Gene 530 (2013) 323–328

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Gene
journal homepage: www.elsevier.com/locate/gene

Short Communication

Sixteen novel mutations in the arylsulfatase A gene causing

metachromatic leukodystrophy
Paola Luzi ⁎, Mohammad A. Rafi, Han Zhi Rao, David A. Wenger
Lysosomal Diseases Testing Laboratory, Department of Neurology, Thomas Jefferson University, 1020 Locust Street, Room 346, Philadelphia, PA 19107, USA

a r t i c l e i n f o a b s t r a c t

Article history: Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder caused mainly by mutations in the
Accepted 17 August 2013 arylsulfatase A (ARSA) gene. In this manuscript we report sixteen novel mutations identified in the ARSA gene
Available online 31 August 2013 of fifteen unrelated patients affected with MLD. Of these 16 mutations nine were missense mutations (p.L11Q,
p.S44P, p.L81P, p.R84L, p.V177D, p.P284S, p.R288S, p.G301R, p.P425S), three were nonsense mutations
Keywords:
(p.Q51X, p.Y149X, p.C156X), three were frame shift mutations (c.28delG, c.105CNA+106_124dup, c.189delC)
Arylsulfatase A
Metachromatic leukodystrophy
and one was a splice-site mutation (c.1102-2ANG). In addition, three previously reported mutations were iden-
Lysosomal storage diseases tified on an allelic background different from the one in the original reports. Two mutations, p.G309S and
MLD p.E312D, were identified on the background of the so-called pseudodeficiency (Pd) allele while previously
Sulfatides they were reported alone. On the other hand, mutation p.R311X was identified in two unrelated patients not
Genotype–phenotype correlations in cis with the Pd mutations, as previously reported.
© 2013 Elsevier B.V. All rights reserved.

1. Introduction A diagnosis of MLD can be reached by detecting low ARSA enzymatic

activity in several tissues (like leukocytes or fibroblast) together with
Metachromatic leukodystrophy (MLD) is a neurodegenerative lyso- excess excretion of sulfatides in the urine. Due to the high frequency
somal storage disease caused, in the majority of cases, by the deficiency of the so-called pseudodeficiency (Pd) allele (Gieselmann et al, 1989)
of arylsulfatase A (ARSA, E.C. 3.1.6.1) activity resulting in accumulation the identification of low ARSA activity alone is not diagnostic of MLD. In-
of sulfatides in the nervous system. The disease is transmitted as an auto- dividuals with two copies of the Pd allele can have the ARSA activity in
somal recessive trait and it is one of the most common among the lyso- the same range of MLD patients, but they are not affected with the dis-
somal storage diseases with an estimated frequency of 1.45:100,000 ease. Also, they do not excrete sulfatides in urine. However, it should be
births (Giugliani, 2012). In our Lysosomal Diseases Testing Laboratory noted that some disease-causing mutations have been found in cis with
nearly 800 cases of MLD have been diagnosed in samples from patients the Pd allele (Gieselmann et al, 1991; Rafi et al, 2003). On the other
with neurological symptoms. Metachromatic leukodystrophy is charac- hand, some individuals with a deficiency of saposin B, an activator pro-
terized by progressive demyelination resulting in a wide range of neuro- tein needed for the proper functioning of arylsulfatase A (Inui et al,
logic symptoms. Three forms of the disease are recognized according to 1983), have a normal ARSA enzymatic activity but are affected with
the age of onset: late infantile, juvenile and adult forms. The late infantile MLD, as confirmed by the presence of sulfatides in urine.
form is the most common, accounting for 50–60% of all cases, with onset Since the cloning of the ARSA cDNA (Stein et al, 1989) and gene
between one and three years of age. The most frequently reported symp- (Kreysing et al, 1990) more than 150 mutations have been reported
toms are loss of acquired skills and difficulty walking. Patients with the ju- (The Human Gene Mutation Database). Two mutations are identified
venile form of MLD present between 4 and 14 years of age, while in the quite frequently: the 459+1GNA change, a splice-site mutation, is associ-
adult form the onset is after puberty. In the juvenile cases the most com- ated with late-infantile onset of the disease and is commonly referred to
monly reported signs of the disease are decreased school performance, as “the common late infantile (LI) mutation” and the p.P426L, a missense
gait abnormality, weakness, and abnormal behavior while in the adults mutation, is associated with a later onset of the disease and is commonly
the presentation can be neuromuscular or behavioral. called “the common adult mutation” (Polten et al, 1991). Patients who
are compound heterozygotes for these two mutations have a juvenile
form of the disease. While an initial diagnosis of MLD is based on finding
Abbreviations: ARSA, arylsulfatase A; DD, developmental delay; LI, late infantile; MLD, low ARSA activity using a synthetic substrate in leukocytes or fibroblasts,
metachromatic leukodystrophy; MRI, magnetic resonance imaging; Pd, pseudodeficiency; the residual activity measured is not predictive of the age of onset or clin-
PCR, polymerase chain reaction; WM, white matter.
⁎ Corresponding author. Tel.: +1 215 503 5715, +1 215 955 1666; fax: +1 215 955
ical course. In general we can hypothesize that a mutation that results in
9554. very low ARSA activity in vivo would indicate an early onset depending
E-mail address: [email protected] (P. Luzi). on the mutation in the second allele. While mutations that have some

