Patients
The study was carried out in 15 MPS IVA patients recruited from pediatric departments
of different hospitals in Sousse, Sfax and Sidi Bouzid. All studied patients were
from a consanguineous marriage, and there were no relationship known between the families.
All procedures were in accordance with the ethical standards of the responsible committee
on human experimentation (institutional and national) and with the Helsinki Declaration
of 1975, as revised in 2000 and approved by the Ethics Committees of the respective
Tunisian hospitals. Informed consent was obtained from all patients and their families
before being included in the study. Additional informed consent was obtained from
all patients for whom identifying information is included in this article.
Biochemical assay
The diagnosis of the MPSIVA disease was based on the following approach after a clinical
and paraclinical suspicion.
Quantitative analysis of total urinary glycosaminoglycans
We first performed the study of urinary GAGs. Urinary GAGs were quantified using a
dimethylmethylene blue (DMB) test 6].
Electrophoresis on cellulose acetate plate was performed to identify the types of
GAGs present in excess (e.g., dermatan sulfate, heparan sulfate, keratan sulfate). Discontinuous electrophoresis
on cellulose acetate plates separated the different GAGs based on their charge and
differential solubility in ethanol. The mucopolysaccharides were visualized by staining
with alcian blue 6].
GALNS enzyme assay
The GALNS enzymatic activity was measured in sonicated fresh leukocyte pellets using the fluorogenic
substrate 4-methylumbelliferyl-?-D-galactoside-6-sulfate, as previously described
7].
Molecular analysis
GALNS mutation analysis
Genomic DNA was isolated from venous blood by the phenol/chloroform procedure, according
to standard protocols as previously described 8].. Genomic DNA of GALNS gene was amplified and sequenced. For patients with a family history of known or
predicted pathogenic mutations or for the index cases’ parents, targeted PCR and sequencing
were performed in the Laboratory of Inherited Metabolic Diseases in Lyon-France.
Haplotype analysis
In order to estimate the origins of the novel missense mutation p.D288G and the other
known mutations, haplotypes were determined using GALNS Sanger sequencing of six amplified genomic fragments, each one including one polymorphism
9], 10].
The mutant and normal alleles were tested for six polymorphisms. These included the
following known polymorphisms in: (1) intron 5 (IVS5?+?134; CCAAGG [allele A] or CCGAGG
[allele a] 11], (2) exon 7 (763 nt from A of the ATG initial codon on the cDNA; GCACGC [allele B]
or GCATGC [allele b] 12], (3) intron 7 (IVS7nt90; GTAC [allele H] or GAAC [allele h] 11], (4) exon 11 at cDNA 1232 (GTCC [allele C] or GGCC [allele c]) 11], (5) exon 13 at cDNA 1487 (AAGCCT [allele D] or AGGCCT [allele d] 11], and (6) exon 14 (CCAG [allele E] or CCGG [allele e] 11].