Souhir Khedhiri

Mucopolysaccharidosis type I: molecular characteristics of two novel alpha-L-iduronidase mutations in Tunisian patients.

BackgroundMucopolysaccharidosis type I (MPS I) is an autosomal storage disease resulting from defective activity of the enzyme α-L-iduronidase (IDUA). This glycosidase is involved in the degradation of heparan sulfate and dermatan sulfate. MPS I has severe and milder phenotypic subtypes.Aim of study: This study was carried out on six newly collected MPS I patients recruited from many regions of Tunisia.Patients and methods: Mutational analysis of the IDUA gene in unrelated MPS I families was performed by sequencing the exons and intron-exon junctions of IDUA gene.ResultsTwo novel IDUA mutations, p.L530fs (1587_1588 insGC) in exon 11 and p.F177S in exon 5 and two previously reported mutations p.P533R and p.Y581X were detected. The patient in family 1 who has the Hurler phenotype was homozygous for the previously described nonsense mutation p.Y581X.The patient in family 2 who also has the Hurler phenotype was homozygous for the novel missense mutation p.F177S. The three patients in families 3, 5 and 6 were homozygous for the p.P533R mutation. The patient in family 4 was homozygous for the novel small insertion 1587_1588 insGC. In addition, eighteen known and one unknown IDUA polymorphisms were identified.ConclusionThe identification of these mutations should facilitate prenatal diagnosis and counseling for MPS I in Tunisia.BackgroundMucopolysaccharidosis type I (MPS I) is an autosomal recessive lysosomal storage disorder caused by the deficient activity of the enzyme of α-L-iduronidase (IDUA, EC 3.2.1.76). This glycosidase is involved in the degradation of heparan sulfate and dermatan sulfate. The clinical phenotype of MPS I ranges from the very severe in Hurler syndrome (MPS IH) to the relatively benign in Scheie syndrome (MPS IS), with an intermediate phenotype designated Hurler/Scheie (MPS IH/S) [1]. Isolation of complementary and genomic DNAs encoding human α -L- iduronidase [2, 3] have enable the identification of mutations underlying the enzyme defect and resulting in MPS I clinical phenotype. More than 100 mutations have been reported in patients with the MPS I subtypes (Human Gene Mutation Database; http://www.hgmd.org). High prevalence of the common mutations p.W402X and p.Q70X has been described; both of them in the severe clinical forms [4, 5]. A high prevalence of common mutation p.P533R has also been described in MPS I patients with various phenotypes [5, 6]. In addition, rare mutations including single base substitution, deletion, insertion and splicing site mutation have been identified [7], indicating a high degree of allelic heterogeneity in IDUA gene.Here, we described two novel IDUA mutations in MPS I Tunisian patients. These lesions were homoallelic in all the patients of the six families investigated as consanguineous marriages are still frequent in Tunisia [8].

Molecular analysis of mucopolysaccharidosis type I in Tunisia: identification of novel mutation and eight Novel polymorphisms

Mucopolysaccharidosis type I (MPS I) is an autosomal recessive lysosomal storage disorder caused by a genetic defect in alpha-L-iduronidase (IDUA) which is involved in the degradation of dermatan and heparan sulfates. The disease has severe and milder phenotypic subtypes. The aim of this study was the detection of mutations in the IDUA gene from 12 additional MPS I patients with various clinical phenotypes (severe, 8 cases; intermediate, 3 cases; mild, 1 case).Patients and methodsIn this study, the IDUA mutations in eight unrelated Tunisian families were performed by amplifying and sequencing the IDUA exons and intron-exon jonctions.ResultsFive IDUA mutations were detected: one is the L578Q, a novel mutation found, in milder patient. The others were the previously described: P533R, Y581X, F602X and R628X that produce a severe and intermediate phenotype. In addition, eighteen variants, including eight previously unreported polymorphisms (IVS6+21c > a, IVS7+79c > t, IVS7-45 g > c, IVS9+36t > c, IVS10+140c > a, IVS11+33c > t, IVS12+13c > t and IVS12-31c > g), were detected.ConclusionThis paper, showed a heterogeneous pattern of mutations and polymorphisms among Tunisian patients.

