Late-Onset Metachromatic Leukodystrophy ...

Late-Onset Metachromatic Leukodystrophy Clinically Presenting as Isolated Peripheral Neuropathy: Compound Heterozygosity for the IVS2⫹1G3 A Mutation and a Newly Identified Missense Mutation (Thr408Ile) in a Spanish Family Manuel Comabella, MD,1 John S. Waye, PhD,2 Nuria Raguer, MD,3 Barry Eng, BSc,4 Carmen Domı´nguez, PhD,5 Carmen Navarro, MD,6 Cecilia Borra´s, PhD,1 William Krivit, MD, PhD,7 and Xavier Montalba´n, MD1

We report the case of a 50-year-old woman and her 32year-old daughter, both of whom are affected with adultonset metachromatic leukodystrophy (MLD) clinically presenting as peripheral neuropathy. Arylsulfatase A (ARSA) activities were markedly reduced, and electrophysiology showed a severe demyelinating neuropathy with features of chronic acquired demyelinating polyneuropathy. Molecular genetic studies of the family revealed that the proband and her affected daughter are compound heterozygotes for the common IVS2ⴙ1G3 A mutation and a newly identified missense mutation, Thr408Ile. This case indicates that adult metachromatic leukodystrophy should be considered in adult patients with demyelinating peripheral neuropathy of unknown etiology. Ann Neurol 2001;50:108 –112

From the Departments of 1Neurology, 3Neurophysiology, and 5Molecular Biology and Biochemistry Research Centre, Hospital Universitari Vall d’Hebron (HUVH), Barcelona, Spain; 2DNA Diagnostic Laboratory, Hamilton Regional Laboratory Medicine Program, and 4 Department of Pathology and Molecular Medicine, McMaster University Medical Centre, Hamilton, Ontario, Canada; 6Department of Pathology, Hospital Do Meixoeiro, Vigo, Spain; and 7Department of Pediatrics, University of Minnesota, Minneapolis, MN. Received Dec 4, 2000, and in revised form Mar 22, 2001. Accepted for publication Mar 28, 2001. Published online 16 May 2001. Address correspondence to Dr Comabella, Unidad de Neuroinmunologı´a Clı´nica, Hospital Universitari Vall d’Hebron, Escuela de Enfermeria 5 Planta, Psg. Vall D’Hebron 119-129, 08035 Barcelona, Spain. E-mail: [email protected]

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Metachromatic leukodystrophy (MLD; MIM 250100) is an autosomal recessive inherited metabolic disease characterized by a deficiency of arylsulfatase A (ARSA; EC 3.1.6.8) activity. This deficiency causes intralysosomal storage of cerebroside sulfate in the white matter of the central nervous system and in the peripheral nerves, leading to progressive demyelination that causes a variety of neurological symptoms. Clinically the disease is heterogeneous, and different forms are classified according to the age at onset of symptoms (late infantile, juvenile, and adult).1 The adult-onset phenotype is quite rare, and its clinical manifestations often include dementia and psychiatric disturbances. There are very few reported cases of adult-onset MLD presenting as peripheral neuropathy, and detailed genetic studies are lacking for most of these patients.2– 4 Here we report a Spanish family with two individuals affected with atypical adult-onset MLD resulting from compound heterozygosity for the IVS2⫹1G3 A splice site mutation and a newly described missense mutation (Thr408Ile). Patients and Methods Patient 1 (III-4) The proband (see Fig 1), a 50-year-old woman, was admitted to our hospital (HUVH) in 1996 with a 2 month history of radicular pain and numbness and paresthesia in extremities, more pronounced in upper limbs. She had been in good health and the family history was negative for neurological diseases. On admission her neurological examination revealed mild proximal weakness of the upper and lower extremities, absent deep tendon reflexes, and glove and stocking sensory loss. No intellectual or behavioral disturbances were clinically detectable. Except for mild hyperglycemia, routine laboratory tests, sedimentation rate, plasma protein electrophoresis, vitamin B12, folic acid, urinary excretion of porphyrins, and thyroid hormones were normal. Cerebral spinal fluid evaluation revealed an increased protein concentration (97 mg/dl). Electrophysiological studies (Table) showed a severe reduction in motor nerve conduction velocities (NCVs) in upper and lower limbs, uniformly affecting all nerves, with marked delay of F responses and prolonged distal motor latencies. Widespread nerve conduction blocks were also present (Fig 2A). Distal compound motor action potential (CMAP) amplitudes were reduced in lower limbs and preserved in upper ones. Sensory nerve action potentials (SNAP) were absent in the sural and radial nerves and had small amplitude and prolonged distal latency in the median and ulnar nerves, with marked reduction in NCVs. Electron microscopy of the sural nerve biopsy showed thin myelin sheaths consistent with demyelination and prismatic and lamellar inclusions in the cytoplasm of Schwann cells and few macrophages (Fig 2B). Assay of ARSA activity in blood leukocytes disclosed a marked deficiency of the enzyme (0.03 nmol/min/mg protein). Study of glycosphingolipids in urine demonstrated a massive excretion of sulfatides. Five years after the onset of clinical symptoms, the patient’s cognitive performance assessed through the Wechsler Adult-Intelligence Scale (WAIS) and Wechsler Memory Scale-Revised (WMS-R) was normal. A magnetic

