ed.14 These observations confirm that REM sleep and dreaming, while linked, may depend on independent generators. In conclusion, our case demonstrates the existence of total dream loss as a distinct neuropsychological dysfunction after deep bilateral occipital lobe damage, in the absence of REM sleep changes. Dr C. Gutbrod performed the neuropsychological testing. Dr K. O. Lövblad performed the brain MRI. Drs P. Brugger, D. Weniger, and B. Schuknecht made helpful comments. References 1. Charcot M. Un cas de suppression brusque et isolée de la vision mentale des signes et des objets (formes et couleurs). Progr Med 1883;2:568 –571. 2. Wilbrand H. Ein Fall von Seelenblindheit und Hemianopsie mit Sectionsbefund. Dtsch Z Nervenheilkd 1887;2:361–387. 3. Grünstein AM. Die Erforschung der Träume als eine Methode der topischen Diagnostik bei Grosshirnerkrankungen. Zeit ges Neurol Psychiatr 1924;93:416 – 420. 4. Nielsen JM. Occipital lobes, dreams and psychosis. J Nervous Ment Dis 1955;121:50 –52. 5. Murri L, Massetani R, Siciliano G, et al. Dream recall after sleep interruption in brain-injured patients. Sleep 1985;8: 356 –362. 6. Solms M. The neuropsychology of dreams. Mahwah, NJ: Erlbaum, 1997. 7. Aserinski E, Kleitman N. Regularly occurring periods of eye motility, and concomitants phenomena during sleep. Science 1953;118:273–274. 8. Hobson JA, Pace-Schott EF. The cognitive neuroscience of sleep: neuronal systems, consciousness and learning. Nat Rev Neurosci 2002;3:679 – 693. 9. Hobson JA. Sleep and dream suppression following a lateral medullary infarct: a first-person account. Conscious Cogn 2002;11:377–390. 10. Lhermitte MJ. Syndrome de la calotte du pédoncule cérébral. Les troubles psychosensoriels dans les lésions mésocéphaliques. Rev Neurol 1922;29:1359 –1365. 11. Manford M, Andermann F. Complex visual hallucinations. Clinical and neurobiological insights. Brain 1998;121: 1819 –1840. 12. Takahashi N, Kawamura M. Pure topographical disorientationthe anatomical basis of landmark agnosia. Cortex 2002;38: 717–725. 13. Taylor SF, Liberzon I, Fig LM, et al. The effect of emotional content on visual recognition memory: a PET activation study. Neuroimage 1998;8:188-197. 14. Solms M. Dreaming and REM sleep are controlled by different brain mechanisms. Behav Brain Sci 2000;23:843– 850. A Novel RAB7 Mutation Associated with UlceroMutilating Neuropathy Henry Houlden, PhD, MRCP,1–3 Rosalind H. M. King, PhD, FRCPath,1 John R. Muddle,2 Thomas T. Warner, PhD, FRCP,1,2 Mary M. Reilly, MD, FRCP,3 Richard W. Orrell, MD, FRCP,1,2 and Lionel Ginsberg, PhD, FRCP1,2 There are two known autosomal dominant genes for the hereditary ulcero-mutilating neuropathies: SPTLC1 (hereditary sensory neuropathy type 1) and RAB7 (Charcot– Marie–Tooth disease type 2B). We report a family with autosomal dominant ulcero-mutilating neuropathy, developing in the teens and characterized by ulcers, amputations, sensory involvement in the feet but no motor features. Sequencing the RAB7 gene showed a novel heterozygous A to C mutation, changing asparagine to threonine at codon 161. The mutation is situated adjacent to a previously identified valine to methionine mutation at codon 162, implying a hotspot for mutations in the highly conserved C terminus of RAB7. Ann Neurol 2004;56:586 –590 The hereditary sensory neuropathies (HSNs) are a heterogeneous group of disorders characterized by prominent sensory loss, often with the development of skin ulcers, arthropathy, osteomyelitis, and amputations.1 Similar features are seen in Charcot–Marie–Tooth disease type 2B (CMT2B), to the extent that some have argued CMT2B would be better classified as a form of HSN. Both HSN type 1 and CMT2B are autosomal dominant conditions and their phenotypes overlap. Age of onset in both disorders is typically in the teens, sensory nerves are prominently affected, and there is a high incidence of ulcero-mutilating complications. One difference between HSN1 and CMT2B is said to be the presence of positive sensory symptoms (paresthesias From the 1University Department of Clinical Neurosciences, Royal Free Campus, Royal Free and University College Medical School, University College London; 2Department of Neurology, Royal Free Hospital; and 3Center for Neuromuscular Diseases, National Hospital for Neurology and Neurosurgery, Queen Square, London, United Kingdom. Received Jun 14, 2004, and in revised form Jul 26. Accepted for publication Jul 29, 2004. Published online Sep 30, 2004, in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/ana.20281 Address correspondence to Dr Ginsberg, Department of Neurology, Royal Free Hospital, Pond Street, London NW3 2QG, United Kingdom. E-mail: email@example.com 586 © 2004 American Neurological