0378-1119/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.gene.2013.08.065
324 P. Luzi et al. / Gene 530 (2013) 323–328

residual activity in vivo are predicted to result in a later-onset form of time of diagnosis, clinical information and history of initial symptoms
MLD. In many cases the assignment of mutation severity can be based provided by the patient's caregivers.
on the combination of alleles found and the clinical presentations of All procedures were in compliance with institutional guidelines and
patients. testing was performed with the consent of the patient's parents.
In our laboratory, once the diagnosis of MLD is confirmed by the de-
tection of sulfatides in urine, mutation analysis of the ARSA gene is 2.2. Determination of arylsulfatase A activity and urine sulfatides
performed if requested by the patient's family and physician. In this
manuscript we report 16 novel mutations identified in the ARSA gene Leukocytes were isolated from whole blood (Wenger and Williams,
of patients with metachromatic leukodystrophy. In addition we de- 1991) and ARSA activity was measured in leukocyte or fibroblast soni-
scribe three previously reported mutations in an allelic background dif- cates using p-nitrocatechol sulfate substrate (Baum et al, 1959).
ferent from the one originally reported. Presence of excess sulfatides in urine was detected as previously de-
scribed (Rafi et al, 2003).

2. Materials and methods 2.3. DNA extraction and molecular analysis

2.1. Patients DNA was extracted from the remaining sonicated samples using a
DNeasy Blood and Tissue Kit (QIAGEN) following the manufacturer's in-
Blood samples from 16 of the 19 patients reported in this manuscript structions. The DNA was then utilized to screen for the common LI mu-
and cultured skin fibroblasts from patient # 15 were sent to our labora- tation, the common adult mutation (p.P426L) and the Pd allele, using a
tory to be screened for a lysosomal storage disorder (Table 1). The rapid detection method based on PCR amplification followed by restric-
screen resulted in a low ARSA value, indicating a possible diagnosis of tion digestion (Ben-Yoseph and Mitchell, 1994; Chabás et al., 1993).
MLD (Table 1). A urine sample was then requested to evaluate the pres- For sequence, the full length ARSA gene was amplified in two
ence of excess sulfatides to confirm the MLD diagnosis (Table 1). Pa- overlapping fragments using a GeneAmp XL PCR Kit (Applied Biosystem).
tients # 8 and # 18 were previously diagnosed elsewhere but samples The fragments were respectively 1980 bp and 1400 bp in size. The first
were sent here for mutation analysis. For patient # 18 we only received one spanned from 100 bp before the initial ATG to the beginning of intron
a DNA sample for sequencing, therefore no ARSA activity or sulfatides 6 and the second fragment spanned from the end of intron 4 to 140 bp
values are available, while for patient # 8 we received both DNA and beyond the polyadenylation signal. The primers used to amplify the
urine to confirm the diagnosis. No urine sample was received for patient first fragment were: sense primer (ctc agg gaa ggg cgg cgc) and antisense
# 15. Blood samples from parents were requested for all diagnosed primer (ggg cca agg atc tgg gat ca); for fragment II: sense primer (cca tgg
patients. ctc atg agc gcc tcc) and antisense primer (cag ttt cct cat tcg tac cac ag).
In Table 1, the clinical characteristics of the patients described in The PCR products were then purified using a QIAquick PCR Purifica-
this report are summarized. The information was provided to us by tion Kit (QIAGEN) following the manufacturer's instructions. Sequenc-
the physician requesting the tests: some descriptions reported a de- ing of the ARSA gene was performed at the Kimmel Cancer Center
tailed record of the presenting symptoms while others contained min- Molecular Analysis Laboratory at Thomas Jefferson University using
imal information. Age of onset was established based on the age at the the BigDye Terminator v3.1 cycle sequencing kit with the ABI 3730xl

Table 1
Clinical and biochemical findings in patients included in this study.

