Diagnostic challenges in Salla disease

doi:10.4236/ojgen.2013.32A3007

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Open Journal of Genetics, 2013, 3, 46-49 OJGen

http://dx.doi.org/10.4236/ojgen.2013.32A3007 Published Online August 2013 (http://www.scirp.org/journal/ojgen/)

Diagnostic challenges in Salla disease

Jessica N. Hartley1,2, Michael S. Salman3,4, Frances A. Booth3,4, Lorne Seargeant3,5,

David A. Wenger6, Jens Wrogemann7, Aizeddin A. Mhanni1,2,3*

1Department of Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, Canada

2Program of Genetics and Metabolism, Winnipeg, Canada

3Department of Pediatrics and Child Health, University of Manitoba, Winnipeg, Canada

4Section of Pediatric Neurology, University of Manitoba, Winnipeg, Canada

5Diagnostic Services Manitoba, University of Manitoba, Winnipeg, Canada

6Lysosomal Diseases Testing Laboratory, Thomas Jefferson University, Philadelphia, USA

7Department of Radiology, Section of Pediatric Radiology, University of Manitoba, Winnipeg, Canada

Email: *[email protected]

Received 27 October 2012; revised 16 November 2012; accepted 9 December 2012

which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ABSTRACT

Sialic acid storage disease (Salla disease) is an auto-

somal recessive disorder caused by mutations in a ly-

sosomal sialic acid export protein, SLC17A5 (OMIM

#604369). This disorder was initially described in

Northern Finland but more recently has been re-

ported in patients of other ethnicities. We describe

the clinical presentation and the neuroimaging find-

ings of two non-Finnish children where a diagnosis of

Salla disease was suspected on the basis of brain mag-

netic resonance imaging. The biochemical confirma-

tion of this diagnosis posed a challenge as both pa-

tients had elevated percent free urine sialic acid but

biochemical analyses in fibroblasts were not conclu-

sive; therefore, molecular testing was necessary for

confirmation of the diagnosis. The described encoun-

ters demonstrate the importance of pursuing confir-

matory molecular diagnostic testing when a sialic acid

storage disorder is suspected.

Keywords: Sialic Acid; Salla Disease; Lysosomal;

SLC17A5

1. BACKGROUND

Free sialic acid storage disease (Salla disease) is an auto-

somal recessive disease caused by mutations in a ly-

sosomal sialic acid export protein, SLC17A5 (OMIM

#604369) which leads to lysosomal accumulation of free

sialic acid [1]. Common features of this disease include

developmental delay, ataxia, subtle coarse facial features

and brain magnetic resonance imaging (MRI) findings of

hypomyelination and hypoplastic corpus callosum [2].

Biochemical findings of a defect in the sialic acid export

protein include elevated total and free sialic acid in urine

and fibroblasts [3,4]. The mild to moderate form of this

condition was initially described in Northern Finland

with the eponym “Salla” referring to the geographically

restricted area where the first family resided [4]. Since

that time, free sialic acid storage disease has been re-

ported in patients of other ethnicities and infantile free

sialic acid storage disease (ISSD) (OMIM# 269920) has

been described to be allelic to Salla, with ISSD being as-

sociated with an infantile onset progressive disease and

extremely elevated total and free sialic acid levels [1,3,

5,6]. We describe the clinical presentation, neuroimaging

and the challenges in confirming this diagnosis in two

children of non-Finnish ancestry.

2. MATERIALS AND PATIENTS

2.1. Methods

Urine Sialic Acid Measurements: Determinations of urine

total and free sialic acid levels were conducted using a

spectrophometric method as described [7,8] and com-

pared to age-matched controls [9].

Sialic Acid Studies in Fibroblasts: A pellet of cultured

skin fibroblasts was sonicated in distilled water and the

protein concentration was determined. The total sialic

acid content were determined after heating an aliquot

containing between 100 and 150 μg protein at 80˚C in

0.1 N sulfuric acid for 1 hour. The same size aliquot in

distilled water and not heated was used to determine the

free sialic acid content. The method of Aminoff was then

followed to measure the sialic acid content [10]. After

reading the absorbance at both 549 and 532 nm, the sialic

*Corresponding author.

OPEN ACCESS

J. N. Hartley et al. / Open Journal of Genetics 3 (2013) 46-49 47

acid content per mg protein was calculated.

This case series has been reviewed by the University

of Manitoba Research Ethics Board to ensure that patient

identity is protected and consent processes were adhered to.