Association Published by Wiley-Liss, Inc., through Wiley Subscription Services and lancinating or burning pain) in the former. More marked distal motor involvement is said to favor a diagnosis of CMT2B. Unlike CMT2B, motor nerve conduction velocities in HSN1 families are occasionally slowed, sometimes into the demyelinating range.2,3 Using linkage analysis, the gene for HSN1 was localized to chromosome 9q22.4 Mutations in the serine palmitoyltransferase, long chain base subunit-1 (SPTLC1) gene were then identified as the cause.5,6 In CMT2B, genetic linkage analysis identified a locus on chromosome 3q13–q22.7–9 The small GTP-ase late endosomal protein RAB7 was subsequently identified as the disease gene.10 Two mutations have been described: a transition Val162Met mutation in exon 4 and a Leu129Phe mutation in exon 3. RAB7 is one of more than 60 Ras-related GTP-ases. These proteins regulate vesicle transport by the recruitment of specific effector proteins with a possible role linking vesicles and target membranes to the cytoskeleton.11,12 RAB7 is involved in transport between late endosomes and lysosomes. The mechanism of action of RAB7 genetic mutations in the development of peripheral neuropathy is as yet unknown, although RAB proteins are known to be involved in neurite outgrowth.13 We report the clinical and pathological findings in a further autosomal dominant ulcero-mutilating neuropathy family, with an Asn161Thr mutation in the RAB7 gene. This mutation is in a highly conserved region, adjacent to the reported Val162Met mutation, suggesting a functionally important hotspot for RAB7 mutations. The mutation leads to a virtual absence of expression of a key RAB7 effector, RILP (Rab interacting lysosomal protein), implying the pathogenetic mechanism for this neuropathy may be through this effector pathway. Materials and Methods Nerve Biopsy A sural nerve fascicular biopsy specimen was obtained from the proband with informed consent and processed according to our standard laboratory procedures.14 The procedure was justified on routine diagnostic grounds, having taken place at a time before molecular genetic testing for HSN1 and CMT2B had become available, and after other affected family members had died. In addition to semithin sections for light microscopy and morphometry, and ultrathin sections for electron microscopy, frozen sections were stained immunocytochemically using a polyclonal antibody against RILP15 (gift from Dr C. Bucci). Genetic Analysis With informed consent, DNA was extracted from blood samples obtained from affected and unaffected family members. The five exons and flanking intronic regions of the RAB7 gene were amplified by polymerase chain reaction (PCR) (primers available on request). PCR products were purified (96-well plate; Millipore, Billerica, MA) and se- quenced using the BigDye Terminator cycle sequencing kit. Sequencing reactions were loaded on an ABI 3100 Genetic Analyzer (Perkin Elmer Applied Biosystems, Foster City, CA). Exons 5 and 6 of the SPTLC1 gene also were sequenced.5,6 Results Clinical Details The family reported here is an autosomal dominant, three-generation, English pedigree with ulceromutilating neuropathy. The proband is a 56-year-old man. At the age of 16 years, he developed a painful ulcer on the left sole. This lesion never healed and he had numerous operations on this foot. At the age of 47 years, he developed progressive right foot pain, with swelling and deformity. There was also spontaneous lancinating pain in the left foot. His mother had also been affected, with scoliosis, ulcers, and operations on her feet, including amputations. His maternal grandfather had a history of gangrene and leg amputation. His brother and son were clinically normal. On examination, the proband had gross deformity of the right foot. The middle toe of his left foot had been amputated. He had mild scoliosis. There was nystagmus to lateral gaze. In the limbs, there was no wasting. Tone, power, and coordination were normal. The ankle jerks were absent. Plantar responses were downgoing. Vibration sensation was reduced to the costal margins. Joint position sense was abnormal at the toes. Pin-prick sensation was impaired in the right foot and later the left. Blood tests were normal including syphilis serology. Plain radiographs of the right foot showed a Charcot– Lisfranc joint. Brain imaging showed cerebellar degeneration. Autonomic function tests were normal. Nerve Conduction Studies Motor nerve conduction studies in the proband were normal in the upper limbs. In the lower limbs, distal motor latency was prolonged in the common peroneal nerve at 9.2 milliseconds, with a compound muscle action potential amplitude of 