Ingrid K. Svenson

A Kinesin Heavy Chain (KIF5A) Mutation in Hereditary Spastic Paraplegia (SPG10)

We have identified a missense mutation in the motor domain of the neuronal kinesin heavy chain gene KIF5A, in a family with hereditary spastic paraplegia. The mutation occurs in the family in which the SPG10 locus was originally identified, at an invariant asparagine residue that, when mutated in orthologous kinesin heavy chain motor proteins, prevents stimulation of the motor ATPase by microtubule-binding. Mutation of kinesin orthologues in various species leads to phenotypes resembling hereditary spastic paraplegia. The conventional kinesin motor powers intracellular movement of membranous organelles and other macromolecular cargo from the neuronal cell body to the distal tip of the axon. This finding suggests that the underlying pathology of SPG10 and possibly of other forms of hereditary spastic paraplegia may involve perturbation of neuronal anterograde (or retrograde) axoplasmic flow, leading to axonal degeneration, especially in the longest axons of the central nervous system.

Serotonin-Related Gene Polymorphisms and Central Nervous System Serotonin Function *

Central nervous system (CNS) serotonergic function affects a wide range of biological and behavioral functions affecting health and disease. Our objective in this study was to determine whether functional polymorphisms of the genes that encode for the serotonin transporter promoter (5HTTLPR) and monoamine oxidase A (MAOA-uVNTR) are associated with CNS serotonin turnover—indexed by cerebrospinal fluid levels of 5-hydroxyindoleacetic acid (5-HIAA)—in a community sample of healthy adults. Subjects were 165 community volunteers without current medical or psychiatric illness, stratified with respect to ethnicity, gender, and socioeconomic status who underwent inpatient evaluation in the General Clinical Research Center of a university medical center. A significant ethnicity×genotype interaction (P=0.008) indicated that, compared to the long/long and long/short genotypes, the 5HTTLPR short/short genotype was associated with higher CSF 5-HIAA levels in African Americans, but with lower levels in Caucasians. A gender×genotype interaction (P=0.04) indicated that 5HTTLPR short/short genotype was associated with higher 5-HIAA levels in women but with lower levels in men. MAOA-uVNTR 3.5 and 4 repeat alleles were associated with higher 5-HIAA (P=0.03) levels in men, but were unrelated to 5-HIAA levels in women. These findings suggest that effects of serotonin-related gene polymorphisms on CNS serotonergic function vary as a function of both ethnicity and gender. Further research will be required to determine the mechanism(s) underlying these differential effects. In the meanwhile, both ethnicity and gender should be taken into account in research evaluating effects of these and related polymorphisms on CNS serotonergic function, as well as the broad range of biological and behavioral functions that are regulated by CNS serotonergic function.

Central nervous system serotonin function and cardiovascular responses to stress.

Objective The objective of this study was to evaluate the impact of indices of central nervous system (CNS) serotonin function on cardiovascular reactivity to mental stress. Methods Lumbar puncture was performed on 54 healthy volunteers to obtain cerebrospinal fluid (CSF) for determination of 5-hydroxyindoleacetic acid (5HIAA) levels. Genotypes were determined with respect to a functional polymorphism of the serotonin transporter gene promoter region (5HTTLPR). Subjects then underwent mental stress testing. Results Persons with one or two long (l) 5HTTLPR alleles had CSF levels of the major serotonin metabolite, 5HIAA, that were 50% higher than those of persons with the s/s 5HTTLPR genotype. Persons with one or two l alleles or higher CSF 5HIAA levels also exhibited greater blood pressure and heart rate responses to a mental stress protocol. Conclusions These findings suggest the 5HTTLPR polymorphism affects CNS serotonin function, and they are consistent with the general hypothesis that CNS serotonin function is involved in the regulation of potentially health-damaging biobehavioral characteristics. In particular, the l allele could contribute, through its association with increased cardiovascular reactivity to stress, to increased risk of cardiovascular disease.

