Molecular characterization of MPS IIIA, MPS IIIB and MPS IIIC in Tunisian patients

Molecular characterization of MPS IIIA, MPS IIIB and MPS IIIC in Tunisian patients

Clinica Chimica Acta 412 (2011) 2326–2331 Contents lists available at SciVerse ScienceDirect Clinica Chimica Acta journal homepage: www.elsevier.com...

386KB Sizes 10 Downloads 50 Views

Clinica Chimica Acta 412 (2011) 2326–2331

Contents lists available at SciVerse ScienceDirect

Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim

Molecular characterization of MPS IIIA, MPS IIIB and MPS IIIC in Tunisian patients☆ S. Ouesleti a, d, V. Brunel b, H. Ben Turkia c, H. Dranguet b, A. Miled a, N. Miladi d, M.F. Ben Dridi c, A. Lavoinne b, P. Saugier-Veber e, S. Bekri b,⁎ a

Laboratoire de Biochimie de l'Hôpital Farhat Hached, Sousse, Tunisia Laboratoire de Biochimie Médicale, CHU de Rouen and EA 4309 NéoVasc, Université de Rouen, France Service de Pédiatrie de la Rabta, Tunis, Tunisia d Unité de recherche des Maladies Neurologiques de L'Enfant, Faculté de Médecine de Tunis, Tunisia e Service de Génétique, CHU de Rouen and Inserm U614, Université de Rouen, France b c

a r t i c l e

i n f o

Article history: Received 9 August 2011 Accepted 30 August 2011 Available online 2 September 2011 Keywords: Sanfilippo disease QMPSF Mutation Large-scale deletion

a b s t r a c t Sanfilippo syndrome (mucopolysaccharidosis type III, MPS III) is a progressive disorder in which patients are characterized by severe central nervous system degeneration together with mild somatic disease. MPS III results from a deficiency in one of the four enzymes involved in the heparan sulfate degradation, with sulfamidase (SGSH), α-N-acetylglucosaminidase (NAGLU), acetyl-coenzyme A: α-glucosaminide N-acetyltransferase (HGSNAT), and N-acetylglucosamine-6-sulfatase (GNS) being deficient respectively in MPS IIIA, MPS IIIB, MPS IIIC and MPS IIID. Mutation screening using PCR reaction/sequencing analysis on genomic DNA fragments was performed in seven Tunisian index cases with MPS IIIA, three with MPS IIIB and two with MPS IIIC. QMPSF (Quantitative Multiplex PCR of Short fluorescent Fragments) analysis was developed for the detection of genomic deletions and duplications in the SGSH gene. These approaches allowed the identification of 11 mutations, 8 of them were novel including a mutation involving the start codon (p.Met1?), one small duplication (p.Leu11AlafsX22), one small deletion (p.Val361SerfsX52) and a large deletion of exon 1 to exon 5 in the SGSH gene, one missense mutation (p.Pro604Leu) and one nonsense mutation (p.Tyr558X) in the NAGLU gene and, finally, one missense mutation (p.Trp627Cys) and one nonsense mutation (p.Trp403X) in the HGSNAT gene. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Mucoplysaccharidoses (MPS) are a group of lysosomal genetic disorders resulting from a deficiency of acid hydrolase which is required for the degradation of sulfated glycosaminoglycans (GAG). They include several subtypes according to affected metabolic pathway and accumulated substrate. Sanfilippo syndrome or MPS III results from the absence of one of four lysosomal enzymes that are sequentially involved in heparan sulfate degradation: heparan sulfate sulfamidase (SGSH, MPS IIIA, MIM #252900), α-N-acetylglucosaminidase (NAGLU, MPS IIIB, MIM #252920), heparan acetylCoA: α-glucosaminide-N-acetyltransferase (HGSNAT, MPS IIIC,MIM #252930), or N-acetylglucosamine-6-sulfatase (GNS, MPS IIID, MIM #252940) [1]. The absence of one of these enzymes leads to the lysosomal accumulation of the heparan sulfate, its urinary excretion and the clinical onset of Sanfilippo syndrome. Each MPS III subtype is inherited as an autosomal recessive disorder.

