Dagmar Nolte

Mutations in voltage-gated potassium channel KCNC3 cause degenerative and developmental central nervous system phenotypes

Potassium channel mutations have been described in episodic neurological diseases. We report that K+ channel mutations cause disease phenotypes with neurodevelopmental and neurodegenerative features. In a Filipino adult-onset ataxia pedigree, the causative gene maps to 19q13, overlapping the SCA13 disease locus described in a French pedigree with childhood-onset ataxia and cognitive delay. This region contains KCNC3 (also known as Kv3.3), encoding a voltage-gated Shaw channel with enriched cerebellar expression. Sequencing revealed two missense mutations, both of which alter KCNC3 function in Xenopus laevis expression systems. KCNC3R420H, located in the voltage-sensing domain, had no channel activity when expressed alone and had a dominant-negative effect when co-expressed with the wild-type channel. KCNC3F448L shifted the activation curve in the negative direction and slowed channel closing. Thus, KCNC3R420H and KCNC3F448L are expected to change the output characteristics of fast-spiking cerebellar neurons, in which KCNC channels confer capacity for high-frequency firing. Our results establish a role for KCNC3 in phenotypes ranging from developmental disorders to adult-onset neurodegeneration and suggest voltage-gated K+ channels as candidates for additional neurodegenerative diseases.

Specific sequence changes in multiple transcript system DYT3 are associated with X-linked dystonia parkinsonism

X-linked dystonia parkinsonism (XDP) is an X-linked recessive adult onset movement disorder characterized by both dystonia and parkinsonism. We report delineation of the disease gene within a 300-kb interval of Xq13.1 by allelic association. Sequencing of this region in a patient revealed five disease-specific single-nucleotide changes (here referred to as DSC) and a 48-bp deletion unique to XDP. One of the DSCs is located within an exon of a not previously described multiple transcript system that is composed of at least 16 exons. There is a minimum of three different transcription start sites that encode four different transcripts. Two of these transcripts include distal portions of the TAF1 gene (TATA-box binding protein-associated factor 1) and are alternatively spliced. Three exons overlap with ING2 (a putative tumor suppressor) and with a homologue of CIS4 (cytokine-inducible SH2 protein 4), both of which are encoded by the opposite strand. Although all DSCs are located within this multiple transcript system, only DSC3 lies within an exon. This exon is used by all alternative transcripts making a pathogenic role of DSC3 in XDP likely. The multiple transcript system is therefore referred to as DYT3 (disease locus in XDP).

Human O-GlcNAc transferase (OGT): genomic structure, analysis of splice variants, fine mapping in Xq13.1.

