Dennis A. Stephenson

Recent developments in neurofibromatosis type 1.

Purpose of reviewThis review summarizes the recent clinical and genetic developments in neurofibromatosis type 1 (NF1) and provides an insight into the possible underlying pathomechanisms. Recent findingsNF1, or von Recklinghausen disease, is one of the most common hereditary neurocutaneous disorders in humans. Clinically, NF1 is characterized by café-au-lait spots, freckling, skin neurofibroma, plexiform neurofibroma, bony defects, Lisch nodules and tumors of the central nervous system. The responsible gene, NF1, encodes a 2818 amino acid protein (neurofibromin). Pathological mutations range from single nucleotide substitutions to large-scale genomic deletions dispersed throughout the gene. In addition to the conventional mutation screening methods, a DNA chip microarray-based technology, combinational sequence-based hybridization, has been introduced to expedite mutation detection. Functional analysis has become more amenable following the development of the following: (1) primary Schwann cell cultures from NF1 patients; (2) mouse models; (3) proteomic technologies; and (4) mRNA silencing by RNA interference. These studies have shown that neurofibromin plays a role in adenylate cyclase and AKT-mTOR mediated pathways. It also appears to affect Ras-GTPase activating protein activity through the phosphorylation of protein kinase C which impacts on cell motility by binding with actin in the cytoskeleton. SummaryRecent advances in the clinical features and molecular genetics of NF1 will be discussed together with insights into the underlying pathomechanisms of NF1.

Mouse Chromosome 5

The information about each locus for a chromosome appears on one line of the map. At the left is a small figure of the whole chromosome. A box on the chromosome figure indicates which portion of the chromosome is expanded for view on that page. To the right of the small figure is the corresponding expanded centimorgan scale. For each locus on the chromosome a line extends from the scale to the marker or group of markers at that position. From left-to-right, the columns are:

Characterization of a familial case with primary erythromelalgia from Taiwan

Familial primary erythromelalgia is a rare autosomal dominant disease characterized by redness and painful episodes of the feet and hands, which is often triggered by heat or exercise. In this report, a Taiwanese family with the characteristic features of erythromelalgia is described. Genetic linkage studies established that the disease locus maps to human chromosome 2. Sequence analysis indicated that the disease segregates with a novel mutation in the alpha subunit of the voltage-gated sodium channel (SCN9A or Nav1.7). The change observed is predicted to cause the substitution of a highly conserved isoluecine 136 for a valine within the first segment of the transmembrane domain (D1S1). Using immuno-histochemistry to stain a skin biopsy specimen from the affected region, we demonstrate that there is a significant reduction in the number of small fibers.

Mutations of the SPG11 gene in patients with autosomal recessive spastic paraparesis and thin corpus callosum

Hereditary spastic paraparesis (HSP) with thin corpus callosum (TCC) is a rare autosomal recessive form of complicated HSP (ARHSP-TCC). The clinical phenotype is characterised by slowly progressive spastic paraparesis and cognitive impairment which usually occurs during the second decade of life.1 However, cognitive impairment is first noticeable during childhood and may precede the occurrence of leg spasticity. The symptoms progress insidiously to severe functional disability over a period of 10–20 years.2 Some affected individuals develop a pseudobulbar involvement, with dysarthria, dysphagia and upper limb spasticity. Additional manifestations include urinary incontinence, sensory deficits in the lower limbs and late distal amyotrophy. Seizures, extrapyramidal signs and cerebellar ataxia can occur occasionally. The disorder is found in several ethnic groups, including five Chinese patients with HSP-TCC reported by Tang et al in 2004.3 The SPG11 or KIAA1840 gene, which maps to chromosome 15q21.1,4 was shown to be responsible for ARHSP-TCC when it was demonstrated that individuals with the disease carried mutational changes of SPG11 that segregates with the phenotype within families.5 The gene encodes a 2443 amino acid protein, spatacsin, of unknown function. We provide genetic and phenotypic data on five patients from two Taiwanese/Chinese families with ARHSP-TCC. Family No …

HMBS Mutations in Chinese Patients with Acute Intermittent Porphyria

Acute intermittent porphyria (AIP), an autosomal dominant disorder, is caused by partial deficiency of hydroxymethylbilane synthase (HMBS) affecting heme biosynthesis. Patients with AIP are characterized by recurrent abdominal pain, port‐wine urine, and motor paresis. The disease can be provoked by changes in hormone levels, drugs and fasting. Molecular analysis for twenty‐four unrelated Chinese AIP patients from Taiwan identified twenty‐five HMBS mutations. There were 10 missense (40%), four nonsense (16%), five frame‐shift (20%) and six splice site (24%) mutations. More than a half (15/25, 60%) of these mutations are predicted to produce a truncated protein. Four (c.33 + 5C>A, Arg26Cys, Arg26His, Arg325X) occurred more than once among the 24 families and one individual carried two mutations in the same allele, a missense (Gly221Asp) and a splice site mutation (c.652‐1G>A). Of the 25 mutations, eleven were novel (Arg149Pro, Gly218Arg, Asn322X, Gly221Asp, Pro313X, c.88‐4_‐16delAAGTCTCTACCCG, c.1008_1019delCAGCCTGGCCAA, c.113delT, c.88‐4_‐16delAAGTCTCTACCCGinsCA, c.160delA, c.902_909delTCCCTGCC). No correlation between genetic defect and phenotype (both clinical and biochemical) was observed in this study.

Molecular markers near two mouse chromosome 13 genes, muted and pearl, which cause platelet storage pool deficiency (SPD).