☆ Research grants and financial support: This work was supported by a grant from VML (Vaincre les maladies lysosomales). ⁎ Corresponding author at: Laboratoire de Biochimie Hôpital Charles Nicolle, 76031 Rouen, France. Tel.: + 33 2 32 88 01 25. E-mail address: [email protected] (S. Bekri). 0009-8981/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2011.08.032

The disease is characterized by a multiple organ involvement and severe central nervous system degeneration. The clinical course manifests in three stages with a period of quite normal development followed by behavioral disturbances and an end-stage neurodegeneration with a short lifespan. Clinical symptoms usually occur between 2 and 6 years of age and include attention deficit, hyperactivity, aggressive behavior, delayed development, coarse hair, hirsutism and sleep disturbance. Somatic and skeletal abnormalities are usually mild [1]. The estimated incidence is 1/66,000 in Germany and Australia [2], 1/89,000 in Ireland [3]. MPS IIIA has been reported to be the most severe form with an early onset, rapid symptoms progression and short survival. The gene locus for human SGSH (heparane sulfate sulfamidase) has been localized to chromosome 17q25.3 [4]. The human SGSH gene has an approximate length of 11 kb with 8 exons [5]. To date, approximately 84 mutations have been described including missense/nonsense mutations, small deletion and insertion and one splicing mutation (Human Gene Mutation Database-HGMD, http://www.hgmd.cf.ac. uk/ac/index.php). MPS IIIA is the most common subtype in Northern Europe [2,3,6–8]. The human gene encoding α-N-acetylglucosaminidase, NAGLU, was isolated, characterized, and localized to chromosome 17q21.3 [9]. This gene spans 8.3 kb, contains 6 exons and encodes a protein

S. Ouesleti et al. / Clinica Chimica Acta 412 (2011) 2326–2331

of 743 amino acids which plays a critical role through the cleavage of heparin glycosilic bounds. At present, over 120 mutations underlying MPS IIIB have been identified, including missense/nonsense mutations, deletions, insertions, point mutations and splicing site mutations (HGMD). MPS IIIB is the most common subtype in Greece [10]. Recently, variations in the HGSNAT gene have been identified in several MPS IIIC patients. This gene is localized to chromosome 8p11.1 with 62.4 kb in length and contains 18 exons coding for a protein of 635 amino acids [11,12]. Fifty four mutations were reported such as missense/nonsense mutations, splicing site mutations, deletions and insertions (HGMD). The deduced HGSNAT aminoacid sequence predicts 11 transmembrane domains with the N-terminus being inside the lysosome and the C-terminus being in the cytosol [12]. The incidence of mucopolysaccharidoses in Tunisia is probably underestimated. A multicentric retrospective study including 96 patients diagnosed from 1988 until 2005 allowed to calculate incidence of 1/44,000. The consanguinity rate, estimated at 32% in the general population and 83% in MPS family, suggests that these pathologies are much more frequent in the Tunisian population [13]. In this study, 13 Tunisian patients from 12 MPS III families including 7 type A, 3 type B and 2 type C were investigated; besides, 32 relatives were also included in the study. The patients have been clinically characterized; molecular analysis of corresponding genes revealed 11 different mutations in the SGSH, NAGLU and HGSNAT genes, of which 8 have never been reported before.

2. Material and methods 2.1. Recruitment of patients In the Tunisian pediatric services registries, records from 13 patients with MPSIII from 12 families were present (patients 1C1 and 1C2 being brothers, Table 1). All have been diagnosed on the basis of enzymatic studies. Written informed consents were obtained from the parents.