2.2. Patients

Patient 1 presented at age twenty-two months with a his-

tory of global developmental delay. The family history

was unremarkable for neurodegenerative disease and

there was no history of consanguinity. The family was of

Irish, English, Ukrainian and Dutch descent (non-Fin-

nish). The pregnancy history was unremarkable with no

history of exposure to known teratogens. The perinatal

and neonatal histories were unremarkable. His birth

weight was 3.486 kg (75th percentile). His Apgar scores

were 5 and 8 at 1 and 5 minutes respectively. His ex-

amination at twenty-two months of age was remarkable

for subtle coarse facial features, a depressed nasal bridge,

a high arched palate and a high forehead. His weight was

12.8 kg (50th - 75th percentile), height was 84.5 cm

(25th - 50th percentile) and OFC was 50.5 cm (90th -

97th percenttile). Hepatomegaly was noted with a palpa-

ble liver edge 3 cm below the right costal margin in the

midclavicular line. His neurologic examination revealed

normal visual behaviour and extraocular movements and

generalized hypotonia (central and peripheral) with nor-

mal muscle strength. His deep tendon reflexes were nor-

mal and symmetric in the upper limbs and slightly in-

creased in the lower limbs. Ataxia was noted in the lower

but not the upper limbs. A formal developmental assess-

ment using the Yale Developmental Schedules at twenty-

seven months of age revealed a developmental quotient

of 65%. The child’s ophthalmologic and echocardiogra-

phic assessments were unrevealing. An electroencepha-

logram revealed left-sided slowing in background activ-

ity and right-sided epileptiform discharges; however there

was no history of clinical seizures. Brain MRI at age

twenty-one months showed abnormalities suggestive of

Salla disease (Figure 1).

Patient 2 presented at age thirty-one months with a

history of ataxia and developmental delay. The preg-

nancy, perinatal, neonatal and family histories were un-

remarkable. No consanguinity was reported and the fam-

ily was not of Finnish ancestry. The child’s examination

at thirty-one months of age was remarkable for subtle

coarse facial features and significant ataxia. The weight

was 14 kg (75th percentile), height was 93.5 cm (50th

percentile) and OFC was 48.8 cm (75th - 90th percentile).

Cranial nerve examination was remarkable for excessive

drooling with an intact gag response. Muscle strength

and tone were normal. The child had prominent truncal

and limb ataxia affecting all four extremities and walked

only with support with a broad-based gait. The child had

a bilateral pincer grasp but had difficulty with fine finger

Figure 1. Coronal T2-weighted MRI in patient 1 at age twenty-

one months showing complete lack of normal myelin signal

and white matter volume loss throughout the cerebral hemi-

spheres (horizontal arrow). Hypomyelination and volume loss

of the cerebellar white matter was also present (vertical arrow).

The cortical gray matter and deep gray matter (not shown) were

preserved.

movements and finger-to-nose testing. Deep tendon re-

flexes were normal and symmetric with down-going

plantar responses. A formal developmental assessment

using the Yale Developmental Schedules at thirty-one

months of age revealed a developmental quotient of 65%.

Follow-up assessment using the Revised Gesell Devel-

opmental Schedules at sixty-nine months of age (five

years) suggested a gross motor developmental quotient

of 26%, a fine motor developmental quotient of 50% and

a language developmental quotient of 35%. An electro-

encephalogram revealed localized epileptiform abnorma-

lities. Brain MRI at age forty months also showed ab-

normalities suggestive of Salla disease (Figure 2).

3. RESULTS

A summary of the results of the laboratory investigations

undertaken in both patients to investigate the possibility

of Salla disease in provided in Table 1.

In patient 1, urine total and free sialic acid were ele-

vated above age-matched control values on two occa-

sions, with the percent free urine sialic acid and skin

electron microscopy being suggestive of a free sialic acid

storage disorder (data not shown). Fibroblast assay re-

vealed elevations of free sialic acid in the diagnostic

range, however total sialic acid values were only mildly

elevated as compared to controls. Molecular analysis of

the SLC17A5 gene revealed compound heterozygosity

J. N. Hartley et al. / Open Journal of Genetics 3 (2013) 46-49

Table 1. Pertinent laboratory investigations for Salla disease.

Patient 1 Patient 2

Urine Average Free Sialic Acid (umol/mmol creatinine) 313* 93.5

Urine Average Total Sialic Acid (umol/mmol creatinine) 387* 146

% Urine Free Sialic Acid

(Salla > 80%)

(Control < 60%) 80.9%* 63.8%*

Fibroblast Sialic Acid (nmol/mg protein)

Free SA

(Salla 10.0 +/− 2.9 SD)**

(Control 1.0 +/− 0.6 SD)

10.4* 2.1

Total SA

(Control 18.0 +/− 4.6 SD) 20 15.3

Skin Electron Microscopy Lysosomal storage of fine fibrillar materialEnlarged lysosomes with fine fibrillar

structures

Molecular Pat c. 250 del C

Mat c. 115C > T Homozygous c. 115C > T

*Diagnostic laboratory values; **Case/control values from Seppala et al. 1991.