Mutation Analysis of the ARSA Gene

Fig 1. Family pedigree. Square symbols represent male members, and circles represent female members. Solid symbols represent neurological symptoms; half-filled symbols represent carriers; diagonal lines represent deceased individuals; arrow represents the proband. Individual genotypes are listed and arylsulfatase A activities expressed as nmol/min/mg protein and percentage of mean control activity are shown in parentheses. *The range of leukocyte arylsulfatase A activity for normal controls is 0.77–1 nmol/min/mg protein. ND ⫽ not done. N ⫽ normal.

resonance imaging of the brain showed small areas of leukomalacia affecting the periventricular white matter of frontal lobes and corpus callosum.

Patient 2 (IV-2) This 32-year-old woman was the firstborn child of consanguineous parents (see Fig 1, III-2 and III-4). Except for occasional muscle cramps in legs, her past medical history was unremarkable. On neurologic examination, deep tendon reflexes were absent at the knees and diminished at the ankles. Motor strength and sensory testing were normal. ARSA activity in blood leukocytes was low (0.02 nmol/min/mg protein). Urinary sulfatide excretion was high. Electrophysiological studies (see Table) showed nonuniform slowing of motor NCVs in upper and lower limbs, with multifocal conduction blocks, increase of distal motor latencies, and marked delay of F responses. Distal CMAP amplitudes were normal, but SNAPs had small amplitudes, and sensory NCVs were slowed. Nerve sural biopsy revealed inclusions in Schwann cell cytoplasm. Cognitive performance assessed through WAIS and WMS-R was normal.

Other Family Members The proband’s husband from the first marriage (III-2) showed no neurological abnormalities. The proband’s youngest daughter from the consanguineous marriage (IV-4) and two daughters born from the unrelated marriage (IV-6 and IV-7) were healthy. Their ARSA activities were mildly decreased to the level seen in carriers (see Fig 1). Electrophysiological study was performed for the proband’s daughters (IV-4, IV-6, and IV-7) and showed no evidence of peripheral neuropathy.

Peripheral blood or plucked scalp hair samples were collected from the various family members and transported to the DNA Diagnostic Laboratory at McMaster University Medical Centre. Genomic DNA was isolated from the patient samples and the ARSA gene was screened for the common MLD alleles using the amplification refractory mutation system (ARMS).5 For comprehensive nucleotide sequencing, the ARSA gene was amplified as 3 overlapping fragments using the following primer sets: exons 1–3, forward primer 5⬘CTGC TGGA GCCA AGTA GCCCT-3⬘, reverse primer 5⬘-CAAA GACT GGAG TTAG CACT-3⬘; exons 3– 6, forward primer 5⬘-ATGC CTCA TGGC CGAC GC-3⬘, reverse primer 5⬘-GAGG ATCC CAGT GCAG GAGG CACT GAGG-3⬘; exons 6 – 8, forward primer 5⬘-TTGA TGGC GAAC TGAG TGAC-3⬘, reverse primer 5⬘-TTCC TCAT TCGT ACCA CAGG-3⬘. The polymerase chain reaction (PCR) products were sequenced using internal primers, the ABI PRISM Big Dye Terminator Cycle Sequencing kit, and the ABI PRISM 310 automated fluorescent sequencer.