0.1mV. Sensory conduction velocities were normal throughout, but some sensory nerve action potentials were reduced in amplitude (right median, 7V; left median, 5V; right ulnar, 3V; left ulnar, 3V; right radial, 23V; left sural, 7V). Thermal thresholds were abnormal at the right thenar eminence (warming, 13°C; cooling, 2.5°C; difference, 15.5°C). At the lateral border of the right foot, thresholds were also abnormal (warming, 13.4°C; cooling, 3.5°C; difference, 16.9°C). Large fiber nerve conduction studies were normal in the proband’s brother. Nerve Biopsy Sural nerve fascicular biopsy from the proband showed moderately severe depletion of the myelinated fiber population (Fig 1A). This was confirmed by mor- Houlden et al: Neuropathy and RAB7 Mutation 587 Fig 1. Sural nerve biopsy from the proband. (A) Semithin resin section (thionin and acridine orange) showing loss of myelinated fibers with prominent regenerative clusters (arrows). Bar ⫽ 50m. (B, C) Immunostaining against RILP in nerve biopsy from proband and control, respectively. (brown) Horseradish peroxidase. Bar ⫽ 50m. 588 Annals of Neurology Vol 56 No 4 October 2004 phometry: total myelinated fiber density was 3,301/mm2 compared with a value of 7,007/mm2 from a normal control of the same age. The myelinated fiber size distribution at first sight appeared relatively normal because the loss of the normal small fiber population was counterbalanced by the presence of many small regenerating fibers. There were very few remaining normal small fibers (see Fig 1A). No abnormal axonal or Schwann cell inclusions were seen by light microscopy, but electron microscopy showed some inclusions in small unmyelinated axons suggesting active axonal loss. This was confirmed by the presence of collections of flattened sheets of Schwann cell processes. Some Remak bundles contained more axons than normal, also suggesting regeneration. Immunostaining of the proband’s nerve biopsy specimen with polyclonal antibody against RILP was markedly reduced compared with control (see Fig 1B, C).15 Genetic Analysis Sequencing the entire RAB7 gene in the proband showed an A to C heterozygous mutation in exon 4 (Fig 2), causing an amino acid change from asparagine to threonine at position 161. This change was absent in the unaffected brother and in 200 chromosomes from English controls on restriction enzyme digest with the enzyme TaiI (Fermentas, Hanover, MD). No other family individual was available for genetic analysis. Sequencing exons 5 and 6 of the SPTLC1 gene in the proband was normal. Discussion The index case in this family showed an early-onset, slowly progressive, ulcero-mutilating neuropathy with amputations and no motor features. There was also evidence of central involvement with nystagmus and cerebellar atrophy on brain imaging. The neuropathy was Fig 2. RAB7 exon 4. A to C heterozygous mutation. Amino acid change is asparagine to threonine at position 161. Arrows indicate mutation position. Fig 3. Amino acid sequence of the RAB7 gene mutation region in a wide variety of species shows that this region is highly conserved, underlining the functional importance of the change. Mutations in the RAB7 gene in the family described here (Asn161Thr) and reported by Verhoeven and colleagues 10 (Val162Met) are indicated in boldface and with arrows. inherited in an autosomal dominant fashion over three generations. Nerve conduction studies supported the diagnosis of a sensory axonal neuropathy, although the electrical features were mild. Sural nerve biopsy showed appearances of a chronic active neuropathy causing axonal degeneration and prominent regeneration. This family expands the phenotypic spectrum of CMT2B. The findings in the proband suggest there are few distinguishing features between CMT2B and HSN1. Clinically, the presence of positive sensory features, the development of ulcers, and the need for amputations would indicate HSN1. The lack of motor findings is more suggestive of HSN1 than CMT2B. These anomalies, and the evidence for central nervous system involvement, illustrate how establishing the genetic basis of a disease may expand the range of phenotypic manifestations. This phenomenon has been recognized in the hereditary neuropathies since molecular genetic techniques became readily available.16 Pathologically, the proband’s sural nerve biopsy did show features typical of CMT2, these being an axonopathy with marked regeneration, although previous histological descriptions have largely been restricted to CMT2A.16 The Asn161Thr mutation identified in this family is only the third to be reported in the RAB7 gene. This mutation is adjacent to the previously reported Val162Met mutation, suggesting clustering at a potential