2327

All patients included in this study were seen at the outpatient clinic by a clinical geneticist and a pediatrician. A questionnaire, requesting information on pregnancy, first clinical signs, mental and motor milestones, behavioral problems, sleeping problems and medical history, was filled out by caregivers. 2.2. Clinical and biochemical diagnoses All patients presented with a severe phenotype. Table 1 summarizes the age, origin and the clinical findings. Clinical diagnosis was confirmed by quantitation and electrophoresis of urinary glycosaminoglycans [14]. Enzyme activity was assayed using fluorogenic substrates revealing low or nondetectable SGSH, or NAGLU or HGSNAT activity. 2.3. Mutation analysis Genomic DNA was extracted from venous blood using QIAamp DNA Blood Mini Kit® Qiagen and was amplified in vitro by PCR. Multiple pairs of primers were synthesized to amplify each of SGSH, NAGLU and HGSNAT exonic regions, including intron/exon boundaries and promoter (primer sequences are available upon request). Primers used to amplify the genomic sequences were designed according to the sequences NM_000199.3 for SGSH, NM_000263.3 for NAGLU and NM_152419.2 for HGSNAT. PCR reactions were carried out in 1× Thermo Scientific Buffer IV (75 mM Tris–HCl pH 8.8, 20 mM (NH4)2SO4, 0.01% Tween 20), 1.5 mM MgCl2, 100 μM dNTPs, 0.025 U/μl Taq polymerase (Thermo Scientific), 0.6 μM of each primer). Touchdown PCR consisted of one cycle of 95 °C for 5 min for the initial denaturation step followed by 12 cycles of denaturation at 95 °C for 25 s, varying annealing (60–48°) for 25 s, and extension at 72 °C. Then, 35 cycles were performed as follows: denaturation at 95° for 25 s, annealing at 48° for 25 s and extension at 72 °C for 25 s. PCR was terminated after a final cycle at 72 °C for 5 min.

Table 1 Origin, consanguinity, and clinical data of the 13 MPS III patients. MPSIII type

Type A

Patient

1A

Origin

Sfax Kairouan Bizerte

Consanguinity Age at diagnosis (year) Mental retardation Behavioral problems Dysmorphic features Macrocrania Hirsutism Organomegaly Skeletal +

+ 3

− 3

+ 8

+

+

+

Deafness Seizures Hernia Diarrhea Recurrent infections Others

+ − + + +

2A

Type B 3A

4A

5A

6A

Tunis

Bizerte Tunis

+ 6

+ 2

+ 3

+

+

+

+

+

+

+

+

+

+ + +

+ + +

7A

1B

Type C 2B

3B

1C1

1C2

2C

N

Kairouan Sousse

Sousse

Mahdia Mahdia Madhia Mahdia

+ 9

+ 1.5

+ 1

+ 9

+ 7

+ 5

+ 8

12/13

+

+

+

+

+

+

+

+

13/13

+

+

+

+

+

+

+

+

+

13/13

+

+

+

+

+

+

+

+

+

+

13/13

+ + + abnormalities

− − + −

− + + +

+ + + +

+ + + +

+ + + +

+ + + +

− + − +

+ − − +

− − − −

+ + + −

9/13 10/13 10/13 +

+ + − + −

+ − − − +

− − − − −

+ + − + −

+ + + − +

+ − + + +

− − − − −

− − − − −

− + − − +

− + − − −

+ + + − −

8/13 5/13 5/13 4/13 6/13

Cerebral atrophy corpus callosum agenesis

Ataxia

+ 9/13 + − + − + Dental abscess

Encephalopathy

Mitral Supra insufficiency ventricular tachycardia

Dental abscess

2328

S. Ouesleti et al. / Clinica Chimica Acta 412 (2011) 2326–2331

Direct DNA fragments sequencing of SGSH, NAGLU, and HGSNAT genes, was performed with an ABI prism bigdye Terminator cycle Sequencing Ready Reaction Kit (PE applied biosystem and ABI model 3130xl Genetic Analyser).

Genetic Analyzer automated sequencer (PE Applied Biosystems, Foster City, CA, USA). Data were analyzed using the Genescan software (PE Applied Biosystems, Foster City, CA, USA). Electropherograms were superimposed to those generated from a normal control DNA by adjusting the peaks obtained for the control amplicon and the heights of the corresponding peaks to the same level. Peaks were then compared between the different samples. Abnormal profiles were confirmed with a locus-specific QMPSF.

2.4. Restriction analysis The presence of a mutation was confirmed when possible by restriction analysis. A 15 μl aliquot of the PCR mix was incubated with the appropriate restriction enzyme according to manufacturer's instructions. Fragments were separated on 2% agarose gel and stained with ethidium bromide. The different enzymes used to identify the mutations are shown in Table 2.