Restriction Endonuclease Detection of the Thr408Ile Mutation Exons 6 – 8 were amplified as a 1,145 bp fragment (see above), and the PCR product was digested with the restriction endonuclease EcoRV. The normal allele contains no EcoRV sites, whereas the Thr408Ile mutation creates an internal EcoRV cleavage site (GATACC3 GATATC) and generates two distinct fragments lengths of 652 bp and 493 bp. The digested PCR products were separated by electrophoresis on 6.0% nondenaturing polyacrylamide gels, and the fragments were visualized by ethidium bromide staining and ultraviolet fluorescence.

Results The proband and her daughter were screened for common MLD alleles, and both were shown to carry the IVS2⫹1G3 A mutation, also known as “c.459⫹ 1G3 A.” Comprehensive nucleotide sequencing of the ARSA gene confirmed that the proband and her daughter are heterozygous for the IVS2⫹1G3 A mutation and revealed that both carry a previously unreported mutation at codon 408 (Thr408Ile; c.1223C3 T). The Thr408Ile mutation was confirmed by sequencing in both directions and by EcoRV restriction digestion (see Fig 2C). The other sequence changes were associated with known structural polymorphisms.6 The proband and her mother were heterozygous for the Trp193Cys (c.579G3 T) polymorphism and homozygous for the Thr391Ser (c.1072C3 G) polymorphism. Both were negative for the two nucleotide changes associated with the so-called pseudodeficiency allele [Asn350Ser (c.1049A3 G) and poly-A (c.1620A3 G)].7 Molecular testing was extended to other family members to determine whether the IVS2⫹1G3 A and Thr408Ile mutations are in cis or trans. The results,

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Table. Nerve Conduction Studies Motor Latency (msec)

Nerve Patient 1 R. median Wrist Elbow Axilla R. ulnar Wrist Elbow Axilla R. peroneal Ankle Below fibular head Popliteal fossa L. tibial Ankle Popliteal fossa Patient 2 R. median Wrist Elbow Axilla R. ulnar Wrist Elbow Axilla R. peroneal Ankle Below fibular head Popliteal fossa L. tibial Ankle Popliteal fossa

5.5

5.1

11.4

12.8

4.9

3.3

7.1

9.9

Amplitude (mV)

Conduction Velocity (m/sec)

8.0 2.7 1.6

21 26

4.6 3.6 0.2

18 7

1.8 1.3 1.3

16 17

1.1 NR

—

7.3 0.2 0.2

22 32

7.1 6.3 2.5

28 25

3.0 1.5 1.3

17 31

5.4 1.3

18

Sensory F-M Latency (msec) (%)

51.3 (80)

61.3 (100)

111 (20)

Nerve

Amplitude (␮V)

Conduction Velocity (m/sec)

R. median

0.6

23

R. ulnar

3.9

23

R. radiala

NR

—

R. surala

NR

—

L. surala

NR

—

R. median

2.7

33

R. ulnar

3.3

28

R. radiala

5.8

32

R. surala

3.9

20

L. surala

5.9

20

94 (10)

37.5 (90)

40.5 (90)

78.5 (10)

71.5 (50)

R ⫽ right. L ⫽ left. NR ⫽ no response. a

Antidromic technique.

summarized in Figure 1, demonstrate that the two mutations segregate independently through the family and therefore are allelic. Simple carriers of the IVS2⫹1G3 A and Thr408Ile mutations had similarly reduced levels of ARSA activity (see Fig 1; IV-6 and IV-7, respectively). Tests were conducted to investigate the possibility that Thr408Ile is a structural polymorphism and not a contributing factor to the MLD phenotype. The EcoRV restriction digestion assay was used to screen normal controls from 110 whites (data not shown). The Thr408Ile mutation was not observed, indicating that this allele is not a common polymorphism. Discussion We report a consanguineous Spanish family among whom two individuals are affected with an atypical adult-onset form of MLD presenting as isolated pe-

