Functional characterization of arylsulfatase B mutations in Indian patients with Maroteaux-Lamy syndrome (mucopolysaccharidosis type VI)

    Functional characterization of arylsulfatase B mutations in Indian patients with Maroteaux-Lamy syndrome (mucopolysaccharidosis type VI) Anusha Uttarilli, Divya Pasumarthi, Prajnya Ranganath, Ashwin B Dalal PII: DOI: Reference:

S0378-1119(16)30883-6 doi: 10.1016/j.gene.2016.11.005 GENE 41657

To appear in:

Gene

Received date: Revised date: Accepted date:

30 June 2016 2 November 2016 4 November 2016

Please cite this article as: Uttarilli, Anusha, Pasumarthi, Divya, Ranganath, Prajnya, Dalal, Ashwin B, Functional characterization of arylsulfatase B mutations in Indian patients with Maroteaux-Lamy syndrome (mucopolysaccharidosis type VI), Gene (2016), doi: 10.1016/j.gene.2016.11.005

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ACCEPTED MANUSCRIPT Title Page

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Maroteaux-Lamy syndrome (Mucopolysaccharidosis Type VI)

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Title: Functional Characterization of Arylsulfatase B Mutations in Indian patients with

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Running title: Functional analysis of pathogenic sequence variations in Indian MPS VI patients

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Authors: Anusha Uttarilli1,2, Divya Pasumarthi1, Prajnya Ranganath1,3, Ashwin B Dalal1

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Affiliations:

1. Diagnostics Division, Centre for DNA Fingerprinting and Diagnostics, Hyderabad,

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Telangana, India.

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2. Graduate Studies, Manipal University, Manipal, Karnataka, India.

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3. Department of Medical Genetics, Nizam’s institute of Medical Sciences, Hyderabad, Telangana, India.

Corresponding Author: Dr. Ashwin B Dalal,

Head, Diagnostics Division,

Centre for DNA Fingerprinting and Diagnostics, 4-1-714, Tuljaguda Complex, Mozamzahi Road, Nampally, Hyderabad, Telangana, 500001, India Tel: +91 040 24749335; Fax: +91 040 24749448 E-mail: [email protected]

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ACCEPTED MANUSCRIPT

Word count for the text (excluding summary, acknowledgements, references and figure

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legends): 2864 No. of figures: Three

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No. of tables: One

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No. of colour figures: None

ACCEPTED MANUSCRIPT Functional Characterization of Arylsulfatase B Mutations in Indian patients with Maroteaux-Lamy syndrome (Mucopolysaccharidosis Type VI)

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Abstract:

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MPS VI is an autosomal recessive disorder which occurs due to the deficiency of N-acetyl galactosamine-4-sulfatase (Arylsulfatase B - ARSB) involved in catabolism of dermatan sulfate

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resulting from disease-causing variations in the ARSB gene. Human Gene Mutation Database

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(HGMD) search revealed 200 different mutations in ARSB worldwide. In the present study we carried out molecular and functional analyses to characterize the mutations reported by us in

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Indian population. Mutation analysis of 19 MPS VI patients revealed presence of a total of

p.Y103Sfs*9),

(c.293T>G;

p.L98R),

p.Tyr103Serfs*9

(c.496delT;

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p.Leu98Arg

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15 different mutations of which twelve were novel [p.Asp53Asn (c.157G>A; p.D53N),

p.Phe166Leufs*18

(c.306_312delCTACCAG+146del;

p.F166Lfs*18),

p.Ile220Serfs*5

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(c.659_660delTA; p.I220Sfs*18), p.Ile350Phe (c.1048A>T; p.I350F), p.Trp353* (c.1059G>A; p.W353*), p.His393Arg (c.1178A>G; p.H393R), p.Ser403Tyrfs* (c.1208delC; p.S403Yfs*), p.Pro445Leu (c.1334C>T; p.P445L), p.Trp450Leu (c.1349G>T; p.W450L) and p.Trp450Cys (c.1350G>C; p.W450C)] and three were known mutations [p.Asp54Asn (c.160G>A; p.D54N), p.Ala237Asp (c.710C>A; p.A237D) and p.Ser320Arg (c.960C>G; p.S320R)]. Functional characterization using site-directed mutagenesis followed by cell transfection assays, immunoblot, reverse transcriptase PCR and immunofluorescence studies for the putative pathogenic variants detected in our MPS VI patient cohort helped us to confirm the pathogenic potential of the variants in ARSB. Keywords: Molecular analysis, Mucopolysaccharidoses type VI, Novel mutation, Functional characterization, pathogenic variant, Genotype-phenotype correlation.