3. Results 3.1. Clinical findings All patients exhibited the severe phenotype of Sanfilippo disease. Table 1 indicates the main patient characteristics at the time of diagnosis. The patients were diagnosed at age 1 to 9 years old. A high consanguinity rate was identified (12/13). All patients presented with multiple clinical signs including mental retardation (13/13), behavioral disturbances (13/13), dysmorphic features (13/13), macrocrania (9/13), hirsutism (10/13), organomegaly (10/13), skeletal abnormalities (9/13), deafness (8/13), seizures (5/13), hernia (5/13), diarrhea (4/13) and recurrent infections (6/13).

2.5. QMPSF analyses Eight amplicons with a size between 146 and 269 bp were designed to cover the entire 8.3 kb coding region of the SGSH gene. Short exonic fragments were simultaneously amplified by PCR and distributed across 3 multiplex reactions (Supplementary Table 1). One additional autosomic fragment, corresponding to the exon 13 of the HMBS gene located on chromosome 11, was coamplified, as a control PCR. Another fragment from the chromosome X was coamplified as quantification control (exon 8 of the NHS gene or exon 4 of the MECP2 gene). The PCR was performed in a 25 μl final volume containing 100 ng of genomic DNA, 0.3–0.9 mM of each primer, 200 mM dNTPs, 25 mM MgCl2, 10% DMSO, 1× Thermo Scientific Buffer IV (75 mM Tris–HCl pH 8.8, 20 mM (NH4)2SO4, 0.01% Tween 20) and 1 U of Taq DNA polymerase (ABgene, Courtaboeuf, France). The PCR consisted of 25 cycles of 94 °C for 15 s, 50 °C for 15 s and 72 °C for 15 s, preceded by a 5 min initial denaturation step at 94 °C and followed by a 5 min final extension at 72 °C. One microliter of the PCR product was resuspended in a mix containing 10 μl of deionized formamide, 0.5 μl of GeneScan-500 Rox (PE Applied Biosystems, Foster City, CA, USA). After denaturation for 2 min 30 s at 94 °C, 2 μl of each sample was loaded on an Applied Biosystems model 3130xl

3.2. Molecular analysis A total of 13 patients from 12 families with MPSIII and 32 relatives were investigated. This study allowed the identification of 100% of the mutant alleles. None of these mutations were present in a panel of 100 control alleles. The presence of all the mutations identified was confirmed by studying the parents and by using restriction analysis when possible (Table 2). Moreover, a polymorphism was characterized in the intron 5 of the SGSH gene (NM_000199.3:c.664-39_66436delinsGC) and identified in 40% of the control individual alleles. DNA samples from 7 MPSIIIA patients were analyzed by sequencing the SGSH gene. QMPSF analysis was performed when indicated. A total of 7 distinct mutations were identified (Table 2). Six out of seven patients

Table 2 Mutations found in the SGSH, NAGLU and HGSNAT genes from the 12 MPS III index cases. Patient

Gene

Allele

Nucleotide alteration

Protein alteration

Method

Restriction enzyme test

References

1A

SGSH NM_000199.3

Sequencing

NlaIII

Novel

SGSH NM_000199.3

3A

SGSH NM_000199.3

p. Arg377Cys p.? p.Gln365X

Sequencing QMPSF Sequencing

4A

SGSH NM_000199.3

p.Leu11AlafsX22

Sequencing

5A

SGSH NM_000199.3

p.Ser66Trp

Sequencing

6A

SGSH NM_000199.3

p.Val361SerfsX52

Sequencing

7A

SGSH NM_000199.3

p.?

Sequencing QMPSF

1B

NAGLU NM_000263.3

p.Tyr558X

2B

NAGLU NM_000263.3

p.Pro604Leu

3B

NAGLU NM_000263.3

1C

HGSNAT NM_152419.2

c.2T N C c.2T N C c.1129C N T g.75802301_75809393del c.1093C N T c.1093C N T c.29dup c.29dup c.197C N G c.197C N G c.1080del c.1080del g.75802301_75809393del g.75802301_75809393del c.1674C N G c.1674C N G c.1811C N T c.1811C N T c.1811C N T c.1811C N T c.1209G N A c.1880A N G c.1209G N A c.1880A N G c.1209G N A c.1880A N G c.1209G N A c.1880A N G

p.Met1?