Patient # Age at Signs and symptoms ARSA Sulfatide Ethnicity

onset activity a Excretionb

1 2.5 y Spasticity, loss of gross motor milestones, cognitive problems, hypertonia 11.2 + Hispanic, 2 more siblings
with same genotype
2 3y Regression of milestones since 1 year of age after high fever, increased tone, spasticity, rigidity, diffuse WM 13.0 + From Kenya
disease on MRI
3 2y Ataxia, motor regression, increased T2 periventricular WM signal on MRI 8.1 + Unknown
4 2.5 y Axial hypotonia, legs extended, hand fisted, swallowing dysfunction with increased choking and gagging, 12.5 + African American
unable to sit, nystagmus, MRI c/w leukodystrophy, nonverbal, irritable
5 2y Altered mental status 10.3 + Adopted from Ethiopia
6 2y Difficulty walking with recent rapid regression of skills 2.7 + Unknown
7 2y Hypotonia and developmental regression, spasticity, unable to sit unassisted, slurred speech, tight heel cords 5.8 + Caucasian
8 2.0 y Motor, speech, and cognitive regression starting at 2 years of age, opisthotic posturing, hypertonia, atrophy of NA + Arabic
muscle, cachectic, diffuse leukodystrophy on MRI
9 2.5 y Progressive gait abnormalities, spasticity, dysarthria, dysmetria, dysphagia, MRI showing WM changes 4.8 + Caucasian
10 6.5 y Cognitive regression in the past 3 months, increasing dystaxia, loss of bowel/bladder function, generalized 7.1 + Unknown
hypotonia, MRI c/w leukodystrophy
11 12 y Developmental delays 7.9 + Caucasian
12 2.5 y DD, difficulty walking, genu valgus, decreased balance and coordination, speech delay, staring episodes 9.2 + Caucasian
13 2.5 y Wide based gait, unsteady, speech delay, absent reflexes, MRI c/w leukodystrophy 15.4 + Chinese
14 15 y Progressive cognitive decline since 9th grade, decrease in IQ, WM changes on MRI. Gait, muscle stretch reflexes, 4.1 + Caucasian
cerebellar testing all normal. No problems with coordination or loss of strength
15 11 y MRI showing diffuse abnormal WM and thin corpus callosum, loss of cerebral volume, kyphoscoliosis, mi 8.3 NA From Saudi Arabia
crocephaly
16 2.5 y Nystagmus noted at 9 months, hypotonia, regression of milestones, legs extended, tremors, spasticity of 5.9 + Unknown
lower limbs, muscle wasting
17 2y Lack of coordination 0 + Caucasian
18 12 y Behavioral problems, dropping IQ, abnormal CT NA NA Unknown
19 3y Progressive gait ataxia, mild speech delay, mild hypotonia, MRI showing WM changes 1.2 + Caucasian

Abbreviations: y = years; NA = not available; WM = white matter; DD = developmental delay; and c/w = consistent with.
a
ARSA activity expressed as nmol/h/mg protein. Normal mean = 70; Affected range 0–16.
b
Presence (+) or absence (−) of excess sulfatides in urine.
 P. Luzi et al. / Gene 530 (2013) 323–328 325

