Pathologie Biologie 62 (2014) 38–40

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Original article

Mutations and polymorphisms in N-acetylgalactosamine-6-sulfate sulfatase gene in Turkish Morquio A patients Mutations et polymorphismes dans le ge`ne N-ace´tylgalactosamine-6-sulfate sulfatase chez des patients turques de type Morquio A S. Khedhiri a,b,*, L. Chkioua a,b, N. Elcioglu c, S. Laradi d, A. Miled a,b a

Laboratory of biochimistry CHU Farhat-Hached, 4000 Sousse, Tunisia Laboratory of biochimistry and molecular biology, Monastir, Tunisia c Department of pediatric genetics, Marmara university medical school, Istambul, Turkey d Laboratory of hereditary diseases of metabolism, biologic centre, Lyon, France b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 23 April 2013 Accepted 8 October 2013

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. ß 2013 Elsevier Masson SAS. All rights reserved.

Keywords: Mucopolysaccharidosis type IVA GALNS Mutations Polymorphisms Consanguinity

R E´ S U M E´

Mots cle´s : Mucopolysaccharidose de type IVA GALNS Mutations Polymorphisme Consanguinite´ Abbreviations: MPS IVA, Mucopolysaccharidosis IVA GALNS, N-acetylgalactosamine-6-sulfate sulfatase KS, Keratan sulfate C6S, Chondroitin-6-sulfate PCR, Polymerase chain reaction

La mucopolysaccharidose de type IVA est une maladie autosomale re´cessive qui re´sulte de la de´ficience du ge`ne N-acetylgalactosamine-6-sulfatase (GALNS). Ce type de de´sordre lysosomal engendre une grande variabilite´ clinique chez les patients de type Morquio A. Les symptoˆmes cliniques varient du cas se´ve`re au cas atte´nue´. Le phe´notype de la maladie Morquio A est caracte´rise´ par une dysplasie osseuse sans de´ficience intellectuelle. Dans cette approche, nous avons e´tudie´ 2 patients Morquio A issus de deux familles turques. Nous avons analyse´ les 14 exons ainsi que les jonctions exons-introns du ge`ne GALNS. Le se´quenc¸age a montre´ la pre´sence de deux mutations de´ja` de´crites: p.L390X dans l’exon 11 et p.W141R dans l’exon 4. La mutation p L390X est associe´e a` 4 nouveaux polymorphismes dans l’intron 2, l’intron 5 et l’intron 6 ainsi qu’a` un polymorphisme anciennement de´crit dans l’exon 7. Nous avons aussi analyse´ les haplotypes associe´s avec les mutations de´crites. Cette analyse mole´culaire permettra d’assurer le de´pistage des anomalies qui peuvent eˆtre associe´es au ge`ne GALNS, ce qui est ne´cessaire pour faire le diagnostic pre´natal et une compre´hension meilleure de la maladie Morquio A en Turquie. ß 2013 Elsevier Masson SAS. Tous droits re´serve´s.

1. Introduction

* Corresponding author. E-mail address: [email protected] (S. Khedhiri). 0369-8114/$ – see front matter ß 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.patbio.2013.10.001

Mucopolysaccharidosis type IVA (MPS IVA; Morquio A disease) is an autosomal recessive lysosomal storage disorder and is characterized by the defective activity of N-acetylgalactosamine6-sulfatase (GALNS, EC 3.1.6.4) [1]. This enzyme hydrolyzes the

S. Khedhiri et al. / Pathologie Biologie 62 (2014) 38–40 Table 1 Clinical data of the MPS IVA patients. The two patients were homozygous for two missense mutations pL390X and pW141R. Features

Patient 1

Patient 2

MPS IVA Genotype Consanguinity of the parents/degree Sex Age (year) Growth retardation Dizostosis multiplex Spinal instability Corneal opacity Genu Valgum GALNS assay (mmoles/h/g prot) Keratane sulphate

pL390X/pL390X 1st degree

pW141R/pW141R Parents far related f rom a small village Female 3 Marked Mild – – – 56 Increased