2A

Allele 1 Allele 2 Allele 1 Allele 2 Allele 1 Allele 2 Allele 1 Allele 2 Allele 1 Allele 2 Allele 1 Allele 2 Allele 1 Allele 2 Allele 1 Allele 2 Allele 1 Allele 2 Allele 1 Allele 2 Allele 1 Allele 2

2C

HGSNAT NM_152419.2

Allele 1 Allele 2

Known [17] Novel Known [18] BssHII

Novel Known [16]

BmgB1

Novel

Sequencing

Eco0109I

Novel

Sequencing

NaeI

Novel

Sequencing Sequencing

BseYI

Novel Novel

p.Pro604Leu p.Trp403X p.Tyr627Cys p.Trp403X p.Tyr627Cys p.Trp403X p.Tyr627Cys p.Trp403X p.Tyr627Cys

S. Ouesleti et al. / Clinica Chimica Acta 412 (2011) 2326–2331

2329

A Wild type allele 8

7

SLC26A11

SGSH

6 5 4

1 1

3 8

7

6

2

3

18

2 2’ 3 18

Mutated allele

B

1

2

3

500 bp

SGSH exon 6

5

6 422 bp

400 bp

C

4

SGSH intron 5

SLC26A11 exon 2

D QMPSF1

QMPSF2

*

Fig. 1. Characterization of the large rearrangement removing exons 1 to 5 of the SGSH gene and the exon 1 and a part of the exon 2 of SLC26A11 gene (NM_000199.3g.75802301_75809393del). A. Schematic representation of the SGSH and SLC26A11 gene rearrangement. Blue boxes represent SGSH exons and pink boxes represent SLC26A11 exons. The 7093 bp deleted region is delineated. The blue arrows correspond to the primers used to amplify and sequence the junction fragment. The SGSH and SLC26A11 exons are numbered. The exon 2′ represented in the junction fragment corresponds to the truncated SLC26A11 exon 2. B. PCR of the deletion breakpoint. Lane 1: DNA 100-bp increment ladder; Lane 2: control; Lane 3: Patient 7A; Lane 4: Patient 7A's father, Lane 5: Patient 2A; Lane 6: Patient 2A's father. The amplification showed a 422 bp product corresponding to the junction fragment in the patients and their relatives. This product was not present in the control DNA sample. C. Sequence analysis of the junctional fragment PCR product identifying the breakpoint in the SGSH intron 5 and in the SLC26A11 exon 2 characterizing the 7093 bp deletion. D. QMPSF profile of a deletion carrier (Patient 2A, in red), and of a normal control (in blue) are superimposed. Fluorescent profiles are normalized using HMBS as control amplicon. A two-fold reduction of exon 1, 2, 3, 5 is observed in Patient 2A profile. Two exon 6 peaks are observed in the control profile (*); this is due to the presence of a polymorphism in the intron 5 (c.664-39_664-36delinsGC). Only one peak is present in the patient profile indicating the absence of one allele.

were homozygous for one of the defective mutations. Three of the seven mutations detected were previously described — c.1129C N T (p.Arg377Cys), c.1093C N T (p.Gln365X), c.197CN G (p.Ser66Trp) —

while four mutations are novel. Among the novel mutations two are point mutations, c.29dup (p.Leu11AlafsX22) and c.1080del (p.Val361SerfsX52), that lead to the putative formation of truncated proteins.

