total RNA samples from the caudate and lymphoblastoid tissues. The hybridization signal seen at ∼7 kb, which represents TAF1, was observed in every lane, but a signal was never seen at the smaller sizes corresponding to MTS transcripts reported elsewhere. This result suggests that the sequences of TAF1 isoforms have such small differences in size, probably less than a few hundred base pairs, that standard northern analysis cannot discriminate between them in 1% agarose gels denatured by 2% formaldehyde. A probe for GAPDH was also hybridized to each membrane as a loading control. M = DIG-labeled molecular-weight marker. c, Relative TAF1 expression. The hybridization signal seen at ∼7 kb, representing TAF1, had a tendency toward slight reduction in patient caudate, so, to confirm this, TaqMan assays were performed.

The SVA insertion in an additional seven patients with XDP and their relatives, from four families. Information on these patients is given in table 1 (patients 14–20).

Long RT-PCR and the alternative exons of TAF1. a, Long RT-PCR analysis. The broken line indicates an expected cDNA fragment of TAF1 with the long RT primer on the end of exon 38 (short arrow). By subsequent PCR with the use of the long cDNA, six lanes—TA02, TA08, TA09, TA14, TA15, and TA18 (red bars)—showed multiple bands. Other lanes (black bars) contained single bands. Also shown is the result obtained using the MTS-specific long RT primer on its 3′ end. There was no difference between the TAF1 and MTS primers or between the patient and the control results (patient data not shown). Scale bar=1 kb. b, Ten alternative exons, including two exon skippings and one deletion, were identified by RT-PCR. The detailed sequence information for these exons is annotated in our AB191243 deposition in DNA Databank of Japan (DDBJ). Of 10 alternative exons, 3 were reported elsewhere as exons of MTS, but a form including both exon 32′ and exon 34′ was not detected. For quantitative RT-PCR, 17 TaqMan probes were designed. Two TAF-series probes (red) and 10 TA-series probes (pink) were designed to detect mainly the TAF1 common forms and alternative splicing isoforms, respectively. The other five MTS-series probes (orange) were designed to detect the MTS transcripts reported elsewhere. Var. = variant. Scale bar=5 kb.

Expression of the TAF1 isoforms in the caudate. a, Expressions are shown relative to the expression of 18S rRNA (as an internal control). The label “relative mRNA expression” means relative mRNA expression level to 1/20 × 18S rRNA. Values are expressed as means ± SEM (n=3). The TA14-391 probe showed a significant reduction in the patient’s caudate. Two-sided P values are shown. Also shown is the expression level of TA14-391 (b) and DRD2 (c) in three brain regions: caudate, accumbens, and cortex. All regions showed a significant decrease in TA14-391 expression. d, Morphometry analysis of TAF1-positive cells in XDP and control caudate nuclei. The number of each type of cell was counted in 1,000,000-μm² areas of the caudate nuclei. These were gliosis and neuronal loss in the XDP caudate nucleus. e, In situ hybridization analysis of TAF1. Although many TAF1-positive neurons were observed in both tissues, the expression level was apparently low in the patient’s caudate neurons, even when a common probe for exon 38 was used. By contrast, the expression level of TAF1 in glial cells was weak in both tissues. ACTB =β-actin; GFAP = glial fibrillary acidic protein. Strong ACTB signals were shown in glial and neuronal cells. The GFAP probe stains active glial cells, especially in the XDP caudate nucleus, because of activation by astrogliosis. In contrast, we observed no signal when using sense probes of these genes (data not shown). Scale bar=25 μm. f, TAF1 immunohistochemical staining. Nearby sections were stained with polyclonal antibodies against TAF1, calcineurin (CALN), and GFAP. The immunoreactivity of TAF1 in the XDP caudate neurons was apparently weak. Moreover, the immunoreactivity of TAF1 in glial cells was originally weak in both tissues. Similar immunoreactivity was observed in three other brain tissues from three different patients with XDP. Scale bar=25 μm.

a, Full-length cloning of a TAF1 isoform sequence containing alternative exon 34′. The 5′ end was obtained from CapSite cDNA from brain. The 3′ end was obtained from Marathon-Ready cDNA from brain. The complete DNA sequences of these long products were determined by a PCR direct-sequencing method by use of 28 redundant internal sequencing primers (red triangles). b, From the long PCR products, products of sufficient quantity and quality for PCR direct sequencing were amplified. NC = negative control without DNA template. c, Each exon 34′–specific primer was designed to have only 3 nt of exon 34′, to prevent erroneous amplification due to slippage.

Detection of methylation around the SVA insertion. a, Restriction sites around the SVA insertion. The HindIII fragment and Southern probe are identical to those in figure 1a. The SVA insertion created 47 new HpaII sites in the HindIII restriction fragment. MspI is a CpG methylase-insensitive isoschizomer of HpaII. b, Southern hybridization of genomic DNA from the patient’s caudate. The 6.1-kb HindIII fragment was shifted, by additional HpaII digestion, to an ∼4.6-kb fragment. By contrast, the HindIII fragment was completely digested, by additional MspI digestion, to a fragment of ∼2.4 kb.