hotspot for RAB7 mutations. Codons 161 and 162 are both highly conserved (Fig 3), implying functional importance throughout evolution, and are located in the C terminus region of RAB7. The other RAB7 mutation is located at codon 129, close to a conserved guanine nucleotide binding domain. RAB7 is a protein involved in vesicle transport between late endosomes and lysosomes and is thought to act by the induction of a number of effectors.17 One of the key effectors of RAB7 is RILP, which induces the Houlden et al: Neuropathy and RAB7 Mutation 589 recruitment of dynein-dynactin motors to the late endosome/lysosome, thereby stimulating the transport of these compartments toward the perinuclear region along microtubules.15,18 The interaction of RAB proteins with effectors such as RILP may underlie the involvement of these proteins in neurite outgrowth.13 The significant reduction of RILP expression (Fig 1B, C) in the proband’s sural nerve biopsy suggests that the mutation lies in an essential part of the gene, crucial for RAB7 and RILP interaction. Alternatively, the RAB7 mutation may cause a dominant negative effect with a reduction in RAB7 production and hence RILP interaction. It seems less likely that mutant RAB7 causes the disease phenotype directly. If this were the case, more widespread clinical abnormalities might be expected, given the probable role of RAB7 in cellular housekeeping. These putative mechanisms are probably oversimplifications, however, because RAB7 may have additional functions and intermolecular relationships. Another RAB7 effector, RABring7, is thought to act in tandem with RILP.19 A recent study has shown that RAB7 may also be a proapoptotic protein.20 In conclusion, the ulcero-mutilating neuropathies are a diverse group of disorders, both clinically and genetically. The family described here expands the range of phenotypic manifestations of CMT2B and may give an initial clue to the pathogenetic mechanism of RAB7 mutations. We thank the family for their help with this project. We are also grateful to Dr C. Bucci for the RILP antibodies, and to J. Workman for technical assistance. We thank the Wellcome Trust for help with laboratory consumables. All DNA analyses were conducted in the Neurogenetics Laboratory at the Institute of Neurology, Queen Square, London, UK. References 1. Dyck P. Neuronal atrophy and degeneration predominantly affecting peripheral sensory and autonomic neurons. In: Dyck, PJ, Thomas PK, Griffin, JW, eds. Peripheral neuropathy. Vol 2. 3rd ed. Philadelphia: Saunders, 1993:1065–1093. 2. Dubourg O, Barhoumi C, Azzedine H, et al. Phenotypic and genetic study of a family with hereditary sensory neuropathy and prominent weakness. Muscle Nerve 2000;23:1508 –1514. 3. Whitaker J, Falchuck ZM, Engel WK, et al. Hereditary sensory neuropathy. Association with increased synthesis of immunoglobulin A. Arch Neurol 1974;30:359 –371. 4. Nicholson GA, Dawkins JL, Blair IP, et al. The gene for hereditary sensory neuropathy type I (HSN-I) maps to chromosome 9q22.1-q22.3. Nat Genet 1996;13:101–104. 5. Bejaoui K, Wu C, Scheffler MD, et al. SPTLC1 is mutated in hereditary sensory neuropathy, type 1. 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Echard A, Jollivet F, Martinez O, et al. Interaction of a Golgiassociated kinesin-like protein with Rab6. Science 1998;279: 580 –585. 12. Nielsen E, Severin F, Backer JM, et al. Rab5 regulates motility of early endosomes on microtubules. Nat Cell Biol 1999;1: 376 –382. 13. Tang BL. Protein trafficking mechanisms associated with neurite outgrowth and polarized sorting in neurons. J Neurochem 2001;79:923–930. 14. Ginsberg L, King R, Orrell R. Nerve biopsy—how to do it. Pract Neurol 2003;3:306 –313. 15. Bucci C, De Gregorio L, Bruni CB. Expression analysis and chromosomal assignment of PRA1 and RILP genes. Biochem Biophys Res Commun 2001;286:815– 819. 16. Dyck P, Chance P, Lebo R, Carney J. Hereditary motor and sensory neuropathies. In: Dyck PJ, Thomas PK, Griffin JW, eds. Peripheral neuropathy. Vol 2. 3rd ed. Philadelphia: Saunders, 1993:1094 –1136. 17. Seachrist JL, Ferguson SS. Regulation of G protein-coupled receptor endocytosis and trafficking by Rab GTPases. Life Sci 2003;74:225–235. 18. Jordens I, Fernandez-Borja M, Marsman M, et al. The Rab7 effector protein RILP controls lysosomal transport by inducing the recruitment of dynein-dynactin motors. Curr Biol 2001;11: 1680 –1685. 19. Mizuno K, Kitamura A, Sasaki T. Rabring7, a novel Rab7 target protein with a RING finger motif. Mol Biol Cell 2003;14: 3741–3752. 20. Edinger AL, Cinalli RM, Thompson CB. Rab7 prevents growth factor-independent survival by inhibiting cell-autonomous nutrient transporter expression. Dev Cell 2003;5:571–582.