2330

S. Ouesleti et al. / Clinica Chimica Acta 412 (2011) 2326–2331

The third novel mutation was detected using direct sequencing and led to a T to C transition mutation in the ATG initiation codon of the SGSH exon 1 in proband 1A (c.2T N C). Proband 7A was homozygous for a large deletion removing not only 5 exons of the SGSH gene (exons 1 to 5) but also the exon 1 and part of the exon 2 of the adjacent gene SLC26A11 (Fig. 1A). Indeed, no PCR product was obtained for exons 1 to 6 of the SGSH gene and exons 1 and 2 of the SLC62A11 gene. Genomic DNA PCR encompassing the exon 6 of the SGSH gene and the exon 2 of the SLC26A11 gene revealed the amplification of an abnormal fragment of approximately 420 bp in the affected patient and his parents, corresponding to the junction fragment comprising the deleted region (Fig. 1B). Sequence analysis revealed that the last 22 bp of the SGSH intron 5 were fused to the last 116 bp of SLC26A11 exon 2 (Fig. 1C), deleting a 7093 bp fragment of the genomic sequence (NM_000199.3: g.75802301_75809393del). The forward and primer sequences of the SGSH exon 6 and the SLC26A11 exon 2 respectively were included in the deleted region, and thus, the PCR amplication of these two exons did not reveal any product in proband 7A. QMPSF analysis of the SGSH gene permitted to confirm the presence of an exon 1 to 5 homozygous deletions in proband 7A and to characterize the presence of heterozygous deletion in his parents. The exon 6 forward QMPSF primer sequence was included in the deleted fragment. A heterozygous nonsense mutation, c.1129CN T (p.Arg377Cys), was identified in proband 2A and sequencing analysis did not allow to characterize the mutation of the second allele. QMPSF analysis was conducted in proband 2A (Fig. 1D) and his parents and permitted to identify a heterozygous deletion of SGSH exons 1 to 6 in the patient and his father. Genomic DNA PCR encompassing the exon 6 of the SGSH gene and the exon 2 of the SLC26A11 gene revealed the amplification of an abnormal fragment similar to that obtained in patient 7A (Fig. 1B). Sequence analysis of the PCR product revealed a junction identical to that described in patient 7A. Analysis of the deletion flanking sequences using the RepeatMasker program did not reveal the presence of repeat elements; thus, the recombination did not involve repeat elements, suggesting a non homologous recombination process. Molecular investigations of 3 MPSIIIB patients lead to the identification of two novel mutations in the NAGLU gene (Table 2). Thus, a homozygous nonsense mutation, c.1674C N G (p.Tyr558X), was characterized in patient 1B. The other mutation, c.1811C N T, resulted in a substitution of leucine for the wild-type proline at amino acid 604 (p.Pro604Leu) of the NAGLU protein. Both patients 2B and 3B were homozygous for this mutation. The HGSNAT gene was sequenced in three patients from two MPSIIIC families (Table 2). A nonsense homozygous mutation, c.1209G N A (p.Trp403X), was identified in both families (patients 1C1, 1C2, and patient 2C). Moreover, a second homozygous missense mutation, c.1880AN G (p.Tyr627Cys) was characterized in both families.

4. Discussion We report on the clinical and molecular characteristics of 13 MPSIII patients belonging to 12 unrelated families. Patients included in this study follow the general severe phenotype as observed in Sanfilippo syndrome [15]. Thus, the clinical picture is predominated by progressive neurological degeneration with mental retardation and behavioral disturbances. A high consanguinity rate is noticed, moreover unrelated families originated from the region presented with common mutations which suggest a founder effect. Indeed, a SGSH deletion of 7 kb was identified in two families (2A and 7A) originated from Kairouan and the same genotype was identified in two unrelated MPSIIIC families (1C and 2C) from Madhia (Tables 1 and 2). Molecular analysis revealed 11 different mutations, of which 8 are reported here for the first time representing a significant contribution to the knowledge of mutational spectrum of the SGSH, NAGLU and HGSNAT genes. Our analysis accounted mutations in both alleles in