DNA analyzer (Applied Biosystem). The results were provided as chro- mutation. To evaluate the effect of the novel mutations on the protein
matograms. If some areas of the sequence were not clear (high back- activity, the mutations reported in this paper were analyzed in silico
ground noise, peaks not well resolved, intensity of the peaks too low) using two different programs, PolyPhen2 and Provean. With the
or even a single base pair was unclear, new sequencing reactions were PolyPhen2 program only missense mutations could be analyzed, uti-
prepared using different sense or antisense primers. lizing a scale between zero (benign mutation) and one (probably
Also, when available, parental DNA was analyzed to confirm the damaging). With this program eight of the reported missense muta-
presence of the mutations found in the patient and to establish, in tions were predicted to be “probably damaging” with scores equal or
cases where more than two changes were found, on which allele they very close to 1.00 indicating high confidence (Table 2). The p.Y39F
occurred. missense mutation had a lower score (0.788) that was indicated as
The mutation database HGMD-professional version 2013.1 (accessed “possibly damaging”. This mutation was found in cis with p.Y149X.
at http://www.hgmd.cf.ac.uk) was consulted to verify that the novel mu- Of these two novel mutations p.Y149X was considered to be the
tations identified were not previously reported. In addition, the exome disease-causing mutation with p.Y39F being regarded as an addi-
variant database (http://evs.gs.washington.edu/EVS/) was consulted to tional change of uncertain significance. With the Protean program
rule out that these novel mutations were polymorphisms. missense and nonsense mutations were analyzed: variants with a
Novel mutations were also analyzed in silico using the program score equal to or below − 2.5 are considered “deleterious”, while
PolyPhen2 (accessed at http://genetics.bwh.harvard.edu/pph2/) and variants with a score above − 2.5 are considered “neutral”. Of the
the program Provean (Protein Variation Effect Analyzer, accessed at novel missense mutations analyzed six were indicated as “deleteri-
http://provean.jcvi.org) to predict the effect on the ARSA protein. ous” by this program, while p.Y39F, p.V177D and p. R288S were con-
sidered “neutral” (Table 2). In addition, of the 3 nonsense mutations
3. Results and discussion reported two were indicated as “deleterious” and p.Q51X was con-
sidered “neutral”. Mutation c.32TNA was predicted to be a “benign”
Currently more than 70 patients with MLD have been characterized mutation by both programs (see patient # 2).
molecularly in our laboratory: 16 previously unreported mutations
have been identified in 15 unrelated patients. All the other patients had - The c.28delG mutation found in patient # 1is located in the leader
previously reported mutations. These new mutations are described in sequence. However, this deletion results in a frame shift in exon 1
this manuscript (Table 2). Also, three previously reported mutations and it is therefore predicted to be severe. This mutation was identi-
were identified on a different allelic background than in the previous re- fied in a family of Hispanic origin with three affected siblings with
ports (Table 3). With the exception of three patients being homozygous the same genotype. The youngest of the three is a 30-month-old pre-
for new mutations, and one (# 8) having two different new mutations, senting with spasticity, cognitive problems and loss of motor mile-
all the others are compound heterozygous with a second mutation previ- stones (Table 1). It is not clear at what age the other two affected
ously reported (Table 2). Of the 19 patients described, the majority had siblings started to show signs of the disease, but both were diag-
the late infantile form of the disease (see age of onset in Table 1) and nosed around 7 years of age. The second mutation identified in
only 5 (patients # 10, 11 14, 15 and 18) had the juvenile form. Six of this family is p.E382K. This mutation was reported by Barth et al
the 19 patients had the common LI mutation as the second allele and (1993) and found homozygous in a juvenile MLD patient in cis
two had the common adult mutation as the second allele. Of the 16 with the Pd allele. However, in our patient this mutation is not asso-
new mutations reported, nine were missense mutations, three were non- ciated with the Pd allele. Other investigators identified this mutation
sense, three were frame shift mutations and one was a splice-site in a typical late infantile patient (Shotelersuk et al, 2004) and

Table 2
Summary of mutations found in patients reported in this study.

Allele I Allele II

Patient cDNA position Location Effect Probability of Comment cDNA position Location Effect
# (gene position) deleterious mutation a (gene position)