Intragenic modifiers of hereditary spastic paraplegia due to spastin gene mutations

Hereditary spastic paraplegia (HSP) is a genetically heterogeneous neurodegenerative disease characterized by wide variability in phenotypic expression, both within and among families. The most-common cause of autosomal dominant HSP is mutation of the gene encoding spastin, a protein of uncertain function. We report the existence of intragenic polymorphisms of spastin that modify the HSP phenotype. One (S44L) is a previously described recessively acting allele and the second is a novel allele affecting the adjacent amino acid residue (P45Q). In 4 HSP families in which either L44 or Q45 segregates independently of a missense or splicing mutation in the AAA domain of spastin, L44 and Q45 are each associated with a striking decrease in age at onset in the presence of the AAA domain mutations. Using a bioinformatics approach, we found that the highly conserved S44 is predicted to be phosphorylated by a number of family members of the proline-directed serine/threonine cyclin-dependent kinases (Cdks). Cdk1 and Cdk5 showed no kinase activity toward synthetic spastin peptide in an in vitro kinase assay, suggesting that this serine residue may be phosphorylated by a different Cdk. Our identification of S44L and P45Q as modifiers of the HSP phenotype suggests a role for spastin phosphorylation by Cdks in the neurodegeneration of the most-common form of HSP.

Childhood socioeconomic status and serotonin transporter gene polymorphism enhance cardiovascular reactivity to mental stress.

Objective: To test the hypothesis that low socioeconomic status (SES) and the 5HTTLPR L allele are associated with increased cardiovascular reactivity (CVR) to stress in a larger sample and that SES and 5HTTLPR genotypes interact to enhance CVR to stress. CVR to mental stress has been proposed as one mechanism linking stress to the pathogenesis of cardiovascular disease. The more transcriptionally efficient long (L) allele of a polymorphism of the serotonin transporter gene promoter (5HTTLPR) has been found associated with increased risk of myocardial infarction. We found the long allele associated with larger CVR to mental stress in a preliminary study of 54 normal volunteers. Methods: Subjects included 165 normal community volunteers stratified for race, gender, and SES, who underwent mental stress testing. Results: Childhood SES as indexed by Father’s Education Level was associated with larger systolic blood pressure (SBP) (p < .05) and diastolic blood pressure (DBP) (p = .01) responses to mental stress. The L allele was associated with larger SBP (p = .04), DBP (p < .0001), and heart rate (p = .04) responses to mental stress compared with the short (S) allele. Subjects with the SS genotype and high Father’s Education exhibited smaller SBP (5.2 mm Hg) and DBP (2.9 mm Hg) responses than subjects with LL genotype and low Father’s Education (SBP = 13.3 mm Hg, p = .002; DBP = 9.7 mm Hg, p < .0001). Conclusions: Both the 5HTTLPR long allele and low SES, particularly during childhood, are associated with increased CVR to mental stress, which could account, at least in part, for the increased cardiovascular disease risk associated with these characteristics. If confirmed in further research, these characteristics could be used to identify persons who might benefit from preventive interventions. CVD = cardiovascular disease; CVR = cardiovascular reactivity; 5HTTLPR = serotonin transporter promoter polymorphism; SBP = systolic blood pressure; DBP = diastolic blood pressure; HR = heart rate; SES = socioeconomic status; GCRC = General Clinical Research Center; CNS = central nervous system.

Allelic differences in the serotonin transporter-linked polymorphic region in geriatric depression.

Previous studies have examined the role of genetic variations in the serotonin transporter-linked polymorphic region (5HTTLPR) in affective disorders. The authors studied 182 older depressed subjects and 107 elderly control subjects and obtained DNA for genotyping at the 5HTTLPR. There were no significant differences in allele frequencies generally or for number of short alleles for the group as a whole, but interesting gender effects emerged. Among men, 23% of depressed men had two short alleles, compared with only 5% of control subjects. Among women, 67% of depressed women with more than one episode had at least one short allele, compared with 41% of single-episode female patients. Also, 74% of women with a positive family history of psychiatric illness in any female relative had at least one short allele, whereas 53% had at least one short allele who did not have such a family history. Our results add to the literature linking this gene to affective illness. The negative association of allele frequency and depression may be related to the relatively small sample size. The findings raise the possibility that this genetic locus may exert differential effects based on gender, increasing risk in men, and increasing risk of recurrence in women.