ACCEPTED MANUSCRIPT Functional Characterization of Arylsulfatase B Mutations in Indian patients with Maroteaux-Lamy syndrome (Mucopolysaccharidosis Type VI)

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Introduction:

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Mucopolysaccharidoses (MPS) are a subgroup of lysosomal storage disorders (LSDs). As a group, MPS disorders occur in approximately 1 in 5000 to 8000 births in the United States,

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Europe and Australia (Wenger et al., 2003) . The exact prevalence in India is not known. MPS

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VI disorder (ARSB gene; Maroteaux-Lamy syndrome: MIM#253200) is caused by the deficiency of the lysosomal hydrolytic enzyme, N-acetylgalactosamine-4-sulfatase (4S; EC: 3.1.6.12)/Aryl

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sulfatase B (ARSB) which is involved in the degradation of dermatan sulfate (DS) (Nuefeld and Muenzer, 2001; Brands et al., 2013). This condition results in over-accumulation of DS in the

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cells, organs and tissues of the body. MPS VI is inherited in an autosomal recessive fashion and presumes a 25% of recurrence risk for the affected families. MPS VI is a systemic disorder

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affecting different organs of the body. The common clinical features of MPS VI disorder include coarse facies, corneal clouding, hepatosplenomegaly, umbilical hernia, inguinal hernia, macrocephaly, macroglossia, severe pulmonary respiratory problems and cardiac dysfunction (Mc Kusick, 1966; Karageorgos et al., 2007). MPS VI disorders also cause various severe skeletal abnormalities including dysostosis multiplex, joint contractures or joint stiffness and short stature, carpal tunnel syndrome, spinal stenosis and sleep apnea (Muenzer, 2011). MPS VI disorder shows great phenotypic variability ranging from severe, intermediate or attenuated and mild phenotypes. The MPS VI disorder is further classified into rapidly progressing and slowly progressing disorder based on the age of onset, severity of the disease and survival period of the patients (Valayannopoulos et al., 2010).

The rapidly progressing MPS VI disorder is

characterized by early onset (before 2 years of age), increased levels of urinary

ACCEPTED MANUSCRIPT glycosaminoglycans (above 200 μg/mg creatinine), severe clinical features such as dysostosis multiplex, respiratory insufficiency, cardiac failure and death by the 2nd or 3rd decade of life

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(Neufeld and Muenzer, 2001; Karageorgos et al., 2007). Slowly progressing MPS VI disorder is characterized by the late onset of the clinical features, manifestation of milder phenotypes in the

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adulthood due to lower rates of accumulation of GAGs (< 100 μg/mg creatinine) and better life

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expectancy in comparison to rapidly progressing MPS VI disorder (Swiedler et al., 2005;

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Thumler et al., 2012). ARSB is located on the long arm of the chromosome 5 (5q14.1) and consists of 8 exons yielding a protein of 533 amino acids in length. To date more than ~200

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pathogenic variations have been reported worldwide (Mathew et al., 2015; Uttarilli et al., 2015) (http://www.hgmd.cf.ac.uk/ac/gene.php?gene=ARSB) (HGMD Professional 2016.3 version

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accessed date – 20.10.16).

Currently, there are very few reports on the mutation spectrum of MPS VI disorder in India

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(Mathew et al., 2015). Molecular analysis conducted by us on 19 MPS VI patients showed the presence of fifteen different disease-causing variations of which twelve were novel [p.Asp53Asn (c.157G>A;

p.D53N),

p.Leu98Arg

(c.306_312delCTACCAG+146del; p.F166Lfs*18), p.I350F),

(c.293T>G;

p.Y103Sfs*9),

p.L98R),

p.Tyr103Serfs*9

p.Phe166Leufs*18

(c.496delT;

p.Ile220Serfs*5 (c.659_660delTA; p.I220Sfs*18), p.Ile350Phe (c.1048A>T;

p.Trp353*

(c.1059G>A;

p.W353*),

p.His393Arg

(c.1178A>G;

p.H393R),

p.Ser403Tyrfs* (c.1208delC; p.S403Yfs*), p.Pro445Leu (c.1334C>T; p.P445L), p.Trp450Leu (c.1349G>T; p.W450L ) and p.Trp450Cys (c.1350G>C; p.W450C) and other three were known pathogenic variations [p.Asp54Asn (c.160G>A; p.D54N), p.Ala237Asp (c.710C>A; p.A237D) and

p.Ser320Arg

(c.960C>G;

p.S320R)]

(http://www.hgvs.org/mutnomen/recs.html)

(Kantaputra et al., 2013; Kantaputra et al., 2014; Uttarilli et al., 2015). Novel variations

ACCEPTED MANUSCRIPT identified in the study were subjected to in silico characterization using different mutation prediction software. The inconsistent prediction results provided by different mutation prediction

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tools for certain mutations create ambiguity in determination of its pathogenic nature, whereas

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functional characterization conclusively confirms the deleterious nature of the pathogenic variations. Hence we conducted this study to functionally characterize the novel variations in

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Indian population and confirm their pathogenic nature.

A) Site-directed mutagenesis and cloning:

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Materials and methods:

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ARSB cDNA was cloned into pcDNA3.1(+) vector to produce pcDNA3.1(+)-ARSB. Sitedirected mutagenesis (SDM) was done for the cDNA mutants detected in our study. Plasmids of

(Supplementary Methods).

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wild-type ARSB gene/clone as well as all the cDNA mutants were isolated and stored

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B) Cell culture and Expression studies:

Transfection studies were performed for all the mutant constructs along with the wild type construct using Lipofectamine 2000 reagent. COS-7 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% Fetal Bovine Serum, 1% penicillin/streptomycin (Gibco, Life technologies) at 370 C and 5% CO2. For transfection, 4x106 cells were seeded and grown to 70 to 90% confluence on 100 mm plates. Cells were transfected with 2ug of each wildtype (WT) as well as cDNA mutant plasmid DNA using 4 ul of Lipofectamine 2000 (Invitrogen, Heidelberg, Germany). Each construct was transfected in triplicates. Cells expressing EGFP were also transfected for assessing the transfection efficiency under the same conditions on different plates. The fluorescence emitted by EGFP cells were visualized using an inverted

ACCEPTED MANUSCRIPT fluorescent microscope. Cells were harvested after 48 hr of transfection and lysates were prepared (Supplementary Methods).