PolyPhen2 Provean

1 28delG (28) Ex1 GGCC N GCC, A10fs NA NA 1144GNA (2133) Ex7 E382K
2 32TNA Ex1 L11Q 0.12 −1.638 Homozygous
3 105CNA+106_124dup (105) Ex1 D35E + P42fs NA NA 1277CNT (2381)+Pd Ex8 P426L
4 130TNC (130) Ex1 S44P 1.00 −2.856 459+1GNA Int2 Loss of splice
donor site
5 151CNT (151) Ex1 Q51X NA −2.251 Homozygous
6 189delC (189) Ex1 TACG N TAG, Y63X NA −6.918 740TNC (1077) Ex4 F247S
7 242TNC (391) Ex2 L81P 1.00 −3.188 459+1GNA Int2 Loss of splice
donor site
8b 251GNT (400) Ex2 R84L 1.00 −6.710 1273CNT (2377) Ex8 P425S
9 116ANT (116)+447CNG (596) Ex1 + Ex2 Y39F + Y149X 0.788 (Y39F) −2.042 Both mutations 634GNC (897) Ex3 A212P
(Y39F) on same allele
−6.156
(Y149X)
10 468CNA (731) Ex3 C156X NA −6.711 1277CNT (2381) Ex8 P426L
11 530TNA (793) Ex3 V177D 1.00 −1.894 459+1GNA Ex8 Loss of splice
donor site
12 850CNT (1499) Ex5 P284S 1.00 −7.482 635CNT (898) Ex3 A212V
13 862CNA (1511) Ex5 R288S 0.999 −1.945 730CNT (1067) Ex4 R244C
14 901GNA (1550) Ex5 G301R 1.0 −7.504 536TNG (799) EX3 I179S
15 1102-2ANG Int6 Loss of splice acceptor site NA NA Homozygous
8b 1273CNT (2377) EX8 P425S 1.0 −6.038 251GNT (400) Ex2 R84L

New mutations are listed in bold. NA = not applicable.

a
Scores obtained by in silico analysis of the mutations using the program PolyPhen2 and the program Provean.
b
This patient has two new mutations.
326 P. Luzi et al. / Gene 530 (2013) 323–328

Table 3
Mutations previously reported on a different allelic background.

Mutations previously found on a different background Second mutation

Patient # cDNA position Location Effect Probability of Presence of Pd Comment cDNA position Location Effect
(gene position) deleterious mutationa mutation in cis (gene position)

PolyPhen2 Provean

16 925GNA (1574) Ex5 G309S 1.00 −5.928 + Previously reported without Pd 459+1GNA Ex8 Loss of splice
donor site
17 931CNT (1580) Ex5 R311X NA −5.928 _ Previously reported on Pd allele 459+1GNA Ex8 Loss of splice
donor site
18 931CNT (1580) Ex5 R311X NA −5.928 _ Previously reported on Pd allele 536TNG (799) Ex3 I179S
19 936GNC (1585) Ex5 E312D 0.983 −2.114 + Previously reported without Pd 459+1GNA Ex8 Loss of splice
and GNT change donor site

NA = not applicable.
a
Scores obtained by in silico analysis of the mutations using the program PolyPhen2 and the program Provean.

