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http://www.fhs.mcmaster.ca/hrlmp/
Issue No. 73
QUARTERLY NEWSLETTER
February 2004
Metachromatic Leukodystrophy:
Molecular Diagnostic Service
Metachromatic leukodystrophy (MLD, OMIM 250100) belongs to a larger group of lysosomal storage
diseases. MLD is caused by deficiency of the enzyme arylsulfatase A (ARSA, EC 3.1.6.8) and the
resulting inability to degrade sulfated glycolipids, especially the galactosyl-3-sulfate ceramides. As is the
case with many other lysosmal storage diseases with progressive neurological involment, MLD has
elements of both demyelination (leukodystrophy) and storage of abnormal lipids. ARSA deficiency causes
intralysosomal storage of cerebroside sulfate in the cells of the white matter of the central nervous system
and of the peripheral nerves, leading to a progressive demyelination and a variety of neurological
symptoms.
Four types of MLD occur with varying ages of onset and courses, namely, late infantile, early juvenile, late
juvenile, and adult forms. All forms of the disease involve a progressive deterioration of motor and
neurocognitive function (1). There is no cure for MLD. Bone marrow transplantation may delay
progression of the disease in some cases (2). Other treatment is symptomatic and supportive.
Clinical characteristics of the Four Forms of Metachromatic Leukodystrophy
Age at Onset
(yrs)
Frequency
Late infantile
<4
Most common
Motor milestones lost,
neurocognitive
functions lost
Death within 5-6 yrs
Early juvenile
4-6
Less common
Motor milestones lost, learning
and behavior impaired
Death within 10-15
yrs
Form
Late juvenile
Adult
6-16
>16
Neurocognitive Deficit
Progression
Rare
Personality changes, behavioral
changes, dementia, psychosis,
Slow
decreased school or work
performance
Rare
Personality changes, behavioral
changes, dementia, psychosis,
Slow
decreased school or work
performance
MLD is a panethnic disease that is inherited in an autosomal recessive manner. The estimated gene
frequency for MLD is 0.5%, corresponding to a carrier frequency of 1 in 100 and an incidence of 1 in
40,000 births in North America. The ARSA gene maps to chromosome 22q13.31 and spans
approximately 3.2 kb. The gene contains eight exons and encodes a protein of 507 amino acid residues
with three N-glycosylation sites (3).
The most common cause of reduced ARSA activity is the so-called pseudodeficiency (PD) allele, a point
mutation in the polyadenylation signal associated with reduced levels of mRNA (4). PD homozygotes
(PD/PD) and compound heterozygotes for PD and more severe MLD mutations (PD/MLD) do not exhibit
the MLD
phenotype, presumably because the PD allele is associated with sufficient residual enzyme activity.
Mutation surveys of MLD patients have identified more than 100 different ARSA mutations, almost all of
which are point mutations. Missense, nonsense, and frame shift mutations are evenly distributed
throughout the eight exons, and numerous splice donor and acceptor site mutations have been reported
(5,6). Two mutations, c.459+1G>A and c.1277C>T (p.Pro426Leu), account for almost one-half of all MLD
alleles in patient surveys (7), with the remaining alleles being rare or private.
Given the broad spectrum of ARSA gene mutations associated with MLD, it is important that molecular
diagnostic strategies be capable of detecting common alleles as well as rare or private alleles. We have
developed rapid and reliable allele-specific assays based on the amplification refractory mutation system
(ARMS) (8) to detect the three most common MLD alleles [c.459+1G>A, c.536T>G (p.Ile179Ser),
c.1277C>T (p.Pro426Leu)], as well as the pseudodeficiency allele (c.1620A>G). This is followed by
comprehensive nucleotide sequence analysis of the entire ARSA gene exons and intron/exon boundaries
(9). Lastly, Southern blot analysis is used to identify rare cases of MLD involving deletion of the ARSA
gene (10).
Since 1998, the Molecular Diagnostic Genetics laboratory at the McMaster site has provided DNA
diagnosis for MLD as Provincial service. The above diagnostic protocol has been used to identify ARSA
gene mutations in more than 50 patients with MLD, with a mutation detection sensitivity of >95%. In our
patient sample, the C.459+1G>A and c.1277C>T (p.Pro426Leu) mutations accounted for almost 40% of
the mutant alleles. The remaining alleles were extremely rare, and included nine point mutations and a
complete ARSA gene deletion that had previously not been reported in the literature (9,10). This
information has been invaluable for identifying couples at risk for having children with MLD, and for
providing prenatal diagnosis for at-risk pregnancies. To date, we have provided prenatal diagnosis for
eight such pregnancies.
References
1)
von Figura K, Gieselmann V, Jaeken J. Metachromatic leukodystrophy. In: Scriver CR, Beaudet AL, Valle D,
Sly WS, Childs B, Kinzler KW, Vogelstein B, eds. The metabolic and molecular bases of inherited diseases. 8th
ed. (2001) New York: McGraw-Hill, 3695-3724.
2)
Koc ON, Day J, Nieder M, Gerson SL, Lazarus HM, Krivit W (2002) Allogeneic mesenchymal stem cell
infusion for treatment of metachromatic leukodystrophy (MLD) and Hurler syndrome (MPS-IH). Bone Marrow
Transplant 30:215-222.
3)
Kreysing J, von Figura K, Gieselmann V (1990) The structure of the arylsulfatase A gene. Eur J Biochem
191:627-631.
4)
Gieselmann V, Polten A, Kreysing J, von Figura K (1989) Arylsulfatase A pseudodeficiency: loss of a
polyadenylation signal and a N-glycosylation site. Proc Natl Acad Sci USA 86:9436-9440.
5)
Gieselmann V, Zlotogora J, Harris A, Wenger DA, Morris CP (1994) Molecular genetics of metachromatic
leukodystrophy. Hum Mutat 4:233-242.
6)
Gieselmann V, Matzner U, Hess B, Lullmann-Rauch R, Coenen R, Hartmann D, D’Hooge R, DeDeyn P, Nagels
G (1998) Metachromatic leukodystrophy: molecular genetics and an animal model. J Inherit Metab Dis 21:564574.
7)
Polten A, Fluharty AL, Fluharty CB, Kappler J, von Figura K, Gieselmann V (1991) Molecular basis of
different forms of metachromatic leukodystrophy. N Engl J Med 324:18-22.
8)
Newton CR, Graham A, Heptinstall LE, Powell SJ, Summers C, Kalsheker N, Smith JC, Markham AF (1989)
Analysis of any pint mutation in DNA: the amplification refractory mutation system (ARMS). Nucleic Acids
Res 17:2506-2516.
9)
Eng B, Nakamura LN, O'Reilly N, Schokman N, Nowaczyk MM, Krivit W, Waye JS (2003) Identification of
nine novel arylsulfatase a (ARSA) gene mutations in patients with metachromatic leukodystrophy (MLD). Hum
Mutat 22:418-419.
10) Eng B, Heshka T, Tarnopolsky MA, Nakamura LM, Nowaczyk MJM, Waye JS (2004) Infantile metachromatic
leukodystrophy (MLD) in a compound heterozygote for the c459+1G>A mutation and a complete deletion of
the ARSA gene. Am J Med Genet (in press).
John S. Waye, PhD
Małgorzata J.M. Nowaczyk, MD, FRCPC, FCCMG
Discipline of Genetics
Hamilton Regional Laboratory Medicine Program