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C) ARSB lysosomal enzyme assay for cell lysates:

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The protein concentration of all the cell lysates was determined by Bradford protein estimation

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method. The ARSB enzyme activities were performed spectrophotometrically for all the cell

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extracts using the colorimetric substrate p-nitrocatechol sulfate. P-nitrocatechol which is produced by hydrolysis of the p-nitrocatechol sulfate was estimated spectrophotometrically at

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515nm. ARSB enzyme assay was performed according to the standard protocol. COS-7 cells transfected with an empty pcDNA3.1(+) was used to correct the enzyme activity of cells

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D) Western blot:

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expressing WT-ARSB gene as well as all other SDM cDNA mutants constructed.

Proteins were subjected to SDS–PAGE (10% polyacrylamide) and transferred onto a PVDF Membrane (GE Healthcare Life Sciences, Amersham, Hybond-P, Buckinghamshire, UK). Western blot experiment was performed using Rabbit anti-ARSB primary antibody (RabMab anti-ARSB-[EPR9409]) and goat-anti-rabbit HRP-labeled peroxidase secondary antibody. Blots were reprobed for Tubulin. E) Reverse Transcriptase PCR (RT-PCR) for p.A237D and p.S320R SDM mutants: COS-7 cells were transfected with pcDNA3.1(+) vector, WT-ARSB gene, p.A237D (p.Ala237Asp), S320R (p.Ser320Arg) and D54N (p.Asp54Asn) plasmid DNAs in 60 mM culture plates (Corning Incorporated, New York, US). Total RNA was isolated and cDNA was synthesized using Superscript III according to manufacturer’s instructions (Invitrogen

ACCEPTED MANUSCRIPT Bioservices Pvt. Limited, India). cDNA was further PCR amplified by using two different primer pairs, one pair specific to exon 7 and 8 of the ARSB CDS (3` to ARSB mRNA) and the other set

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specific to exon 1 of ARSB CDS (5` to ARSB mRNA). Primer sequences for 3` specific primer

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set are RT_FP: GACTCTTCACCGTGTCCCAG and RT_RP: GTTGGTGGGTCTGATGAGGG whereas the primer set for 5` to ARSB mRNA is 2RT_FP: GCGAGCTTGCCCCGAG and

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2RT_RP: CGTGCGGATGCGGGAG. cDNA amplified with GAPDH primers was used as a

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control and the RNA stability was checked using semi-quantitative RT-PCR.

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F) Subcellular localization studies by Confocal microscopy: Subcellular localization studies were performed in wild-type ARSB clone and p.D53N

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(p.Asp53Asn) mutant transfected COS-7 cells using standard Immunofluorescence protocol

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using rabbit anti-ARSB and mouse anti-LAMP2 primary antibodies. Anti-mouse Alexa flour 568

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and anti-rabbit Alexa flour 488 antibodies were used as secondary anti-bodies. The cells fixed and mounted on glass slides were visualized using Confocal microscopy (Supplementary Methods). Results:

Molecular analysis of 19 Indian patients with MPS VI disorder revealed the presence of fifteen different pathogenic sequence variations. All the variations detected were predicted to be disease causing using pathogenicity – prediction tools such as HANSA (www.cdfd.org.in/HANSA/), MutationTaster (www.mutationtaster.org), Polyphen2 (genetics.bwh.harvard.edu/pph2/), SIFT (sift.jcvi.org/) and Mutation Assessor (www.ngrl.org.uk). Database search in 1000 Genome Browser (browser.1000genomes.org/), ExAC server (exac.broadinstitute.org/) and dbSNP (http://www.ncbi.nlm.nih.gov/projects/SNP/) excluded the possibility of these variations to be

ACCEPTED MANUSCRIPT polymorphisms. Functional characterization was conducted on the fifteen different sequence

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variations previously reported by us.

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A) In vitro characterization of novel mutations by lysosomal ARSB enzyme assay estimated in cell extracts:

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The effect of the mutations on the function of ARSB protein was characterized by transient

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expression of wild-type ARSB clone as well as all the different mutant clones in COS-7 cells separately. Transfection was also done for the polymorphic clone p.Val358Met which was found

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in our study. Two known mutations, p.Pro313Ala and p.Trp322*clones from previous literature were taken as positive controls. The wild-type ARSB clone transfected COS-7 cells showed

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over-expressed enzyme activity which was approximately 12 times more than that of the vector transfected cells (i.e., pcDNA3.1(+) COS-7 cells) (Supp. Fig 1). The enzyme activity of

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untransfected COS-7 cells was almost equivalent to the enzyme activity of pcDNA3.1(+) vector transfected COS-7 cells. The endogenous enzyme activity levels observed in all the cell lysates were corrected by subtracting the enzyme activity of pcDNA3.1(+) vector transfected cells from all the wild type as well as mutant enzyme activities. A graph was plotted for wild type and all other mutants after correcting the endogenous enzyme activity levels (Figure 1A). The ARSB enzyme activity of p.Val358Met polymorphism was found to be 82% of the wild type. The cells expressing p.Pro313Ala and p.Trp322* showed a drastic reduction to approximately 4-7% of the wild-type ARSB enzyme activities. Although p.Asp54Asn, p.Ala237Asp and p.Ser320Arg were reported to be disease causing in HGMD, there was no information available describing their functional consequences. cDNA mutants were created for fourteen (missense, nonsense, and small deletion) variations detected in the study and were functionally characterized. The cells

ACCEPTED MANUSCRIPT expressing the mutant ARSB cDNAs revealed enzyme activities in the range of 3-15% of the wild type ARSB enzyme activity with the exception of two mutant clones, p.His393Arg and

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p.Trp450Leu showing significant residual enzyme activities in comparison to all other mutant

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clones (Figure 1A). COS-7 cells transfected with the cDNA constructs of p.His393Arg and p.Trp450Leu mutants were showing the enzyme activities of 38 and 41% respectively to that of

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the wild-type ARSB enzyme activity.