described that the pathogenic effect is not due to the amino acid sub- activity. This seems highly unlikely and raises concerns about the re-
stitution but to the disruption of a potential exonic splicing enhancer liability of such programs.
resulting in a complete exon 7 skipping. - The deletion of a C at position 189, found in patient # 6, results in a
- Patient # 2 from Kenya was found to be homozygous for a c.32TNA premature stop codon (p.Y63X). This mutation was identified in a
mutation (p.L11Q). In silico analysis gives a“neutral” score with 2-year-old patient (Table 1) presenting with difficulty walking and
both programs used predicting a “benign” effect on the protein ac- rapid regression of skills. The second mutation, p.F247S, was previ-
tivity. This mutation results in a substitution in the leader sequence ously reported in a juvenile patient in trans with the common adult
of a leucine with a glutamine at position 11. No other changes were mutation (Olkhovich et al, 2003). The early onset and the rapid course
identified in this patient after repeated sequencing of the ARSA of the disease in this patient indicate that not only the new reported
gene. In addition, one copy of this mutation was confirmed to be deletion is severe (as predicted by the Provean program) but that
present in both parents. The father's ARSA gene was also sequenced also p.F247S must produce a protein with very little residual activity.
completely and no other changes were identified. The patient has - Mutation c.242TNC (p.L81P) was identified in a patient with a typical
an enzymatic activity for the arylsulfatase A in the affected range, late infantile presentation (Table 1, # 7) in trans with the common LI
excretes sulfatides and has a clinical presentation consistent with mutation. Based on the clinical presentation this new mutation is
late infantile MLD (Table 1). The ARSA activities for both parents therefore severe. Both in silico analysis programs predict a “deleteri-
are in the carrier range. The 18-amino acid signal peptide for the ous ”effect on the protein.
ARSA gene has the typical structure of a leader sequence: the core - Patient # 8 was a patient of Arabic origin who was diagnosed at
of the peptide contains a long stretch of 12 hydrophobic amino 4.5 years of age. He started to show signs of regression at 2 years
acids. Mutation p.L11Q is located in the middle of this structure of age and at the time of diagnosis the stage of disease was very ad-
and introduces a charged amino acid that disrupts the hydrophobic vanced (Table 1). Two previously unreported mutations were iden-
core. The hydrophobic core in signal peptides is required for secret- tified in this patient: c.251GNT (p.R84L) in exon 2 and c.1273CNT
ed proteins to be efficiently translocated across the endoplasmic (p.P425S) in exon 8. Both in silico analysis resulted in a score
reticulum (von Heijne, 1985). We hypothesize that this mutation predicting a “damaging” effect for both mutations indicating high
is disease-causing due to the fact that the ARSA protein is ineffi- confidence in the deleterious outcome on the ARSA protein activity.
ciently processed. Mutations in the signal peptides of other pro- Also one copy of the Pd allele was detected in this patient. Parental
teins have been reported as the cause of the disease due the DNA was not available, therefore it was not possible to establish
deficiency of that specific protein (Arnold et al, 1990; Rosenblum which of the two mutations was in cis with the Pd mutation. It is in-
et al, 1996; Zschenker et al, 2001). teresting to note that different mutations have been reported on the
- The duplication of 19 base pairs starting at cDNA position 106 same nucleotides, resulting in a different amino acid substitution:
proceeded by a substitution c.105CNA (patient # 3) also results in c.251GNA (p.R84Q) (Kappler et al, 1992) and c.1273CNA (p.P425H)
a frame shift beginning at amino acid position 42. This mutation (Marcão et al., 2003). Mutation p.R84Q has been reported with
is therefore predicted to be “severe”. This mutation was found in and without the Pd allele (Coulter-Mackie and Gagnier, 2003; Rafi
trans with the common adult mutation which is in cis with the Pd et al, 2003).
allele. As reported before (Regis et al, 2002), the common adult - Patient # 9 presented at 2.5 years of age with characteristic symptoms
mutation in cis with the Pd mutation results in a more severe phe- of late infantile MLD. Mutation analysis showed the presence of two
notype than the adult mutation alone. Patient # 3 in fact presented new mutations in cis: p.Y39F in exon 1 and p.Y149X in exon 2. The
at 2 years of age with a typical late infantile form of the disease disease-causing mutation was considered to be p.Y149X since it cre-
(Table 1). ates a premature stop codon, with p.Y39F being regarded as a change
- Mutation c.130TNC found in patient # 4 results in a substitution of a of unknown significance. This is also in agreement with the results of
serine at position 44 with a proline. This mutation in an African the in silico analysis (Table 2). The second mutation identified in this
American patient was found together with the common LI mutation patient (p.A212P) was previously reported and is considered a severe
(Table 2). The patient had the typical late infantile presentation mutation (Grossi et al, 2008). This is in agreement with the clinical
(Table 1) indicating that this new mutation is indeed severe as pre- picture of this patient.
dicted by both in silico analysis programs. - Mutation c.468CNA (p.C156X) was found in a 6.5-year-old patient
- Mutation c.151CNT (patient # 5) results in a premature stop codon in (patient # 10) having the common adult mutation on the other allele.
exon 1 (p.Q51X). This mutation was found homozygous in a patient This new mutation is expected to be severe since it creates a prema-
from Ethiopia with an early presentation of the disease. This is consis- ture stop codon in exon 3, as also predicted by the Provean program.
tent with the expected severity of this mutation. The Provean pro- Very recently, mutation p.C156X was identified in our laboratory ho-
gram predicts this mutation to have a “neutral” effect on the protein mozygous in a typical late infantile patient of Hispanic origin
 P. Luzi et al. / Gene 530 (2013) 323–328 327

