Mucopolysaccharidosis type I (MPS I) is an autosomal recessive lysosomal storage disorder that results from a deficiency in the alpha-L-iduronidase (IDUA; E.C.3.2.1.76). This glycosidase is involved in the degradation of dermatan and heparan sulfates. IDUA deficiency causes a progressive accumulation of undegraded mucopolysaccharides leading to characterized clinical phenotypes 1 . MPS I has three major clinical phenotypes, ranging from the severe Hurler form to the Scheie phenotype:

The severe phenotype (Hurler syndrome, MPS IH) involves mental retardation, skeletal of deformities, stiff joints, hepatosplenomegaly, corneal clouding, and a shortened life expectancy. The attenuated phenotype (Scheie syndrome, MPS IS) is characterized by mild skeletal deformities, stiff joints, corneal clouding and a long life span without mental retardation. The intermediate phenotype (Hurler / Scheie syndrome, MPS IH/S) involves skeletal deformities, severe organomegaly, usually limited bone involvement and a variable life span with neurological involvement. The wide spectrum of clinical severity in MPS I suggests the presence of allelic heterogeneity.

The IDUA gene is located on chromosome 4p16.3. It contains 14 exons spanning 19 kb. It is transcribed into 2.3 kb cDNA, which encodes a 653 amino acids protein 2 3 . Human Gene Mutation Database described more than 100 mutations and 32 polymorphisms (http://www.hgmd.org). Previous mutations including missense and large deletion have been found in patients with the severe phenotype. The others gender such as nonsense, insertion-deletion and splice site have been identified in both severe and mild phenotypes.

In Tunisia, first cousin inbreeding is the most represented with an estimated incidence about 32% 4 . Furthermore, Tunisian population is characterized by a heterogeneous genotype resulting from social assets created by invasions and migrations. For example, it exists a large mutations spectrum of hemoglobinopathies such as beta-thalassemia identified as a result of rare alleles selection 5 . Thus, screening IDUA mutation in MPS I patients is essential to identify the specific mutation of our country, to detect heterozygote and prenatal diagnosis.

In this study, we investigated IDUA mutation in MPS I Tunisian families based on a complete scan of IDUA gene.

MPS I is the most common subtype of mucopolysaccharidosis with an estimated of about 1.7 in 100. 000 live births for the severe and mild forms 8 . The incidence of MPS I in Tunisia is also high, estimated at 0.63 in 100.000 live births 9 . MPS I patients show a wide spectrum of clinical phenotype ranging from the severe Hurler to the attenuated Scheie form 10 . Currently, over 100 mutations have been reported in patients with the MPS subtypes (Human Gene Mutation Database; http://www.hgmd.org).

In our investigation, all patients were born from to consanguineous marriages between first, second and third cousins (Figure 1). The molecular analysis of the MPS I patients in eight Tunisian families, identified five mutations including a novel mutation: L578Q and four reported mutations: P533R, Y581X, F602X and R628X (Figure 2). Of note, the common mutations, Q70X and W402X in European populations were not found 11 .

Patients 1, 2 and 3 from family one were homozygous for the F602X mutation. This predicts the synthesis of a premature chain termination of the IDUA glycopeptide. These patients have undetectable IDUA activity. F602X mutation was firstly reported in Tunisian patient as deletion-insertion complex in exon 13 of the IDUA gene 12 . Besides, our study confirmed the absence of F602X mutation in 50 DNAs of our normal population.

Patients 4, 5, 6, 7, 8 and 10 from families two, three, four and six were homozygous for the P533R mutation in exon 11 of IDUA gene. The P533R missens mutation was first described in a group of 73 MPS I patient (3%) 13 . Alif et al 2002 14 reported a high frequency of this mutation in a group of 13 MPS I patients from Morocco (92%). This mutation has been identified also in 10 MPS I patients (62.5%) from Tunisia 15 . The P533R mutation has been identified in a group of 27 MPS I patients (11%) from Sicily 16 and not identified in 3 MPS I patients from Egypt 17 .

Some studies of genetic and population background for different inherited diseases such as Niemann-Pick 18 , metachromatic leukodystrophy 19 , Gaucher disease 20 and mucopolysaccharidosis type I 21 succeed to distinguish the origin of frequent mutation. Haplotype analysis may distinguish whether a relatively common mutation is resulted from a founder effect or from recurrent mutations or multiple haplotypes. Thus for P533R origin estimation we think that haplotypes analysis will be determined for different MPS I populations.

The patient of family five was homozogous for the previously reported R628X mutation. This patient had the severe Hurler phenotype and she deceased at five years of age. We showed in a previous study that this patient was probably homozygous for this mutation. This finding is a confirmation of previous hypothesis 15 .

The patient of family seven was heteroallelic for the novel L578Q mutation and the previous reported P533R mutation. This patient had the typical Scheie phenotype. The P533R mutation resulted in a non conservative substitution of a neutral proline for a basic arginine and associated with a low level of residual IDUA enzymatic activity in CHOK I cells 22 . The novel L578Q mutation presumably also causes a mild instability or conserves of function since the patient have the attenuated phenotype. She goes at school and she had a good note in scientific subjects. Our study showed that L578Q mutation is not found in 50 DNAs of normal population.

The patient of family eight was homozygous for the previously described Y581X mutation. This patient had the severe Hurler phenotype and she deceased at five years of age.

A large number of polymorphisms and non-pathogenic sequence variants have been described in the IDUA gene 23 22 24 . The effect of these sequence variants on the IDUA activity has not been clearly defined, especially when they are associated with specific mutations. However, it has been speculated through structural effects on IDUA gene that such polymorphisms may modify the patient clinical phenotype 25 26 , particularly for patients who have missense mutations causing the Hurler / Scheie and Scheie phenotypes 11 . We found the same previously described polymorphisms associated to different phenotypes: Hurler and Hurler / Scheie form for patients of family one and three. Besides, the novel polymorphisms were associated to severe phenotype for patient of family five and six.

Mutations which permit some enzymatic function may be more susceptible to modulation by variables factors such as novel combinations of mutant alleles, attenuating polymorphisms, genetic background, and environmental factor (Tables 2, 4).

Screening of polymorphisms in IDUA gene and determination of the haplotypes is necessary to search the origin of mutations within MPS I patients. Individuals resulting from a common ancestor are likely to have inherited both copies of the mutated gene and also in the haplotypes which will be transmitted to descent and thus rare genotypes are maintained 27 .

Further studies in determination of haplotypes within MPS I patients might help in finding out whether these haplotypes are associated with the reported mutations and polymorphisms.

In the present paper, eighteen IDUA sequence variants have been identified of MPS I Tunisian patients, including eight novel polymorphisms. The effect of noncoding and coding polymorphisms on IDUA expression in the MPS I patients from Tunisia is unclear leading to ambiguous correlation genotype / phenotype establishment. Further studies on large number of MPS I patients and normal population might help in finding out whether these novel and reported polymorphisms are associated with a specific phenotype.

MPS I: Mucopolysaccharidosis I; IDUA: Alpha L iduronidase; PCR: polymerase chain reaction.

The authors declare that they have no competing interests.

Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.

LC and SK have done all the work (PCR, sequencing...) in the laboratory. AK and AB have done the analysis of the results. RF and CVS interpret the results. S F, S L and A M have given final approval of the version to be published. All authors read and approved the final manuscript