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B) Western blot analysis:

Immunoblot analysis was performed for untransfected COS-7 cells, vector transfected, wild-type

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ARSB protein along with one polymorphic mutant clone p.Val358Met and all other cDNA mutants created by SDM. The wild-type human-ARSB protein lysate extracted from COS-7 cells

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interacted with the rabbit anti-ARSB antibody and yielded a specific band at 60 KD which corresponds to the precursor ARSB protein. There was no significant amount of ARSB protein detected in untransfected COS-7 cells and pcDNA3.1 (+) vector transfected COS-7 cells. ARSB protein expression similar to wild type ARSB protein was present in all the mutant ARSB proteins constructed, except for mutants p.Ala237Asp and p.Ser320Arg. In comparison to other mutants, p.Ala237Asp there was a drastic reduction (very faint signal intensity) in the expression of full-length ARSB protein whereas in p.Ser320Arg cell lysate, ARSB precursor protein was not detected at all (Figure 1B). ARSB enzyme activity estimated in cell lysates for p.Ala237Asp and p.Ser320Arg was almost equivalent to the enzyme activity of ARSB protein in untransfected and vector transfected COS-7 cells. C) Reverse-Transcriptase PCR (RT-PCR):

ACCEPTED MANUSCRIPT Cultured COS-7 cells were transfected with the plasmid DNAs of pcDNA3.1(+), wild-type ARSB clone, p.Ser320Arg, p.Ala237Asp and p.Asp54Asn. p.Asp54Asn mutant construct was

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used as positive control for the RT-PCR experiment. RT-PCR with the RT_FP and RT_RP

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primer pair (specific to exon 6 & 7 of ARSB mRNA) revealed that the mutant p.Ala237Asp showed the presence of ARSB cDNA whereas p.Ser320Arg showed the absence of ARSB

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specific cDNA (Figure 2A). The absence of ARSB specific cDNA could be due to an exonic

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missense mutation leading to splice site changes in the transcript. Human splice finder (http://www.umd.be/HSF3/) predicted that the CG substitution in the mutation c.960C>G

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leads to the creation of an exonic splicing silencer (ESS) site resulting in potential alteration of the splicing event (Figure 2B). To conclusively prove that the mutant p.Ser320Arg results in

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alternative splicing, RT-PCR performed with another pair of primers 2RT_FP and 2RT_RP (specific to exon 1 and 2 of ARSB mRNA) showed the presence of ARSB cDNA in both the

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mutants, p.Ala237Asp and p.Ser320Arg (Figure 2C). D) Immunofluorescence by Confocal microscopy analysis: The subcellular localization of ARSB protein was studied by immunofluorescence technique using Lamp2 as an endogenous marker for lysosomes. ARSB protein was found to be colocalized with Lamp2 in wild type overexpressed ARSB transfected cells. p.Asp53Asn mutant transfected COS-7 cells also showed co-localization of ARSB with lysosomes indicating that the mutation was not leading to any problems in the ARSB protein sorting to lysosomes (Figure 2D).

ACCEPTED MANUSCRIPT Discussion: We identified 15 different disease-causing mutations in 19 MPS VI patients in our cohort

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(Kantaputra et al., 2014; Uttarilli et al., 2015). Fourteen of the nineteen families recruited for the

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study showed the presence of consanguinity whereas one family showed no consanguinity. The

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consanguinity data was not available for four of the families recruited and analyzed (Table 1). Parental mutation analysis was done for 11 families out of the 19 families recruited for the study.

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Mutation characterization for the parents revealed that they were heterozygous carriers of the

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specific mutation found in the probands. One of the MPSVI patients showed the presence of a compound heterozygous mutation. Mutation analysis in their parents showed that one parent was

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heterozygous for one mutation and other parent was heterozygous for another mutation. Prenatal

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diagnosis was done in subsequent pregnancies, for five different families with affected probands. Two pregnancies were found to be affected as the fetal DNA showed homozygosity for the

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mutations p.Trp450Cys and p.Asp54Asn respectively. Of the three unaffected fetuses, one was homozygous for the wild-type allele (unaffected) and the other two were heterozygous carriers for one of the parental mutation, p.His393Arg and p.Trp353* respectively. The parents opted to terminate the affected pregnancies; the unaffected pregnancies were continued and were confirmed to be normal on postnatal follow-up. Prenatal diagnosis through a combination of enzyme assay and targeted mutation analysis in the fetal sample is considered to be more reliable for lysosomal storage disorders as the enzyme analysis results can be at times uninformative especially for heterozygous carriers (Table 1). All the novel variations were predicted as pathogenic variations based on in silico characterization using different mutation prediction software such as MutationTaster, Polyphen2,

ACCEPTED MANUSCRIPT SIFT, and HANSA. In the present study, hence functional characterization was done for all the variations detected in the study.