Female 19 Marked Marked Marked Marked Marked 41 Increased

sulfate ester bonds at the non-reducing end of keratan sulfate, chondroitin-6-sulfate and galactose-6-sulfate. This deficiency results in a progressive accumulation of the undegraded substrate within the lysosomes of affected tissues, leading to progressive and chronic course with visceral attacks whose cardinal feature is bone dysplasia. Clinical phenotypes of MPS IVA patients vary from the classical form with severe skeletal (spondyloepiphyseal) odontoid dysplasia, such as cyphoscoliosis, short trunk dwarfism or/and coxa valga also with corneal clouding, hepatosplenomegaly and a life span of 20–30 years for an attenuated form with mild involvement of the skeleton [2] but without any mental retardation whatever the clinical form. The diagnosis of MPS IV A is based on clinical features, then confirmed by GALNS assay in leukocytes or cultured cells. The human GALNS enzyme has been purified; it is a dimer whose molecular mass is 120 kDa, derived from two 60 kDa polypeptides and processed to respectively 40 KDa and 15 KDa, linked by disulfide bonds [3]. The cDNA of 1566 bp open frame encodes 522 amino acids. The gene for GALNS is located on chromosome 16q 24.3 and is split into 14 exons spanning 50 Kb in length (GDB accession ID: 129085) [4,5]. To date, over 148 mutations in GALNS gene have been recorded [6]. The allelic heterogeneity is very important and some authors assume a correlation with the Morquio A clinical variability. 2. Materials and methods

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measured in sonicated fresh leukocyte pellets and compared with controls. The results were expressed as mmol/h/g GALNS activity. 2.3. Mutation analysis of the GALNS gene Genomic DNA was isolated from venous blood by the phenol/chloroform procedure, according to standard protocols as described previously [8]. The 14 exons and flanking intron–exon junctions were polymerase chain reaction (PCR) amplified from the patients and their parents. PCR reactions were carried out in a volume of 25 mL containing 1 ng/mL of genomic DNA, 0.2 mmol/L dNTPs, 0.4 pmol of each primers, 1,5 mmol/L MgCl2, 1% DMSO, 50 mmol/L KCL, 10 mmol/L Tris–HCl pH 8.8 and 1 unit of Taq polymerase (Promega Corp, Madison, WI). Cycling conditions designed in our laboratory started with 5 min of initial denaturation step at 95 ?C, followed by 31 cycles of 45 s at annealing temperature (ranged between 61 ? and 66 ?C), 45 s at 72 ?C followed by a final extension step for 10 min at 72 ?C. The PCR products were utilized as a template for mutation identification by direct sequencing in both forward and reverse directions. GALNS haplotyping for the mutant and normal alleles were constructed by sequencing six amplified genomic fragments, each one including one polymorphism. The first polymorphism is located within the fifth intron (IVS 5 + 134; CCAAGG [allele A] or CCGAGG [allele a]) [9]. The second polymorphism is found within exon 7 (763 nt) from A of the ATG initial codon on the cDNA; GCACGC [allele B] or GCATGC [allele b] [10]. The third polymorphism is positioned within the seventh intron (IVS7nt 90; GTAC [allele H] or GAAC [allele h]) [9]. The fourth polymorphism is located at 1232 nt from A of the ATG initial codon within exon 11 (GTCC [allele C] or GGCC [allele c]) [9]. The fifth polymorphism is found at 1487 nt from A of the ATG initial codon within exon 13 (AAGCCT [allele D] or AGGCCT [allele d]) [9]. Finally, the sixth polymorphism (CCAG [allele E] or CCGG [allele e]) is located in exon 14 [11].