all patients and the presence of these variations was confirmed in their parents; thus, 32 relatives were investigated in this study. Seven MPSIIIA patients were characterized and the molecular analysis succeeded to identify seven mutations. Three of these mutations (c.1129C N T (p.Arg377Cys), c.1093C N T (p.Gln365X), c.197C N G (p.Ser66Trp)) were described previously [16–18] whereas four [c.29dup (p.Leu11AlafsX22), c.1080del (p.Val361SerfsX52), c.2T N C (p.Met1?) and g.75802301_75809393del] are novel. Two novel mutations [c.29dup (p.Leu11AlafsX22); c.1080del (p.Val361SerfsX52)] may lead to the formation of truncated proteins, which are very likely non-functional products, or to nonsense-mediated mRNA decay. The third novel mutation occurs in the ATG initiation codon (c.2T N C; ATGN ACG). The sequence surrounding the first initiation codon in the SGSH gene (GCCGCCatgAG) matches well with the known optimal recognition sequence (GCCRCCatgG; where R = purine) with the −3R being particularly important [19]. It was reported that the +4G is not required for an efficient translation [20]. The initiation site is reached via a ribosome-mediated scanning mechanism. Thus, the ribosome scans in a 5′ to 3′ direction until it encounters the consensus sequence containing the AUG codon. When a mutation alters the start codon, the scanning mechanism should activate initiation from the next downstream AUG [21]. Fukao et al. examined the translation efficiency of ACAT1 cDNA mutant harboring a single-base substitution of the initiation codon using in vitro transient expression [22]. All mutants produced normal protein with a lower efficiency when compared to the wild-type sequence. The c.2TN C mutant lowered the protein production to 22%. Considering these reports, the mutation identified in the SGSH initiation codon might abolish or lower the protein production. The fourth novel SGSH mutation resulted in 7093 bp deletion encompassing SGSH exons 1–5 and the SLC26A11 exon 1 and a part of the exon 2. So far, this is the first description of a large-scale rearrangement of the SGSH gene. QMPSF has been successfully used to screen for exonic rearrangements of many genes [23–26] and its use in this report allowed to identify the presence of a SGSH–SLC26A11 deletion in heterozygous individuals. This deletion by eliminating the SGSH and SLC26A11 promoters and initiation codons is predicted to abolish SGSH and SLC26A11 function. SLC26A11, a member of the SLC26 sulfate/anion exchanger family is widely expressed in particular in kidney, placenta and brain. However, physiological role of SLC26A11 is poorly characterized [27]. We observed, thus, a pronounced allelic heterogeneity in Tunisian MPS IIIA patients with 7 distinct mutations identified in 7 patients. Two novel mutations were identified in the NAGLU gene. The first variation is a nonsense mutation, c.1674CN G (p.Tyr558X), which is consistent with the predicted protein truncation or nonsensemediated mRNA decay. The second variation, a missense mutation c.1811C N T (p.Pro604Leu), involved a proline residue which is well known as a helix breaker and is commonly found in turns. Thus, its replacement by a leucine, would be expected to yield considerable destabilizing conformational changes to the native NAGLU fold [28]. Finally, two novel mutations were characterized in the HGSNAT gene. The nonsense mutation, c.1209G N A (p.Trp403X) involved a codon in which a missense mutation, c.1209G N T (p.Trp403Cys) has been described [12], suggesting that the codon 403 may be a mutational hotspot. The missense mutation, c.1880A N G (p.Tyr627Cys), is adjacent to the predicted last transmembrane domain on the cytoplasmic side of HGSNAT. This change might have a drastic effect on protein folding [29]. Thus, all the above-mentioned mutations are therefore likely to have a significant functional impact on the corresponding proteins. In conclusion, this is the first study on molecular defects in Tunisian MPSIII patients; our study reveals that MPSIIIA, B and C presented with a severe phenotype in the studied cohort and contributes to the description of novel mutations including a large-scale deletion. Supplementary materials related to this article can be found online at doi:10.1016/j.cca.2011.08.032.