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Lysosomal ARSB enzyme activity was performed in COS-7 cell lysates expressing

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wild-type ARSB gene and 14 different pathogenic variations. The wild-type ARSB clone showed highly increased ARSB activity (~ 12 times increase in comparison to untransfected and

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vector transfected COS-7 cell lysates) (Supp. Fig 1) due to the overexpression whereas most of

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the SDM variations like p.Asp53Asn, p.Asp54Asn, p.Leu98Arg, p.Phe166Leufs*18, p.Ile220Serfs*5, p.Ala237Asp, p.Ser320Arg, p.Ile350Phe, p.Ser403Tyrfs, p.Pro445Leu,

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p.Trp450Cys and p.Trp533* resulted in significantly low ARSB activity (3-15% to that of wildtype ARSB enzyme activity) except for p.His393Arg and p.Trp450Leu (38-41% to that of wild-

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type ARSB enzyme activity) cell lysates (Figure 1A). This helped us to confirm the pathogenic potential of these variations identified in our cohort of MPS VI patients.

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It is known that during transport from the ER to Golgi apparatus, Arylsulfatase B is cleaved and processed from a 60 KD precursor polypeptide to three disulfide-linked polypeptides 43, 8 and 7 KD (Garrido et al., 2008). Immunoblot analysis showed that 60 KD full-length ARSB protein was expressed in the cell lysates expressing WT-ARSB and all the SDM mutants except for the mutant p.Ser320Arg and p.Ala237Asp (Figure 1B). The mutants p.Ala237Asp and p.Ser320Arg showed significantly lower residual ARSB enzyme activity when compared to other mutants. A significant decrease in the ARSB protein expression levels was observed in p.Ala237Asp lysate compared to WT-ARSB and other SDM mutants and no protein expression was observed in p.Ser320Arg lysate (Figure 1B). This protein decrease may be either due to problems in the translation of the ARSB protein, maturation of precursor ARSB protein to mature ARSB protein, hindrance at the level of post-translation modifications such as addition of

ACCEPTED MANUSCRIPT proper signal peptide, problems at the protein targeting to the lysosomes which may be due to improper recognition by mannose-6-phosphate receptor, loss of large C-terminal region of ARSB

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protein and misfolding of the ARSB protein due to the pathogenic variations occurring at

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functionally important domains of ARSB protein. The five deletion mutants and the two nonsense pathogenic variations detected in our study could not be subjected to western blot

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specific immunogen for the ARSB protein to bind.

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analysis with the available Rabbit Monoclonal ARSB antibodies as they lack the C-terminal

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Reverse-Transcriptase PCR analysis with two different primer pairs showed that there was likely possibility that the missense mutation p.Ser320Arg abolishes the exonic splicing enhancer site

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(ESE) due to CG substitution. The missense mutant p.Ser320Arg was predicted to be involved

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in the creation of an ESS site resulting in the occurrence of an alternative splicing event. This was confirmed by the absence of notable RNA expression in the SDM mutant p.Ser320Arg

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whereas p.Ala237Asp showed significant RNA expression, when the RT-PCR analysis was done using the primers specific to the ARSB cDNA (exon 7 and 8) downstream to the p.Ser320Arg mutation occuring in exon 5 (Figure 2A). Interestingly, it was found that there was a presence of significant RNA expression in p.Ser320Arg mutant, when RT-PCR analysis was performed with the primers specific to the ARSB cDNA (exon 1 region) upstream to the p.Ser320Arg mutation (Figure 2B). This indicates that transcription step of ARSB mRNA is intact and is not hindered by the mutations in ARSB gene, i.e. in the mutant p.Ala237Asp whereas ARSB mRNA stability was likely to be affected in the mutant p.Ser320Arg. Hence ARSB protein was not detected for p.Ser320Arg cDNA mutant in western blot analysis. This absence of ARSB cDNA in p.Ser320Arg mutant (Figure 2A, 2B & 2C) may be due to ARSB mRNA degradation or possibility of alternative splicing event, but the exact reason for the same is unknown. The exact

ACCEPTED MANUSCRIPT mechanism by which a missense mutation affects or reduces mRNA expression is not known clearly, but there was a report available from the previous study showing that ~9% of the single

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base substitutions in IDS gene affect splice site signals (Matos et al., 2015). There is a growing

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evidence that missense, nonsense and silent mutations affect the consensus sequence of exonic cis-elements such as exonic splice enhancers (ESE) and exonic splice silencers (ESS) that are

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important for correct splice-site identification (Cartegni et al., 2002; Gorlov et al., 2003; Gorlov

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et al., 2004; Bashyam et al., 2010; Jin et al., 2012). A missense mutation in hematopoieticrestricted Src homology 2-containing inositol-5`-phosphatase (SHIP1) resulted in markedly

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reduced SHIP1 protein and thus reduced RNA levels as this presumably affected the RNA stability (Nguyen et al., 2011). Thus it is important to evaluate the exonic single nucleotide

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substitutions for assessing their possible consequences on pre-mRNA processing (Cartegni et al., 2002). Immunofluorescence experiment revealed that there was reduced expression of ARSB