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ripheral neuropathy. Electrophysiological studies in the proband and her affected daughter showed a severe demyelinating neuropathy with marked reduction of NCVs and widespread conduction blocks. The presence of conduction blocks and the nonuniform slowing of NCVs in different nerve segments are features more suggestive of an acquired demyelinating neuropathy than of a hereditary disorder.8 Biochemical and molecular studies of the family demonstrated that both affected individuals are compound heterozygotes for the IVS2⫹1G3 A mutation and a newly identified missense mutation, Thr408Ile. The IVS2⫹1G3 A mutation is one of the most common MLD alleles, affecting the IVS2 donor splice consensus sequence and thereby abolishing proper processing of the ARSA premRNA.6,9 Homozygotes for severe MLD alleles such as IVS2⫹1G3 A generally have the most severe, infantile-onset form of the disease.6 Given the rela-

Fig 2. (A) Right median motor nerve and right ulnar motor nerve conduction studies demonstrating conduction blocks in elbow and axilla, respectively. (B) Electron microscopy of the sural nerve biopsy showing prismatic inclusion of 1.5 ␮m diameter formed of stacks of polyhedric structures within a Schwann cell cytoplasm of a myelinated fiber. Note myelin sheath on the right and basal membrane along the Schwann cell cytoplasm. Original magnification ⫻18,000. (C) Detection of the novel Thr408Ile missense mutation by direct nucelotide sequencing and EcoRV restriction endonuclease digestion.

tively mild clinical course of the proband and her daughter, it is reasonable to conclude that the Thr408Ile missense mutation is a mild MLD allele. Almost 100 different MLD alleles have been reported to date, approximately 70% of which are missense mutations associated with reduced levels of ARSA activity.10,11 Several lines of evidence lend support to the notion that Thr408Ile is an MLD allele. First, apart from known polymorphic variants, the only nucleotide changes found in the proband and her daughter were IVS2⫹1G3 A and Thr408Ile. Second, family studies demonstrated that the IVS2⫹1G3 A and Thr408Ile mutations are allelic. Third, Thr408Ile was not found in a screen of 110 normal control individuals. Fourth, a simple carrier of Thr408Ile (see Fig 1; IV-7) had reduced ARSA activity consistent with that of MLD carriers. Fifth, the mutation involves a residue that is conserved among many members of the sulfatase enzyme family [http://maple.bioc.columbia.edu/ predictprotein/predictprotein.html (the PredictProtein Server)], with substitution of a polar residue (Tyr) with a nonpolar aliphatic residue (Ile).

Based on previous reports, adult-onset MLD patients presenting with polyneuropathy have a mild disease phenotype, and, as in our case, polyneuropathy may be the only symptom for many years.2 To our knowledge, genetic studies have been performed for only one other case of adult-onset MLD and isolated polyneuropathy.4 In that particular case, the affected individual was shown to be homozygous for a novel missense mutation, Thr286Pro.4 This demonstrates that the genetic causes of late-onset MLD presenting as isolated polyneuropathy are heterogeneous. Moreover, these cases indicate that adult-onset metachromatic leukodystrophy should be considered in adult patients with demyelinating peripheral neuropathy of unknown etiology. Analysis of urinary sulfatide excretion was performed by Dr Marie Vanier, Centre Hospitalier Lyon-Sud, Lyon, France.

References 1. Kolodny EH, Fluharty AL. Metachromatic leukodystrophy and multiple sulfatase deficiency: sulfatide lipidosis. In: Scriver CR, Beaudet AL, Sly WS, Valle D, Stanbury JD, Wyngearden JB,