Mucopolysaccharidoses type I and IVA: clinical features and consanguinity in Tunisia.

UNLABELLED Mucopolysaccharidoses (MPS) are a group of lysosomal storage disorders caused by the deficiency of specific enzymes which leads to the lysosomal accumulation of glycosaminoglycanes. Mucopolysaccharidosis type I or Hurler disease is characterized by the deficiency of alpha-l-iduronidase enzyme. Mucopolysaccharidosis type IVA or Morquio A disease is due to the lack of N-acetylgalactosamine-6-sulfate-sulfatase. Theses deficiencies result in a progressive accumulation of the substrates: dermatan and heparan sulfates for Mucopolysaccharidosis type I and keratan sulfate for MPS type IVA. This process leads to progressive and chronic course for visceral attacks of the affected organs such as lungs and heart. In the Hurler disease, the nervous system is particularly affected while in Morquio a disease, a skeletal dysplasia and a normal intelligence are characteristic. AIM OF THE STUDY This study was carried out on MPS type I and MPS type IVA unrelated families recruited from many regions of Tunisia in order to determine the relation between consanguinity and these types of disorders. PATIENTS AND METHODS Clinical and molecular analyses confirmed the diagnosis for four MPS type I and five MPS type IVA studied families. RESULTS First cousins unions characterize all families except one Hurler family and one Morquio A family where the consanguinity is third cousin degree. CONCLUSION MPS type I and type IVA seems to be associated with consanguinity in Tunisia.

Polymorphisms in Tunisian patients with N-acetylgalactosamine-6-sulfate sulfatase gene deficiency: Implication in Morquio A disease

AbstractMucopolysaccharidosis type IVA or Morquio A syndrome is characterized by the lack of N-acetylgalactosamine-6-sulfate-sulfatase and the accumulation of keratan sulfate and chondroitin-6-sulfate in the lysosomes. At least, 148 mutations and 16 polymorphisms were identified in the GALNS gene.The aim of this study was the screening of polymorphisms within 7 patients recruited from many regions of Tunisia in order to determine the haplotypes and their association with the mutations previously reported.Patients and methodsWe have used the PCR sequencing to analyse the different haplotypes and to identify the polymorphisms within 7 affected MPS IVA patients.ResultsNine GALNS polymorphisms were detected in the 7 studied patients. Five of these polymorphisms are within the GALNS gene exons. Six polymorphisms have been previously described and used for linkage analysis in MPS IVA patients and determination of haplotypes. We have identified two novel heterozygous polymorphisms in intron 13 and intron 3ConclusionPolymorphisms may be useful for carrier detection and prenatal diagnosis in informative families whose specific mutations have not been identified. The determination of haplotypes can also determine the origin of some mutations in a population.

Mucopolysaccharidosis IVA within Tunisian patients: Confirmation of the two novel GALNS gene mutations.

UNLABELLED Mucopolysaccharidosis type IVA or Morquio A disease is an autosomal recessive disease resulting from a deficiency of the lysosomal enzyme N-acetylgalactosamine-6-sulfate-sulfatase, which hydrolyses N-acetylgalactosamine-6-sulfate and galactose-6-sulfate in glycosaminoglycans. Phenotypes in Morquio A disease vary from the classical form with severe bone dysplasia, heart valve involvement, corneal opacity, short trunk dwarfism and a life span of 20 to 30 years, to attenuated forms with normal life span, mild bone involvement and mild visceral organ involvement. Unlike the other forms of mucopolysaccharidoses, Morquio A disease is characterized by normal intelligence. AIM OF THE STUDY The aims of this study were to determine if the novel GALNS anomalies IVS1+1G-A and G66R identified in Tunisia are mutations or polymorphisms. PATIENTS AND METHODS This study was carried out on six Morquio A patients recruited from many regions of Tunisia. We have used SCCP, sequencing and enzymatic digestion. RESULTS IVS1+1G-A and G66R were two deleterious mutations and not polymorphisms. CONCLUSION Screening of mutations and polymorphisms in GALNS gene provide useful information on genotype/phenotype correlations. It should also facilitate more accurate genetic counselling of newly diagnosed cases and their family members.