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protein in p.Asp53Asn variant even though the ARSB was localizing to lysosomes. We attempted to analyze the genotype-phenotype associations and their correlation with the results of functional studies. All the pathogenic variations located in exon 1, viz., p.Asp53Asn, p.Asp54Asn, p.Leu98Arg and p.Tyr103Serfs*8 detected in our study were found to cause severe phenotype and this was in turn confirmed by functional studies (Uttarilli et al., 2015). Literature survey revealed that the mutations p.Asp53Asn and p.Asp54Asn are known to be residing in the catalytically active site and these sequence variations result in severe phenotypes which supported our findings in the present study, but the variation p.Asp53Asn in other Indian reports showed moderate or mild phenotype (Karageorgos et al., 2007; Saito et al., 2008; Valayannopoulos et al., 2010; Mathew et al., 2015). Two other pathogenic variations were reported at Leu98 position: p.Leu98Pro and p.Leu98Gln. Patients with p.Leu98Pro showed

ACCEPTED MANUSCRIPT intermediate or attenuated phenotype whereas the variation p.Leu98Gln resulted in rapidly progressing MPS VI phenotype (Karageorgos et al., 2007; Saito et al., 2008; Valayannopoulos et

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al., 2010). The variation p.Leu98Arg in other Indian MPS VI patient group showed the presence

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of attenuated phenotype, unlike our patients who showed rapidly progressing disorder (Mathew et al., 2015). Thus a clear genotype-phenotype association could not be established.

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In the present study, all the unrelated patients with p.Trp450Cys mutation showed severe

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phenotype (rapidly progressing MPS VI phenotype) whereas patients with the same variation in another Indian group of patients showed attenuated phenotype (Mathew et al., 2015). Hence,

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genotype-phenotype correlation cannot be established for these variations in our MPS VI patients.

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The residue Trp450 was found to be highly conserved across different ARSB homologs and any substitution at this position is likely to be pathogenic. Interestingly, we found that two

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different mutations at the same position, Trp450, led to drastic phenotypic variability in MPS VI patients. Patient with p.Trp450Leu showed very mild clinical features and transfection studies also showed that p.Trp450Leu transfected COS-7 cells were showing almost half (45%) of the enzyme activity as compared to wild-type overexpressed ARSB (Figure 3A, 3B & 3C). All our patients with p.Trp450Cys showed severe dysostosis multiplex and lysosomal ARSB enzyme assay performed in transfected cells showed drastic reduction (~2-4% to wild-type construct) in ARSB enzyme activity (Figure 3D, 3E & 3F). The patients with p.His393Pro were reported to exhibit severe to mild phenotypes in MPS VI patients from Australia (Litjens et al., 1996). There are no reports on the sequence variation p.His393Arg in Indian population. We also found that patients with p.His393Arg showed mild clinical features and ARSB enzyme assay performed in cell lysates of transfected COS-7 cells

ACCEPTED MANUSCRIPT revealed the presence of 41% of ARSB residual enzyme activity. Review of data regarding clinical features, age of presentation and disease progression rates, in these patients with

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p.His393Arg as well as p.Trp450Leu revealed a mild phenotype which was correlating with the

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respective enzyme activities (Figure 3D, 3E & 3F). Thus a genotype-phenotype correlation has been established for these two pathogenic variations [p.His393Arg and p.Trp450Leu] in the

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current study. This information will be useful in genetic counseling of families with this

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pathogenic variation in future.

Genotype-phenotype correlation was found to be shown in two families affected with different

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mutation (p.His393Arg in one family and p.Trp450Leu in other family) in each family. Mild clinical symptoms of the patients were correlated with the presence of half of the residual

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enzyme activities when compared to the other mutants. In all other mutants, it was difficult to establish genotype-phenotype due to the rarity of MPS disorders, presence of unique mutation

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spectrum in each affected family and presence of a large number of mutations identified in MPS VI patients, with less recurrence in other families (Saito et al., 2012) (Figure 3).

Conclusions:

Molecular analysis of nineteen Indian MPS VI patients showed the presence of 15 mutations of which twelve were novel. Molecular and functional analysis of more number of patients can provides a more comprehensive mutation spectrum data which can be useful to set a diagnostic panel of mutations which are commonly found in MPS VI patients. Although mutation prediction software provide some idea about disease-causing potential of novel variants, it is imperative to develop simpler methods of functional analysis of novel variants for clinical significance. Thus, functional analysis of novel mutations in ARSB gene using site-directed

ACCEPTED MANUSCRIPT mutagenesis followed by cell transfection and enzyme assay, immunoblot studies using western blot, reverse transcriptase PCR for expression studies and localization studies helped us to

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confirm the pathogenic nature of novel mutations identified in our study.