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Fredrickson DS, editors. The metabolic and molecular bases of inherited diseases, 7th ed. New York: McGraw-Hill, 1995: 2693–2739. Fressinaud C, Vallat JM, Masson M, et al. Adult-onset metachromatic leukodystrophy presenting as isolated peripheral neuropathy. Neurology 1992;42:1396 –1398. Hageman ATM, Gabree¨ls FJM, de Jong JGN, et al. Clinical symptoms of adult metachromatic leukodystrophy and arylsulfatase A pseudodeficiency. Arch Neurol 1995;52:408 – 413. Felice KJ, Gomez Lira M, Natowicz M, et al. Adult-onset MLD: a gene mutation with isolated polyneuropathy. Neurology 2000;55:1036 –1039. Newton CR, Graham A, Heptinstall LE, et al. Analysis of any point mutation in DNA: the amplification refractory mutation system (ARMS). Nucleic Acids Res 1989;17:2506 –2516. Polten A, Fluharty AL, Fluharty CB, et al. Molecular basis of different forms of metachromatic leukodystrophy. N Engl J Med 1991;324:18 –22. Gieselmann V, Polten A, Kreysing J, von Figura K. Arylsulfatase A pseudodeficiency: loss of a polyadenylation signal and a N-glycosylation site. Proc Natl Acad Sci USA 1989;86:9436 – 9440. Miller RG, Gutmann L, Lewis RA, Sumner AJ. Acquired versus familial demyelinative neuropathies in children. Muscle Nerve 1985;8:205–210. Zlotogora J, Furman-Shaharabani Y, Harris A, et al. A single origin for the most frequent mutation causing late infantile metachromatic leucodystrophy. J Med Genet 1994;31:672– 674. Gieselmann V, Zlotogora J, Harris A, et al. Molecular genetics of metachromatic leukodystrophy. Hum Mutat 1994;4:233– 242. Gieselmann V, Matzner U, Hess B, et al. Metachromatic leukodystrophy: molecular genetics and an animal model. J Inherit Metab Dis 1998;21:564 –574.

Dichloroacetate Exerts Therapeutic Effects in Transgenic Mouse Models of Huntington’s Disease Ole A. Andreassen, MD,1 Robert J. Ferrante, PhD,2–5 Hsueh-Meei Huang, PhD,6 Alpaslan Dedeoglu, PhD,1 Larry Park, PhD,6 Kimberly L. Ferrante, BA,6 Jennifer Kwon, BS,6 David R. Borchelt, PhD,7 Christopher A. Ross, MD, PhD,7 Gary E. Gibson, PhD,8 and M. Flint Beal, MD6

Dichloroacetate (DCA) stimulates pyruvate dehydrogenase complex (PDHC) activity and lowers cerebral lactate concentrations. In the R6/2 and N171-82Q transgenic mouse models of Huntington’s disease (HD), DCA significantly increased survival, improved motor function, delayed loss of body weight, attenuated the development of striatal neuron atrophy, and prevented diabetes. The percentage of PDHC in the active form was significantly reduced in R6/2 mice at 12 weeks of age, and DCA ameliorated the deficit. These results provide further evidence for a role of energy dysfunction in HD pathogenesis and suggest that DCA may exert therapeutic benefits in HD. Ann Neurol 2001;50:112–117

Defects in energy metabolism are linked to the pathogenesis and progression of neurodegenerative diseases. Considerable evidence suggests that excitotoxicity, oxidative stress, adenosine triphosphate (ATP) depletion, and mitochondrial dysfunction may play a role in the pathogenesis of Huntington’s disease (HD).1 We and others have shown that lactate is elevated in the cortex of HD patients,2 that there is a reduced phosphocreatine/inorganic phosphate ratio in resting muscle of HD patients,3 that the maximal rate of mitochondrial

From the 1Neurochemistry Laboratory, Neurology Service, Massachusetts General Hospital and Harvard Medical School, Boston, 2 Geriatric Research Education and Clinical Center, Bedford VA Medical Center, Bedford, 3Department of Neurology, 4Department of Pathology, and 5Department of Psychiatry, Boston University School of Medicine, Boston, MA; 6Department of Neurology and Neuroscience, Weill Medical College of Cornell University, New York Presbyterian Hospital, New York, NY; 7Department of Pathology, Johns Hopkins Medical School, Baltimore, MD; and 8Burke Medical Research Institute, White Plains, NY. Received Jan 31, 2001, and in revised form Mar 29. Accepted for publication Mar 29, 2001. Published online 25 May 2001. Address correspondence to Dr M. Flint Beal, Department of Neurology and Neuroscience, New York Presbyterian Hospital/Weill Medical College of Cornell University, 525 East 68th Street, New York, NY 10021. E-mail: [email protected]

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