OPEN ACCESS

Figure 2. Coronal T2-weighted MRI in patient 2 at age forty

months showing marked volume loss and increased T2 signal

intensity of the cerebral hemispheric white matter (horizontal

arrow) and corpus callosum (oblique arrow), reflecting a lack

of myelin. The cortical gray matter and deep gray matter were

preserved. There was no evidence of volume loss or signal

abnormality in the posterior limbs of the internal capsule, cere-

bellum, and brainstem (not shown).

for the common Finnish mutation, 115C > T (R39C), and

another mutation, 250delC, previously described in a

French patient [11] confirming the diagnosis of Salla dis-

ease.

In patient 2, four independent urine samples revealed

borderline elevations of free and total sialic acid. Urine

percent free sialic acid was mildly elevated compared to

age-matched control values, but not in the diagnostic

range for Salla disease. Skin electron microscopy was

consistent with a free sialic acid storage disorder (data

not shown). Fibroblast assay showed normal total and

free sialic acid. Molecular analysis of the SLC17A5 gene

revealed homozygosity for the common Finnish mutation,

115C > T (R39C), confirming a diagnosis of Salla dis-

ease.

4. DISCUSSION

The clinical phenotype of both patients is consistent with

Salla disease, rather than infantile free sialic acid storage

disease (ISSD) given that symptoms developed during

the first year of life and that both patients continued to

make developmental progress, albeit slowly, without any

evidence of developmental regression [5]. The elevated

total but not free sialic acid in fibroblast culture in pa-

tient one and the normal total and free sialic acid in fi-

broblast culture for patient two posed a diagnostic chal-

lenge. The more severe ISSD is associated with ex-

tremely high total and free sialic acid levels in cultured

fibroblasts, leukocytes and urine as compared to the

milder Salla disease [3]. To our knowledge, there are no

published reports of individuals with phenotypic Salla

disease with normal fibroblast free sialic acid, even

among the Finnish population where the condition was

originally described. A pair of siblings with a Salla phe-

notype and homozygosity for a different mutation, K136E,

also had non-diagnostic biochemical findings, with nor-

mal urine free sialic acid (percent free sialic acid not

reported), elevated sialic acid in cerebrospinal fluid and

mildly elevated free sialic acid in fibroblasts [12]. Nor-

mal leukocyte total and free sialic acid levels in mildly

affected children of Mennonite descent, who are homo-

zygous for the R39C Finnish mutation, like patient 2,

have been noted. In the urine of these patients, the total

sialic acid content was noted to be normal, however the

percent free was elevated above normal (greater than

80% free versus less than 60% in non-Salla individuals)

J. N. Hartley et al. / Open Journal of Genetics 3 (2013) 46-49 49

(Wenger, personal communication 2012). Our patient

two was comparable, with mildly elevated urine free si-

alic acid, however, 63% free sialic acid is not consid-

ered to be in the diagnostic range.

Molecular diagnostic testing was required for confir-

mation of the diagnosis in both our patients as well as

those noted by Wenger (personal communication 2012)

and reported by Mochel [12]. Patient 2 was homozygous

for the Finnish mutation associated with the Salla disease

phenotype. It is interesting to note that neither of our

patients is of Finnish ancestry but the R39C mutation is

present in both. Salla disease due to the originally de-

scribed R39C mutation appears to be pan-ethnic; homo-

zygosity for this mutation has been reported in individu-

als of other ethnicities, including those of Old Order

Mennonite extraction [1].

Our report demonstrates the importance not only by

considering the diagnosis of Salla disease in children

with subtle coarse facial features, developmental delay

and hypomyelination on brain MRI who are not of Fin-

nish ancestry, but also of the importance of pursuing

molecular testing for Salla disease for confirmation of

the diagnosis in the face of non-diagnostic biochemical

analyses.

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ONLINE DATABASES CITED

Online Mendelian Inheritance in Man, OMIM®. Johns

Hopkins University, Baltimore, MD. MIM Number:

604369: 03/07/2012. World Wide Web URL:

http://omim.org/

Online Mendelian Inheritance in Man, OMIM®. Johns

Hopkins University, Baltimore, MD. MIM Number:

269920: 07/22/2010. World Wide Web URL:

http://omim.org/