Molecular analysis in a GALNS study cohort of 15 Tunisian patients: description of a novel mutation

BackgroundMucopolysaccharidosis type IVA (MPS IVA) is an autosomal recessive disease caused by the deficiency of the lysosomal enzyme N-acetylgalactosamine-6-sulfate sulfatase (GALNS). The purpose of this study was to analyze the GALNS mutations and the haplotypes associated.MethodsMutation screening of the GALNS gene was performed by direct sequence analysis using DNA samples from 15 unrelated Tunisian MPS IVA patients. We also analyzed the haplotypes associated with the novel mutation and with the other reported GALNS mutations.ResultsWe have identified an unreported missense mutation p.D288G (c.863A > G) in one patient, the most frequently c.120 + 1G > A (IVS1 + 1G > A) mutation in eleven MPS IVA patients and three previously reported mutations p.G66R, p.A85T and p.R386C on the other MPS IVA patients. All the studied patients were homozygous for these identified mutations. Bioinformatics analysis predicted the novel mutation as being probably pathogenic. These findings with the unobserved p.D288G mutation in controls subjects, suggested that it is a disease-causing mutation, which was correlated with the severe phenotype observed in the patients. We have found that the two GALNS unreported and reported mutations, respectively p.D288G and p.R386C, were associated with a common and specific haplotype.ConclusionOur results were in agreement with previous reports from Tunisia, suggesting, on one hand the genotype/phenotype correlations in MPS IVA patients and the other hand the haplotype analyses were useful for determination of mutation origin in Tunisian population.RésuméLa mucopolysccharidose de type IV A (MPS IVA) est une maladie autosomique récessive due à la déficience de l’enzyme lysosomale N-acétylgalactosamine-6-sulfate sulfatase (GALNS). Le but de cette étude est d’analyser les mutations du gène GALNS et les haplotypes associés.MéthodesLe dépistage des mutations dans le gène GALNS a été réalisé par l’analyse directe des séquences en utilisant des échantillons d’ADN issus de 15 patients non apparentés atteints de la MPS IVA. Par ailleurs, nous avons analysé les haplotypes associés, d’une part à la mutation nouvelle, et d’autre part, aux autres mutations déjà rapportées.RésultatsNous avons identifié une nouvelle mutation faux-sens p.D288G (c.863A > G) chez un patient, la mutation la plus fréquente c.120 + 1G > A chez onze patients et trois mutations déjà rapportées : p.G66R, p.A85T et p.R386C chez trois patients. Tous les patients étudiés sont homozygotes pour ces mutations identifiées. Les analyses bioinformatiques prédisent que la mutation, p.D288G, décrite pour la première fois pourrait être pathogénique. L’absence de cette nouvelle mutation chez la population témoin suggère que la mutation p.D288G, corrélée au phénotype sévère chez le patient étudié, est responsable de la maladie. Par ailleurs, nous avons constaté que les deux mutations du gène GALNS, nouvelle et déjà rapportée, respectivement, p.D288G et p.R386C sont associées à un haplotype commun et spécifique.ConclusionNos résultats sont en accord avec les rapports précédents de la Tunisie, ce qui suggère, d’une part les corrélations génotype/phénotype chez les patients MPS IVA et d’autre part l’analyse des haplotypes ont été utiles pour la détermination de l’origine de la mutation dans la population tunisienne.

Genetic basis of cystinosis in Tunisian patients: Identification of novel mutation in CTNS gene

Nephropathic cystinosis (NC) is an autosomal recessive disorder characterized by defective transport of cystine across the lysosomal membrane and resulting in renal, ophthalmic, and other organ abnormalities. Mutations in the CTNS gene cause a deficiency of the transport protein, cystinosin. This study was performed to investigate mutations of the CTNS gene in three Tunisian families with NC. Polymerase chain reaction (PCR), ARMS multiplex PCR and direct sequencing were performed for molecular characterization of the CTNS gene in 3 unrelated Tunisian patients and their parents. Based on family history, prenatal diagnosis (PND) was performed in fetal DNA isolated from chorionic villi obtained at 10–12 weeks of gestation. None of the patients showed the most common 57-kb deletion in heterozygous or homozygous status. One patient was homozygous for the previously reported mutation c.1515G > A (p.G308R). One patient presented the novel gross deletion of 20,327 bp. One was homozygote for the previously reported mutation c.771_793del (p.Gly258Serfs*30). In addition, eight polymorphisms were identified in the 3 patients and their parents. The prenatal diagnosis in one family showed that the fetus DNA was heterozygous for the c.771_793del (p.Gly258Serfs*30) mutation. This study expands the mutational and population spectrum of NC, representing the first molecular diagnosis of NC in Tunisian population. The mutation screening of the CTNS gene was used for prenatal diagnosis to prevent and/or limit this inheritable disease in our country where the families are particularly large and have a high rate of consanguinity.