presenting at 2 years of age with regression of milestones, hypoto- matter changes on MRI and mild speech delay. His phenotype is clearly
nia and evidence of white matter disease (ARSA activity = more severe than the one described by Hermann et al (2000), indicat-
8.6 nmol/h/mg protein and presence of sulfatides in the urine). ing that probably the presence of the Pd allele increases the pathoge-
The juvenile onset in patient #10 is explained by the presence of nicity of the mutation. This is not surprising since the association of
the p.P426L mutation in trans with p.C156X. one otherwise mild mutation with the Pd mutation is being shown
- Mutation c.530TNA (p.V177D) was identified in a 12-year-old pa- to result in a more severe clinical phenotype like in the case of the
tient (# 11). The provided clinical history for this patient was mini- common adult mutation (Regis et al, 2002). However, other factors,
mal, with the major complaint being “developmental delays”. The such as genetic background and environment, could also be responsi-
second mutation is the common LI mutation, indicating that ble for the more severe phenotype of this patient. The PolyPhen2 pro-
p.V177D is a milder mutation able to mitigate the effect of a severe gram predicts mutation p.E312D to be “damaging” while according to
mutation and to result in a juvenile form of the disease. The the Provean program this mutation is “neutral”. The third mutation re-
PolyPhen2 program predicts this mutation to be “damaging” with ported in Table 3 (p.R311X) was identified in two unrelated patients
a score of 1 while according to the Provean program this mutation and in both of them it was not in cis with the Pd allele, as was found
is “neutral”. previously (Rafi et al, 2003).
- Mutation c.850CNT (p.P284S) was identified in a 3-year-old patient In patient # 17 the p.R311X was in trans with the common late in-
(# 12) in trans with a previously reported severe mutation fantile mutation and the patient had the typical late infantile presenta-
(p.A212V) (Barth et al, 1993; Coulter-Mackie et al, 1997). In silico tion of the disease as expected from two severe mutations.
analysis predicts a damaging mutation with both program used, In patient # 18 the p.R311X mutation was instead associated with
and the clinical picture of this patient confirms the severity of the the known late-onset mutation p.I179 (Fluharty et al, 1991) on the
change. other allele and the patient presented around 12 years of age with
- Mutation c.862CNA resulting in a substitution of an arginine with a mostly behavioral problems.
serine at position 288 was identified in a 2.5-year-old patient (#
13) of Chinese ancestry. It is interesting to note that a mutation of 4. Conclusions
the C at position c.862 with a T, resulting in a substitution of the ar-
ginine with a cysteine, has been reported before and was also iden- In this manuscript we report 16 novel mutations associated
tified in a Chinese patient (Gieselmann et al, 1994; Wang et al, with metachromatic leukodystrophy. For the purpose of genotype–
2007). The second mutation identified in this patient, p.R244C, phenotype correlation, we tried to classify, when possible, these muta-
was reported before (Draghia et al, 1997) and it is considered se- tions as “severe” or “mild” based on the patient's clinical presentation
vere. The PolyPhen2 program predicts mutation p.R288S to be and age of onset and considering the “severity” of the second mutation,
“damaging”, while according to the Provean program this mutation when it was known. In silico analysis of novel mutations was of limited
is “neutral”. value in predicting the severity of a mutation. In addition, the two differ-
- Mutation c.901GNA (p.G301R) was identified in a juvenile/adult ent methods employed were not in agreement regarding the possible
patient (# 14) with symptoms presenting around 15 years of age. damaging effect of a mutation in three of the reported cases. It is impor-
The second mutation is a known later-onset mutation, p.I179S tant to have mutations identified in more patients to assist in patient
(Fluharty et al, 1991), characterized mostly by cognitive decline management. This information will be useful for both families and phy-
more than motor involvement, consistent with the disease presen- sicians caring for these patients. Very recent preliminary results from a
tation in this patient. In silico analysis with both programs predicts hematopoietic stem cell gene therapy clinical trial involving MLD pa-
the new mutation p.G301S to be “damaging”, but no prediction of tients have been published (Biffi et al, 2013). As approaches to treat-
severity can be done for this mutation. ment become more available it will be important to have more
- The only splice-site mutation reported in this manuscript (1102- mutations characterized.
2ANG) was found homozygous in an 11-year-old patient from
Saudi Arabia (# 15). The effect of this mutation is a loss of a splice
acceptor site in intron 6 probably resulting in abnormal splicing. Conflict of interest
Since this mutation results in a juvenile form of the disease, some
residual activity may be present. The authors declare they have no conflicting interest.

In Table 3 are reported three mutations previously identified on a

different allelic background.
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