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Acknowledgements:

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The authors thank the patients and their families who volunteered in the study, for their kind cooperation. The authors acknowledge the support provided by the Centre for DNA

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Fingerprinting and Diagnostics, Hyderabad, and the Indian Council of Medical Research (ICMR), New Delhi for financial support. The first author (AU) acknowledges the Council of

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Scientific and Industrial Research, New Delhi, for providing Junior and Senior Research Fellowships towards the pursuit of a Ph.D. degree of the Manipal University, India. AU

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acknowledges the theoretical support provided by Mr. S. Jamal Md Nurul Jain for standardizing the Arylsulfatase B enzyme assay in leukocytes in the laboratory. AU is also thankful to Mr. M. Raveendra Babu for his suggestions and support in the molecular techniques like cloning, sitedirected mutagenesis and Immunoblot assays. Conflicts of Interests: The authors declare that there is no conflict of interest. References: 1. Bashyam MD, Chaudhary AK, Reddy EC, Devi AR, Savithri GR, Ratheesh R, Bashyam L, Mahesh E, SenD, Puri R, Verma IC, Nampoothiri S, Vaidyanathan S, Chandrasekhar MD, Kantheti P. Phenylalanine hydroxylase gene mutations in phenylketonuria patients from India: Identification of novel mutations that affect PAH RNA. Mol Genet Metab.,

ACCEPTED MANUSCRIPT 100 (2010), pp. 96–99. doi: 10.1016/j.ymgme.2010.01.016. 2. Brands MM, Oussoren E, Ruijter GJG, Vollebregt AA, van den Hout HM, Joosten KF,

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Hop WC, Plug I, van der Ploeg AT. Up to five years experience with 11

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mucopolysaccharidosis type VI patients. Mol Genet Metab., 109 (2013), pp. 70–76. doi: 10.1016/j.ymgme.2013.02.013.

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ACCEPTED MANUSCRIPT VI. J Inherit Metab Dis., 37 (2013), pp. 263–268. doi: 10.1007/s 10545-013-9645-8. 9. Kantaputra PN, Kayserili H, Guven Y, Kantaputra W, Balci MC, Tanpaiboon P,

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Tananuvat N, Uttarilli A, Dalal A. Clinical manifestations of 17 patients affected with

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mucopolysaccharidosis type VI and eight novel ARSB mutations. Am J Med Genet Part A., 164 (2014), pp.1443–1453. doi: 10.1002/ajmg.a.36489.

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10. Karageorgos L, Brooks D, Pollard A, Melville E, Hein L, Clements P. Mutational

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12. Mathew J, Jagadeesh SM, Bhat M, Udhaya Kumar S, Thiyagarajan S, Srinivasan S. Mutations in ARSB in MPS VI patients in India. Mol Genet Metab Reports., 4 (2015), pp. 53–61.doi: 10.1016/j.ymgmr.2015.06.002. 13. Matos L, Gonçalves V, Pinto E, Laranjeira F, Prata MJ, Jordan P, Desviat LR, Perez B, Alves S. Functional analysis of splicing mutations in the IDS gene and the use of antisense oligonucleotides to exploit an alternative therapy for MPS II. Biochim Biophys Acta., 1852 (2015), pp. 2712–2721.doi: 10.1016/j.bbadis.2015.09.011. 14. Mc. Kusicks VMD, Beighton P. Genetics of Mucopolysaccharidoses. Mc Kusicks Heritable disorders of connective tissues, Mosby-year book 5 (1996), pp. 1153-1198. 15. Muenzer J. Overview of the mucopolysaccharidoses. Rheumatology (Oxford), 50 (2011), pp. v4-v12. doi: 10.1093/rheumatology/ker394.

ACCEPTED MANUSCRIPT 16. Nguyen NY, Maxwell MJ, Ooms LM, Davies EM, Hilton AA, Collinge JE, Hilton DJ, Kile BT, Mitchell CA, Hibbs ML, Jane SM, Curtis DJ. An ENU-induced mouse mutant

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18. Swiedler SJ, Beck M, Bajbouj M, Giugliani R, Schwartz I, Harmatz P, Wraith JE, Roberts J, Ketteridge D, Hopwood JJ, Guffon N, Sa Miranda MC, Teles EL, Berger KI,

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Piscia-Nichols C. Threshold effect of urinary glycosaminoglycans and the walk test as indicators of disease progression in a survey of subjects with Mucopolysaccharidosis VI

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(Maroteaux-Lamy syndrome). Am J Med Genet A., 134A (2005), pp. 144-150. doi: 10.1002/ajmg.a.30579.

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19. Saito S, Ohno K, Sugawara K, Sakuraba H. Structural and clinical implications of amino substitutions

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mucopolysaccharidosis type VI. Mol Genet Metab., 93 (2008), pp. 419–425. doi: 10. 1016/j.ymgme.2007.11.017. 20. Saito S, Ohno K, Sekijima M, Suzuki T, Sakuraba H. Database of the clinical phenotypes, genotypes and mutant arylsulfatase B structures in mucopolysaccharidosis type VI. J Hum Genet., 57 (2012), pp. 280-282. doi: 10.1038/jhg.2012.6. 21. Thumler A, Miebach E, Lampe C, Pitz S, Kamin W, Kampmann C, Link B, Mengel E. Clinical characteristics of adults with slowly progressing mucopolysaccharidosis VI: a case series. J Inherit Metab Dis., 35 (2012), pp. 1071-1079. doi: 10.1007/s 10545-0129474-1.

ACCEPTED MANUSCRIPT 22. Uttarilli A, Ranganath P, Jain SJ, Prasad CK, Sinha A, Verma IC, Phadke SR, Puri RD, Danda S, Muranjan MN, Jevalikar G, Nagarajaram HA, Dalal AB. Novel mutations of

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the arylsulphatase B (ARSB) gene in Indian patients with mucopolysaccharidosis type

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VI. Indian J Med Res., 142 (2015), pp. 414–425. doi: 10.4103/0971-5916.169201. 23. Valayannopoulos V, Nicely H, Harmatz P, Turbeville S. Mucopolysaccharidosis VI.