A Second Leaky Splice-Site Mutation in the Spastin Gene

To the Editor: Mutations in the gene encoding spastin, an ATPase of unknown function, cause the most common form of autosomal dominant hereditary spastic paraplegia (SPG4 [MIM 182601]; Hazan et al. 1999), a neurodegenerative disorder characterized by progressive spasticity of the lower limbs. Recently, we described 11 mutations in this gene, 7 of which cause missplicing of the spastin transcript (Svenson et al. 2001). Because of the presence of a rare spastin gene–coding region polymorphism in the proband of one family with an IVS9+4a→g mutation in the spastin gene, we were able to determine that this mutation is only partially penetrant—that is, both normally and aberrantly spliced (skipped exon 9) transcripts are produced from the mutant allele. This “leakiness” of the mutation was not recapitulated in in vitro splicing experiments with minigene constructs containing only exon 9 and flanking intronic sequence. However, when we included additional sequence (exons 8–12, with flanking intronic sequence), the leakiness of the mutation was recapitulated. These findings demonstrate the importance of genomic-sequence context in the determination of splice-site selection and the extent of missplicing caused by this mutation. Because of the absence of any other spastin gene–coding region polymorphisms, we were unable to test several other splice-site mutations for leakiness by reverse-transcriptase (RT)–PCR analysis of patient-derived RNA. None of these other mutations appeared leaky in our in vitro splicing experiments with minigene constructs, even in the larger genomic-sequence context that included flanking exons. We have since found that at least one of these other splice-site mutations is, in fact, leaky in a much larger genomic-sequence context—that is, in the complete human chromosome 2 that harbors the mutation. This mutation, an IVS11+2t insertion, causes skipping of exon 11, as determined by RT-PCR analysis of patient-derived RNA (see the report by Svenson et al. [2001], for pedigree information and experimental data). In our in vitro splicing experiments with a minigene construct that carries this mutation as well as the complete sequence of both flanking introns and exons on each side of the skipped exon, we detected only aberrantly spliced (skipped exon 11) transcript (fig. 1A). To determine whether the presence of additional genomic-sequence context would affect the change in splice-site selection caused by this mutation, we performed RT-PCR analysis of RNA extracted from mouse-human hybrid cell lines containing either the wild-type or the mutant chromosome 2 of a patient with SPG who is heterozygous for this mutation. As shown in figure 1B, we detected both normally and aberrantly spliced transcripts in the RNA extracted from the hybrid cell line containing the mutant—but not the wild-type—chromosome 2. We confirmed through sequencing that the transcripts were derived from the human and not the mouse spastin gene. We were unable to test the other splice-site mutations for leakiness by this method, because of the lack of availability of appropriate patient-derived cell lines. Figure 1 A, The pSPL3 exon-trapping vector was used to assay splice-site selection. COS1 cells were transfected with vector alone (pSPL3), with wild-type exons 10–12 and flanking intronic sequence (wild type), or with the same construct but containing ... These findings provide further evidence of the dependence of splice-site selection on genomic-sequence context. The exon-definition model of splice-site selection proposes that an exon is defined by the binding of splicing factors to the 3′ and 5′ splice sites of the same exon (Berget 1995). Pairing of U1 small nuclear RNA (snRNA) to the 5′ splice junction includes at least six nucleotides beyond the invariant gt. The IVS11+2t insertion mutation would shift the base pairing by one nucleotide, resulting in a net loss of four base pairs relative to the pairing with the wild-type sequence. Despite this drastic alteration in U1 snRNA:hnRNA (i.e., heterogeneous nuclear RNA) pairing, this mutation, in its full genomic-sequence context, is only partially penetrant. Given these observations, we suggest that, in the case of splicing mutations outside the invariant gt–ag splice-site sequences, splice-site selection might also depend on the definition of exons and the strength of intron-exon junction sequences not directly adjacent to the mutant splice junction. In addition, splice-site selection might be influenced, in part, by intronic sequences distant from the intron-exon junctions. Although the function and normal range of expression level of spastin are still unknown, our findings reported here provide additional support for the hypothesis that the function of spastin is highly concentration dependent. Our finding that normally spliced transcript is produced from at least two different mutant alleles is consistent with the threshold of spastin required for transition from normal function to disease state lying within a narrower interval than the 50% decrease predicted by a disease model of haploinsufficiency. Thus, as we have speculated elsewhere (Svenson et al. 2001), even slight variation in spastin expression level might have dramatic phenotypic consequences, accounting, at least in part, for the wide variability in age at onset, symptom severity, and rate of symptom progression reported to occur in SPG caused by spastin gene mutations.

A new locus for dominant hereditary spastic paraplegia maps to chromosome 2p12.