O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) catalyzes polypeptide glycosylation by the addition of Nacetylglucosamine to serine and threonine residues. This process is referred to as O-GlcNAcylation and has been observed to occur on many nuclear and cytosolic proteins. These include RNApolymerase II, nuclear pore proteins, neurofilaments, microtubuleassociated proteins such as tau, synapsin I, and both oncoand tumor suppressor proteins. O-GlcNAcylation is a dynamic process and appears to play an important regulatory role in the cell comparable to phosphorylation. In some cases, O-GlcNAcylation and phosphorylation even have reciprocal functions on the same molecule, e.g., in the regulation of transcription by modification of RNA polymerase II (reviewed by Wells et al. 2001). OGT has been reported to be a heterotrimer composed of two catalytic 110-kDa subunits and of one 78-kDa subunit. There is evidence that the 78-kDa subunit originates from the larger subunit by a combination of alternative RNA splicing and specific proteolysis (Kreppel et al. 1997). An important characteristic of OGT are tetratricopeptide repeats (TPR) in its N-terminal portion. TPRs are composed of subunits of 34 amino acids that contain the loosely conserved residues W-L-G-Y-A-F-A-P (Lamb et al. 1995; Das et al. 1998). Crystal structure analysis suggests that each repeat forms a pair of antiparallel -helices that act as superhelical structure in protein-protein interactions (Das et al. 1998). OGT is highly conserved in all eukaryotes tested, including cenorhabditis, mouse, rat, and humans. While the transcripts reported are highly homologous in their 3 portions among the various species, there appear to be discrepancies at the 5 ends. Thus, the purported full-length human OGT (hOGT) cDNA codes for a protein of 920 amino acids (Lubas et al. 1997). This is significantly smaller than the protein of 1037 amino acids encoded by the fulllength rat cDNA (Kreppel et al. 1997). OGT is ubiquitously expressed, with the highest levels of mRNA detected in human pancreas (Lubas et al. 1997). There are several alternative transcripts of OGT. Four transcripts of 8.0 kb, 6.0 kb, 4.2 kb, and 1.7 kb have been described in the rat (Kreppel et al. 1997), and transcripts of 9.3 kb, 7.9 kb, 6.3 kb, and 4.4 kb were reported in humans (Lubas et al. 1997). OGT is encoded by one gene that has been assigned to the proximal long arm of the X Chromosome (Chr) (Xq13.1) in humans (Nemeth et al. 1999; Shafi et al. 2000). In order to clarify the apparent differences in size between rat and human OGT transcripts, we analyzed the human gene at both the genomic and transcript level. We describe the complete genomic structure of human OGT, give the composition of its alternative transcripts, and describe its precise location in Xq13.1 with respect to flanking genes. The DNA of a considerable portion of Xq13.1 has been sequenced (AL590763), and 18 exons corresponding to the reported full-length hOGT cDNA (Lubas et al. 1997) were identified in silico (exons 6–23 of Fig. 1). We screened a human fetal brain 5 stretch plus cDNA library (Clontech, Cat. HL5015b) with a 490-bp probe corresponding to exons 8–11 (Fig. 1). Three different clones with inserts of 1.6 kb, 2.0 kb, and 2.3 kb were obtained. The 2.0-kb insert revealed an additional 700-bp 5 sequence, but did not contain the published start codon. Comparison of this 700-bp cDNA sequence with the genomic sequence (AL590763) revealed additional four exons (exons 1–4 of Fig. 1), with exon 1 containing an ATG start codon. These four exons have not been described as part of hOGT previously. In an attempt to isolate phage clones with larger inserts of hOGT cDNA, we screened the human fetal brain large insert cDNA library (Clontech, Cat. HL5504U), using exons 1–3 (Fig. 1) as a probe. We purified seven different phage clones with insert sizes ranging from 4.2 kb to 5.6 kb. Analysis of the cDNA inserts revealed alternative splicing of exon 2. This exon exists as a 181bp variant (exon 2a) and a truncated in-frame variant of 151 bp (exon 2b), in which the first 10 amino acids encoded by exon 2a are deleted. We also discovered phage inserts with and without the 3.27-kb intervening sequence (IVS4) between exons 4 and 6 (Fig. 1). Presence and absence of this IVS was independent of the type of exon 2 used. Although this IVS4 (Fig. 1) does not include an open reading frame (ORF), several ESTs (i.e., T78105, L44477) have been mapped to this region. Since IVS4 can be transcribed (also see below), we alternatively refer to it as exon 5 (Fig. 1). In order to analyze all splice variants of OGT, we performed Northern blot analyses using a commercially available blot (Clontech, Cat # 7760-1). Hybridization with a probe derived from exons 13–16 (Fig. 1) revealed five transcripts of 9.5 kb, 8.0 kb, 6.4 kb, 4.4 kb, and 4.2 kb (Fig. 2A). This corresponds to the previously described alternative transcripts (Kreppel et al. 1997; Lubas et al. 1997). Unlike previous studies that reported one transcript of about 4.4 kb (Lubas et al. 1997) or about 4.2 kb (Kreppel et al. 1997) only, we detected two transcripts of 4.2 kb and 4.4 kb, possibly owing to a higher resolution. Expression was highest in pancreas, with the 9.5-kb and 8.0-kb transcripts being most abundant. In heart, brain, skeletal muscle, kidney, lung, and liver the overall expression was less and most pronounced for the 9.5-kb and 6.4-kb transcripts (Fig. 2A). Using exons 1–3 as a probe (Fig. 2B), a hybridization pattern comparable to that obtained with a probe derived from exons 13– 16 (Fig. 2A) was detected. The exception was very faint hybridization to the 4.4-kb and 4.2-kb transcripts. The results clearly demonstrate that the human 9.5-kb, 8.0-kb, and 6.4-kb transcripts contain the proximal exons that we reported here (see above). A strikingly different hybridization pattern was obtained with a probe generated from EST T78105 in the distal portion of exon 5 that is devoid of an ORF (Fig. 1; see above). This probe detects only the two largest transcripts of 9.5 kb and 8.0 kb (Fig. 2C). Another probe from a more proximal region of exon 5 resulted in The nucleotide sequence data reported in this paper have been submitted to GenBank and have been assigned accession number AJ315767.

First case of X-linked dystonia-parkinsonism (“Lubag”) to demonstrate a response to bilateral pallidal stimulation

“Lubag” or X‐linked dystonia‐parkinsonism (XDP) is a genetic syndrome afflicting Filipino men. Intracranial surgical procedures for Lubag have been unsuccessful. We report a 45‐year‐old Filipino male with genetically confirmed XDP who underwent bilateral pallidal deep brain stimulation (DBS) surgery. The patient started to exhibit improvement on initial programming, most notably of his severe jaw‐opening dystonia. At 1‐year follow‐up, his Burke‐Fahn‐Marsden dystonia score and motor Unified Parkinsons Disease Rating Scale score were improved by 71% and 62%, respectively, with the stimulators on compared to stimulators off state. Bilateral pallidal DBS may be a viable option for Lubag patients with medically refractory symptoms.