The recessive muted (mu) and pearl (pe) mutations on Chromosome (Chr) 13 cause pigment dilution and platelet storage pool deficiency (SPD) in mice. In addition, mu causes inner ear abnormalities and pe has symptoms associated with night blindness. Using an interspecific backcross involving the wild-derived Mus musculus musculus (PWK) stock, we have mapped 33 microsatellite markers and four cDNAs relative to mu, pe, and another recessive mutation, satin (sa). Analyzing a total of 528 backcross offspring, we found tight linkage between the pigment loci and several microsatellite markers (D13Mit87, D13Mit88, D13Mit137 with mu; and D13Mit104, D13Mit160, D13Mit161, and D13Mit169 with pe). These markers should aid the eventual molecular identification of these specific SPD genes.

Longitudinal Evaluation of an N-Ethyl-N-Nitrosourea- Created Murine Model with Normal Pressure Hydrocephalus

Background Normal-pressure hydrocephalus (NPH) is a neurodegenerative disorder that usually occurs late in adult life. Clinically, the cardinal features include gait disturbances, urinary incontinence, and cognitive decline. Methodology/Principal Findings Herein we report the characterization of a novel mouse model of NPH (designated p23-ST1), created by N-ethyl-N-nitrosourea (ENU)-induced mutagenesis. The ventricular size in the brain was measured by 3-dimensional micro-magnetic resonance imaging (3D-MRI) and was found to be enlarged. Intracranial pressure was measured and was found to fall within a normal range. A histological assessment and tracer flow study revealed that the cerebral spinal fluid (CSF) pathway of p23-ST1 mice was normal without obstruction. Motor functions were assessed using a rotarod apparatus and a CatWalk gait automatic analyzer. Mutant mice showed poor rotarod performance and gait disturbances. Cognitive function was evaluated using auditory fear-conditioned responses with the mutant displaying both short- and long-term memory deficits. With an increase in urination frequency and volume, the mutant showed features of incontinence. Nissl substance staining and cell-type-specific markers were used to examine the brain pathology. These studies revealed concurrent glial activation and neuronal loss in the periventricular regions of mutant animals. In particular, chronically activated microglia were found in septal areas at a relatively young age, implying that microglial activation might contribute to the pathogenesis of NPH. These defects were transmitted in an autosomal dominant mode with reduced penetrance. Using a whole-genome scan employing 287 single-nucleotide polymorphic (SNP) markers and further refinement using six additional SNP markers and four microsatellite markers, the causative mutation was mapped to a 5.3-cM region on chromosome 4. Conclusions/Significance Our results collectively demonstrate that the p23-ST1 mouse is a novel mouse model of human NPH. Clinical observations suggest that dysfunctions and alterations in the brains of patients with NPH might occur much earlier than the appearance of clinical signs. p23-ST1 mice provide a unique opportunity to characterize molecular changes and the pathogenic mechanism of NPH.

The isolation and mapping of PCR markers specific to mouse Chromosome 2

The isolation of variable Sequence Tagged Sites (STSs) is the first step towards the construction of a dense genetic map, as a prelude to the construction of a physical map of the mouse genome. Over 4000 highly variable simple sequence repeats (SSRs) have so far been mapped across the whole of the mouse genome; these provide an enormous source of both genetic and physical markers (Dietrich et al. 1992, 1994). Chromosomeand region-specific DNA markers can be isolated from mouse-hamster somatic cell hybrids that carry the whole or fragments of a mouse chromosome. All approaches employed take advantage of the interspecific sequence divergence of short and long interspersed elements (SINES and LINES) between mouse and hamster. We applied a strategy that was previously described (Hochgeschwender et al. 1989; Hoglund et al. 1992) to isolate molecular markers originating from the whole of mouse Chromosome (Chr) 2 (MMU2). The probe was derived by radiolabeling total mouse genomic DNA. The mouse-hamster hybrid cell line EBS-18/AZ was derived from line EBS-18 (Lalley et al. 1987) as previously described (Minna et al. 1975; Franke et al. 1977) by back-selecting against the X Chr with minimal essential medium supplemented with azaguanine. Cytogenetic analysis demonstrated the presence of the whole of MMU2, proximal MMU15, and other submicroscopic mouse chromosome fragments (Peter Lalley, unpublished observations). Southern blot and PCR analysis detected the presence of nine loci across the whole of MMU2. These were: Vim (vimentin), Abl (Abelson leukemia oncogene), Gsn (gelsolin), Acra (acetylcholine receptor alpha subunit), lllb (interleukin 1 beta), Pcna (proliferating cell nuclear antigen), Ahcy (S-adenosyl homocysteine hydrolase), Ada (adenosine deaminase), and Acra4 (nicotinic acetylcholine receptor alpha 4 subunit, neural). Although we could not exclude submicroscopic deletions, it seemed likely that most regions from MMU2 were present. The presence of MMU15 was not confirmed by molecular analysis. The library was constructed from size-fractionated hybrid DNA with the vector LambdaGEM11 (Promega). The mouse and hamster bacteriophage )~ libraries were constructed in vector )~FIX II. Duplicate library filters were prehybridized overnight in 5x SSPE; 5x Denhardts solution; 0.5% SDS, supplemented with 100 gg/ml of sonicated hamster and salmon-sperm DNA before adding the radiolabeled mouse DNA; hybridization was also performed overnight. The washes were performed at 68~ in 2x SSPE/0.5% SDS twice for 10 rain followed by one wash in 2x SSPE/0.5% SDS at 68~ for 30 rain. The positive bacteriophage )~ clones were hybridized to

Abstracts of papers presented at the eighth Mammalian Genetics and Development Workshop held at the Wellcome Trust Building, Euston Road, London on 19–21 November 1997

Sponsored by: The Wellcome Trust, The Genetical Society and B&K Universal Group Limited