Sir: Hereditary spastic paraplegia (SPG) is clinically heterogeneous with both pure and complicated forms. The complicated forms are distinguished by the presence of additional neurological symptoms such as optic neuropathy, dementia, ataxia, and mental retardation [1]. SPG is also genetically heterogeneous with autosomal dominant, autosomal recessive, and X-linked forms reported. So far, 11 different chromosomal loci have been identified for autosomal dominant SPG [2, 3]. Five genes underlying these subtypes have been discovered: spastin, atlastin, HSP60, KIF5A, and BSCL2 [2, 4]. However, further genetic heterogeneity has been suggested [5]. Therefore, we conducted a genome wide linkage screen on two multigenerational Caucasian families with an autosomal dominant form of uncomplicated SPG. All patients studied had typical signs of spastic paraplegia mainly characterized by proximal weakness of the lower extremities with brisk reflexes and spastic gait abnormalities. The upper extremities showed normal tone and only very mild weakness of the small hand muscles. The sensory system was not involved. No additional neurological symptoms were present that suggested cerebellar or visual involvement. Median motor nerve conduction velocity was 46 m/s (normal: >42 m/s) in the index patient. Magnet resonance imaging studies of the spinal cord of the index patient revealed no evidence for central canal stenosis or intrinsic spinal cord abnormalities. The average age of onset in the large pedigree DUK2299 was 27.7 years (Fig. 1a). The smaller family, DUK2036 (not shown), demonstrated an average age of onset at 11.8 years. Remarkably, some affected individuals developed symptoms in their fifties and sixties. Significant logarithm of the odds ratio (LOD) scores were obtained at a new locus on chromosome 2p12. The two pedigrees produced a combined two-point LOD score of 4.7 at marker D2S1777 (θ=0; Fig. 1b). The peak twopoint LOD score for DUK2299 was 3.62 at D2S2951 and for the small pedigree DUK2036, it was 1.33 at D2S1777. Multipoint analysis provided a combined (both families) peak LOD score of 5.67 at D2S2951. Haplotype analysis also supported the linkage data. Further fine mapping with additional microsatellite markers identified recombination events that narrowed the minimal genomic distance shared by all affected individuals to 9 Mb between D2S139 and D2S2181 (Fig. 1a). We also excluded linkage to SPG3A, SPG4, SPG6, SPG8, SPG10, SPG12, SPG13, and SPG19 chromosomal loci. Mutations in spastin and atlastin were excluded by sequencing analysis. Somewhat unusual for SPG, both families showed full penetrance of the phenotype according to the measured haplotypes. We considered the emerging pathological themes in SPG [2] and selected four candidate genes within this new chromosomal locus: vesicle-associated membrane protein 5 S. Züchner . M. E. Kail . P. C. Gaskell . M. A. Pericak-Vance . A. E. Ashley-Koch Center for Human Genetics, Duke University Medical Center, Durham, NC, USA

Fine mapping and genetic heterogeneity in the pure form of autosomal dominant familial spastic paraplegia

Abstract We evaluated seven families segregating pure, autosomal dominant familial spastic paraplegia (SPG) for linkage to four recently identified SPG loci on chromosomes 2q (1), 8q (2), 12q (3), and 19q (4). These families were previously shown to be unlinked to SPG loci on chromosomes 2p, 14q, and 15q. Two families demonstrated linkage to the new loci. One family (family 3) showed significant evidence for linkage to chromosome 12q, peaking at D12S1691 (maximum lod=3.22). Haplotype analysis of family 3 did not identify any recombinants among affected individuals in the 12q candidate region. Family 5 yielded a peak lod score of 2.02 at marker D19S868 and excluded linkage to other known SPG loci. Haplotype analysis of family 5 revealed several crossovers in affected individuals, thereby potentially narrowing the SPG12 candidate region to a 5-cM region between markers D19S868 and D19S220. Three of the families definitively excluded all four loci examined, providing evidence for further genetic heterogeneity of pure, autosomal dominant SPG. In conclusion, these data confirm the presence of SPG10 (chromosome 12), potentially reduce the minimum candidate region for SPG12 (chromosome 19q), and suggest there is at least one additional autosomal dominant SPG locus.

Subcellular localization of spastin: implications for the pathogenesis of hereditary spastic paraplegia

Hereditary spastic paraplegia (HSP) is a group of clinically and genetically heterogeneous diseases characterized by neuronal degeneration that is maximal at the distal ends of the longest axons of the central nervous system. The most common cause of autosomal dominant HSP is mutation of a novel gene encoding spastin, a protein whose function is still being elucidated. One clue concerning spastin function is its intracellular localization. Here, we describe a novel anti-spastin antiserum designed to a unique epitope contained within all splicing isoforms. The antiserum exhibits specific immunostaining of recombinant spastin in intact, fixed cells. Using this reagent, we find that endogenous spastin is located at the centrosome in a variety of cell types at all points in the cell cycle. This localization is resistant to microtubule disruption, suggesting that spastin may be an integral centrosomal protein. In addition to the centrosome, spastin also localizes at discrete focal regions along the axons of primary cultured neurons. These data lend additional support to the emerging hypothesis that spastin plays a role in microtubule dynamics, with a crucial role in microtubule organization.