Subthalamic-thalamic DBS in a case with spinocerebellar ataxia type 2 and severe tremor—A unusual clinical benefit

This is a single case report of a patient with spinocerebellar ataxia type 2 (SCA2) and severe tremor. Whereas disease progression with prevailing ataxia and dysmetria was slow over the first symptomatic 6 years, 6 months prior to operation were characterized by the development of a severe, debilitating postural tremor rendering the patient unable to independently sit, stand, speak, or swallow. Deep brain stimulation (DBS) at a subthalamic–thalamic electrode position almost completely arrested her tremor. The patient regained the functional state prior to her rapid disease progression allowing a restricted range of daily activities. Her condition has remained approximately stable over the two postoperative years to date. In addition to the efficacy of DBS on cerebellar tremor, the results illustrate a remarkable improvement of the patients general condition and independence.

An autosomal dominant ataxia maps to 19q13: Allelic heterogeneity of SCA13 or novel locus?

The autosomal dominant spinocerebellar ataxias (ADCAs) represent a growing and heterogeneous disease phenotype. Clinical characterization of a three-generation Filipino family segregating a dominant ataxia revealed cerebellar signs and symptoms. After elimination of known spinocerebellar ataxia (SCA) loci, a genome-wide linkage scan revealed a disease locus in a 4-cM region of 19q13, with a 3.89 lod score. This region overlaps and reduces the SCA13 locus. However, this ADCA is clinically distinguishable from SCA13.

AFX1 and p54nrb: fine mapping, genomic structure, and exclusion as candidate genes of X-linked dystonia parkinsonism.

Abstract We have mapped AFX1 and p54nrb to a yeast artificial chromosome (YAC) contig of Xq13.1 that harbors the X-linked dystonia parkinsonism (XDP) locus DYT3. AFX1 is flanked by loci DXS7116 and Il2Rγ, and p54nrb by loci DXS6673E and DXS7120. The exon-intron structure of both genes was analyzed. AFX1 is composed of three exons with most of exon 3 being untranslated. p54nrb is made up of 12 exons ranging in size from 40 bp to 1227 bp. The start codon is in exon 3 and the stop codon in exon 12. Both genes are expressed in the brain, among other tissues. AFX1 and p54nrb were excluded as candidates of DYT3 by sequencing of the exons and the flanking intronic sequences in an XDP patient and a control, and by Northern blot analysis.

Structural and functional analysis of the human TAF1/DYT3 multiple transcript system.

We analyzed TAF1/DYT3, a complex transcript system that is composed of at least 43 exons. Thirty-eight exons code for TATA box binding protein associated factor I (TAF1). Five downstream exons (d1–d5) of yet unknown function can either form transcripts with TAF1 exons or be transcribed independently. Splice variants can include d (notably d3 and d4) plus at least 12 TAF1 exons (exons 26–37 but not exon 38). These splice variants are highly polymorphic and include alternative exons (e.g., exons 30b, 31b, 32′, 34′, 35′). The frequency of these splice variants differs greatly in human fetal brain. Data were obtained by both RT-PCR and construction of a plasmid cDNA library. Promoter assays performed in NT2/D1 and in U87 cells demonstrate that TAF1-independent transcription of exons d2–d4 is driven by a TATA box-less promoter that is regulated by transcription factor Ikaros. Antisense transcription of exon d4 is under the control of a LTR promoter. While the 38 exons encoding TAF1 have been highly conserved in eukaryotes, the downstream exons d1–d5 were added to the transcript system much later during evolution and first appear in primates. The study demonstrates the structural and functional evolution of a complex transcript system.

A Novel Missense Mutation in AFG3L2 Associated with Late Onset and Slow Progression of Spinocerebellar Ataxia Type 28

SCA28 is caused by mutations in the AFG3L2 gene. This gene encodes a subunit of the mitochondrial metalloprotease AFG3L2 (AFG3-like protein 2). Clinical features of SCA28 include slow to moderate progressive ataxia, dysarthria, and additional symptoms such as nystagmus, slow saccades, and increased deep tendon reflexes. Here, we report on a novel AFG3L2 mutation in a patient with slowly progressive ataxia and a positive family history. The nucleotide change results in the substitution of an evolutionarily highly conserved tyrosine by histidine (p.Y689H) in the M41 peptidase domain of AFG3L2.

Spinocerebellar ataxia 14: Novel mutation in exon 2 of PRKCG in a German family

We describe a novel mutation in the gene coding for protein kinase C gamma (PRKCG) in patients of a German family affected with slowly progressive gait ataxia, dysarthria, and nystagmus. The G/T missense mutation occurred in exon 2 of PRKCG and results in a substitution of glycine by valine (G63V) in the evolutionarily highly conserved cysteine‐rich region 1/C1 domain of PRKCG. Among the 20 mutations described to date, this is the first mutation located in exon 2 of PRKCG.