3. Results and discussion 3.1. Clinical features and GALNS activity measurement The clinical course of each subject at the last follow-up is described in Table 1. The two Turkish patients were homozygous for two missense mutations previously described: p L390X in exon 11 and pW141R 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. Haplotype analysis revealed that pW141R mutation occurred in the haplotype a/B/h/C/d/e. The haplotype associated with the p L390X mutation was A/b/h/C/D/E. The results were summarized in Table 2. 3.2. Clinical features and phenotype–genotype correlation

2.1. Patients The study was carried out on two Morquio A patients recruited in Marmara University Medical School, Department of Pediatric Genetics of Turkey. Assignment of the MPS IV A phenotype was based on clinical observations (Table 1). The families gave their informed consent before withdrawal of blood. 2.2. GALNS activity measurements Enzyme assay was performed as described by [7] using the fluorogenic substrate 4-methylumbellyferyl-b d-galactose-6-sulfate sulfatase. The GALNS activity was

The two Turkish patients studied were extremely affected. The negligible GALNS activity correlated with the severe phenotype described in these two patients. 3.3. Mutation analysis The pW141R mutation previously reported [12] was detected in exon 4 of GALNS gene corresponded to T-A missense homozygous substitution, leading to the replacement of an aromatique amino

Table 2 Mutations and polymorphisms in Turkish Morquio A patients. The p L390X mutation was associated with four novel polymorphisms in intron 2 (IVS2+86), intron 5(IVS5+105) and intron 6(IVS6+125 and IVS6+138) and one polymorphism previously described in exon 7 (p.H236H). Patient

1 2

a

Mutations

Polymorphisms

Haplotypes

Codon Allele1 Change

Allele 2

Exon

421T > A 421T > A 1168 delC 1168 delC

Exon 4

pW141R

No polymorphisms

a/B/h/C/d/e

Exon 11

pL390X

IVS2 + 86 (c244 + 86 G > A)a; IVS5 + 105 (c567–105 T > C)a; IVS6 + 125(c633 + 125A > G)a; IVS6 + 138(c633 + 138C > A)a; p.H236H (c708C > T)

A/b/h/C/D/E

Novel sequence changes described in our patients.

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S. Khedhiri et al. / Pathologie Biologie 62 (2014) 38–40

the patients of this study. Haplotype analysis revealed that pW141R mutation occurred in the haplotype a/B/h/C/d/e. The haplotype associated with the p L390X mutation was A/b/h/C/D/E. Associations between a specific mutation and a specific haplotype can be demonstrated in genetic disorders. To understand the origin of a mutation, it is very useful to identify whether a certain recurrent mutation is associated with the same haplotype.

4. Conclusion The identification of polymorphisms and mutations in the GALNS gene significantly promoted the understanding of correlation with genotype and phenotype within MPS IVA patients.

Disclosure of interest

Fig. 1. Position of the 2 mutations: p.W141R and p.L390X in the tertiary model of the human GALNS. p.W141R mutation is located in the ß-sheets of the larger N terminal GALNS domain. p.L390X mutation is located in the ß-sheets of the larger C terminal GALNS domain.

acid to a basic residue at codon 141 (pW141R). The severity of the clinical phenotype is believed to be correlated with the significance of the residue exchange (Trp is an essential amino acid) [13]. The tertiary structural model of the human GALNS was constructed by homology with human N-acetylgalactosamine 4 sulfatase (4S) and arylsulfatase A (ASA) [14]. The larger N terminal domain has a/ß topology with a 10 stranded mixed ß-sheet flanked by a-helices. The smaller C terminal domain has four stranded ßsheets and one a-helix. It is of interest to notice the inside position of W141 in the protein because the substitution of a buried residue seems more likely to result in severe phenotype [13]. This mutation is located in the ß-sheets of the larger N terminal GALNS domain (Fig. 1). It is likely that this mutation may cause the destruction of the hydrophobic core or the modification of the packing. The p l390X nonsense mutation was detected in exon 11 of GALNS gene. It corresponded to the c.1168delC. This mutation should result in the synthesis of truncated proteins without catalytic activity, predicting a severe phenotype in MPS IVA [6]. It is located in the ß-sheets of the larger C terminal GALNS domain (Fig. 1). This mutation and the p.W141R mutation observed only in the Turkish population affected with Morquio A disease [12]. Thus, we could suggest that a possible founder effect for these mutations should exist. 3.4. Polymorphism analysis The screening of polymorphisms in GALNS gene revealed that there are at least 16 polymorphisms detected [6]. 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. These polymorphisms are in the heterozygous status. The frequent haplotype identified in normal individuals is a/b/ h/c/D/E [9]. This haplotype was not detected within any one from

The authors declare that they have no conflicts of interest concerning this article.

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