Mutations and polymorphisms in N-acetylgalactosamine-6-sulfate sulfatase gene in Turkish Morquio A patients

Mucopolysaccharidosis type IVA (MPS IVA) is an autosomal recessive inherited metabolic disease resulting from deficiency of N-acetylgalactosamine-6-sulfatase (GALNS). This lysosomal storage disorder leads to a wide range of clinical variability ranging from severe, through intermediate to mild forms. The classical phenotype of Morquio A disease is characterized by severe bone dysplasia without intellectual impairment. Two severe MPS IVA patients from two unrelated Turkish families have been investigated. The 14 exons and intron-exon junctions of the GALNS gene were sequenced after amplification from genomic DNA. Direct sequencing revealed two homozygous mutations previously described: p.L390X in exon 11 and p.W141R in exon 4. The p L390X mutation was associated with four novel polymorphisms in intron 2, intron 5 and intron 6 and one polymorphism previously described in exon 7. We have analysed the haplotypes associated with the two identified mutations. These molecular findings will permit accurate carrier detection, prenatal diagnosis and counseling for Morquio A syndrome in Turkey.

Molecular analysis of iduronate -2- sulfatase gene in Tunisian patients with mucopolysaccharidosis type II

Mucopolysaccharidosis type II (MPS II, Hunter syndrome) is X-linked recessive lysosomal storage disorder resulting from the defective activity of the enzyme iduronate-2-sulfatase (IDS). Hunter disease can vary from mild to severe, depending on the level of enzyme deficiency. We report the IDS mutation and polymorphisms causing the Hunter syndrome in patients from one family in TunisiaPatients and methodsA preliminary diagnosis was made by qualitative detection of urinary glycosaminoglycans of the suspected MPS II probands. The IDS mutation and polymorphisms were determined on these probands and their family members by amplifying and sequencing each of the exons and intron-exon junctions of IDS gene.ResultsThe studied probands were homoallelic for p.R88P mutation. In addition, three known polymorphisms/sequence variants: IVS3-16 (c.419-16 delT), T214M (c.641C > T), T146T (c.438 C > T), IVS5-87(c.709-87G > A) and one previously unknown: IVS7+38(c.1006+38T > C were identified in the MPS II patients. These are the first Tunisian MPS II patients to be genotyped.ConclusionThe identification of these mutation and polymorphisms and their genotype-phenotype correlation should facilitate prenatal diagnosis and counseling for MPS II in Tunisia, where a very high rate of consanguinity exists.

Morquio A disease: clinical and molecular study of Tunisian patients

Type IVA mucopolysaccharidosis or Morquio A disease is a lysosomal storage disease, autosomal recessive, caused by deficiency of the N-acetylgalactosamine-6-sulfate sulfatase or GALNS. The severe phenotype is characterized by a severe skeletal dysplasia without any mental retardation. The aim of this study was to propose a strategy of molecular and prenatal diagnosis of this pathology. A molecular study was carried out on 7 patients MPS IVA issued from 5 unrelated families recruited from different Tunisian regions. All the patients were offspring of consanguineous marriages. The clinical and biologic study confirmed the diagnosis of MPS IVA within the 7 studied patients. Three GALNS mutations were identified by molecular analysis: IVS1+1G>A, G66R and A85T. The unions between Tunisian relatives are important and increase the Morquio A incidence in Tunisia. The identification of GALNS mutations in the Tunisian population permits better understanding of the Morquio A phenotype, a reliable genetic counselling and a molecular prenatal diagnosis to Tunisian at-risk relatives.