S. Ouesleti et al. / Clinica Chimica Acta 412 (2011) 2326–2331

References [1] Neufeld EF, Muenzer J. The mucopolysaccharidoses8th ed. . New York: McGrawHill; 2001. [2] Baehner F, Schmiedeskamp C, Krummenauer F, et al. Cumulative incidence rates of the mucopolysaccharidoses in Germany. J Inherit Metab Dis 2005;28:1011–7. [3] Nelson J. Incidence of the mucopolysaccharidoses in Northern Ireland. Hum Genet 1997;101:355–8. [4] Scott HS, Blanch L, Guo XH, et al. Cloning of the sulphamidase gene and identification of mutations in Sanfilippo A syndrome. Nat Genet 1995;11:465–7. [5] Karageorgos LE, Guo XH, Blanch L, et al. Structure and sequence of the human sulphamidase gene. DNA Res 1996;3:269–71. [6] Beratis NG, Sklower SL, Wilbur L, Matalon R. Sanfilippo disease in Greece. Clin Genet 1986;29:129–32. [7] Michelakakis H, Dimitriou E, Tsagaraki S, Giouroukos S, Schulpis K, Bartsocas CS. Lysosomal storage diseases in Greece. Genet Couns 1995;6:43–7. [8] Poorthuis BJ, Wevers RA, Kleijer WJ, et al. The frequency of lysosomal storage diseases in The Netherlands. Hum Genet 1999;105:151–6. [9] Zhao HG, Li HH, Bach G, Schmidtchen A, Neufeld EF. The molecular basis of Sanfilippo syndrome type B. Proc Natl Acad Sci USA 1996;93:6101–5. [10] Beesley C, Moraitou M, Winchester B, Schulpis K, Dimitriou E, Michelakakis H. Sanfilippo B syndrome: molecular defects in Greek patients. Clin Genet 2004;65:143–9. [11] Fan X, Zhang H, Zhang S, et al. Identification of the gene encoding the enzyme deficient in mucopolysaccharidosis IIIC (Sanfilippo disease type C). Am J Hum Genet 2006;79:738–44. [12] Hrebicek M, Mrazova L, Seyrantepe V, et al. Mutations in TMEM76* cause mucopolysaccharidosis IIIC (Sanfilippo C syndrome). Am J Hum Genet 2006;79:807–19. [13] Ben Turkia H, Tebib N, Azzouz H, et al. Incidence of mucopolysaccharidoses in Tunisia. Tunis Med 2009;87:782–5. [14] Whiteman P, Henderson H. A method for the determination of amniotic-fluid glycosaminoglycans and its application to the prenatal diagnosis of Hurler and Sanfilippo diseases. Clin Chim Acta 1977;79:99–105. [15] Valstar MJ, Ruijter GJ, van Diggelen OP, Poorthuis BJ, Wijburg FA. Sanfilippo syndrome: a mini-review. J Inherit Metab Dis 2008;31:230–9.

2331

[16] Blanch L, Weber B, Guo XH, Scott HS, Hopwood JJ. Molecular defects in Sanfilippo syndrome type A. Hum Mol Genet 1997;6:787–91. [17] Di Natale P, Balzano N, Esposito S, Villani GR. Identification of molecular defects in Italian Sanfilippo A patients including 13 novel mutations. Hum Mutat 1998;11: 313–20. [18] Esposito S, Balzano N, Daniele A, et al. Heparan N-sulfatase gene: two novel mutations and transient expression of 15 defects. Biochim Biophys Acta 2000;1501: 1–11. [19] Kozak M. At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells. J Mol Biol 1987;196:947–50. [20] Xia X. The + 4G site in Kozak consensus is not related to the efficiency of translation initiation. PLoS One 2007;2:e188. [21] Kozak M. Pushing the limits of the scanning mechanism for initiation of translation. Gene 2002;299:1–34. [22] Fukao T, Matsuo N, Zhang GX, et al. Single base substitutions at the initiator codon in the mitochondrial acetoacetyl-CoA thiolase (ACAT1/T2) gene result in production of varying amounts of wild-type T2 polypeptide. Hum Mutat 2003;21:587–92. [23] Baert-Desurmont S, Buisine MP, Bessenay E, et al. Partial duplications of the MSH2 and MLH1 genes in hereditary nonpolyposis colorectal cancer. Eur J Hum Genet 2007;15:383–6. [24] Killian A, Di Fiore F, Le Pessot F, et al. A simple method for the routine detection of somatic quantitative genetic alterations in colorectal cancer. Gastroenterology 2007;132:645–53. [25] Saugier-Veber P, Bonnet C, Afenjar A, et al. Heterogeneity of NSD1 alterations in 116 patients with Sotos syndrome. Hum Mutat 2007;28:1098–107. [26] Saugier-Veber P, Goldenberg A, Drouin-Garraud V, Rossi A, Tosi M, Frebourg T. Simple detection of genomic microdeletions and microduplications using QMPSF in patients with idiopathic mental retardation. Eur J Hum Genet 2006;14:1009–17. [27] Vincourt JB, Jullien D, Amalric F, Girard JP. Molecular and functional characterization of SLC26A11, a sodium-independent sulfate transporter from high endothelial venules. FASEB J 2003;17:890–2. [28] Fersht AR. Protein stability. New York, NY: Freeman, W.H.; 1999. [29] Feldhammer M, Durand S, Pshezhetsky AV. Protein misfolding as an underlying molecular defect in mucopolysaccharidosis III type C. PLoS One 2009;4:e7434.