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Orphanet J Rare Dis., 5 (2010), pp. 5. doi: 10.1186/1750-1172-5-5.

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24. Wenger DA, Coppola S, Liu S-L. Insights into the diagnosis and treatment of lysosomal

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storage diseases. Arch Neurol., 60 (2003), pp. 322–328. doi: 10.1001/archneur.60.3.322.

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Figure Legends:

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Figure 1A: ARSB enzyme assay in cultured COS-7 cells. X-axis denotes the COS-7 cell

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lysates expressing either wild-type ARSB protein and different mutant proteins identified in the study (including p.Pro313Ala and p.Trp322* mutant proteins taken as positive controls for the study from literature). Y-axis denotes relative increase in ARSB enzyme activity with respect to vector control cell lysate (pcDNA3.1+), expressed in nmol/hr/mg protein; Bars correspond to the mean of three different experiments. Standard errors are indicated. 1B: Immunoblot analysis in transfected COS-7 cell lysates. 50 µg of protein lysate was loaded per well. COS-7 cells were transfected with 2 µg of different plasmids. ARSB precursor protein corresponds to 60 KD. Tubulin (55 KD) was used as a loading control; Figure 2A: Semi-Quantitative RT-PCR. COS-7 cells were transiently transfected with 1 µg of five different plasmid DNAs. Total RNA was isolated and total cDNA was synthesized from 1 µg of total RNA as starting material. ARSB exon 7 and 8 specific primers were used to

ACCEPTED MANUSCRIPT synthesize ARSB specific cDNA. GAPDH primers were used to synthesize GAPDH specific cDNA which was used as a loading control for the semi-quantitative RT-PCR experiment. 2B:

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Human splice finder analysis. Prediction of the effect of the missense mutant, p.Ser320Arg on

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the splicing event. 2C: RT_PCR. ARSB exon 1 specific primers were used to synthesize ARSB specific cDNA. GAPDH specific cDNA was used as a loading control for the RT-PCR

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experiment. 2D: Subcellular localization studies using indirect immunofluorescence

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experiment in wild-type (WT) and mutant cDNA-transfected COS-7 cells. Co-localization of ARSB and LAMP2 (an endogenous marker of lysosomes) is shown in the overlay.

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Magnification: 100X.

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Figure 3: Pathogenic effects of the variants p.Trp450Cys and p.Trp450Leu. 3A) Clinical

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features of the patient with p.Trp450Leu variation (11 years old girl); 3B) Sequence chromatogram of MPS VI patient affected with p.Trp450Leu mutation and a control sample; 3C)

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Lysosomal ARSB enzyme assay in p.Trp450Leu transfected COS-7 cells; 3D) Clinical features of the patient with p.Trp450Cys variation (10 years old girl); 3E) Sequence chromatogram of MPS VI patient affected with p.Trp450Cys mutation and a control sample; 3F) Lysosomal ARSB enzyme assay in p.Trp450Cys transfected COS-7 cells.

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Figure 1

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Figure 2A

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Figure 2B

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Figure 2C

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Figure 2D

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Figure 3

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Table 1: Genotype and phenotypes of the sequence variations identified in Indian MPS VI patients. Gender

Age at presentation

Cons angu inity

Mutation (HGVS nomenclature)

Location on the gene

ARSB & control enzyme activity(nmol/hr/m g ptn)

Phenotype

Reference

Novel /Known

1

M

2 Yrs 6 mths

Yes

Exon 1

17.36 (115)

Severe

2

M

4 Yrs 7 mths

Yes

p.Asp53Asn]/[p.A sp53Asn] [p.Asp53Asn]/[p. Asp53Asn]

53.5 (235)

Severe

3

M

6 Yrs

Yes

[p.Asp54Asn]/[p. Asp54Asn]

Exon 1

NA

Severe

Uttarilli et al., 2015; Kantaputr a et al., 2014A Uttarilli et al., 2015

4

M

1 Yr 3 mths

NA

Exon 1

NA

Severe

5

F

5 Yrs

NA

Exon 1

NA

Severe

6

M

7 Yrs

NA

Exon 1

0.86 (70)

Severe

7

M

21 mths

Yes

Exon 1_Intron 1

21.9 (182)

Severe

8

M

2 Yrs 6 mths

Yes

Exon 2

18.9 (273)

9

M

4Yrs

NA

[p.Leu98Arg]/[p. Leu98Arg] [p.Leu98Arg]/ [p.Leu98Arg] [p.Leu98Arg]/[p. Leu98Arg] [c.306_312delCT ACCAG+146del]/ [c.306_312delCT ACCAG+146del] [p.Phe166Leufs*1 8]/ [p.Phe166Leufs*1 8] [p.Ile220Serfs*5+ p.Ser320Arg]/ [p.Ile220Serfs*5+ p.Ser320Arg]

[Exon 3+Exon5]

10

M

3 Yrs

Yes

[p.Ala237Asp]/[p. Ala237Asp]

Exon 4

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Novel

No

No

Known

Yes

Uttarilli et al., 2015 Uttarilli et al., 2015 Uttarilli et al., 2015 Uttarilli et al., 2015

Novel

No

Yes Affected fetus No

Novel

Yes

No

Novel

Yes

No

Novel

Yes

No

Severe

Uttarilli et al., 2015

Novel

Yes

Yes Unaffected fetus

NA

Severe

Uttarilli et al., 2015

Yes

No

11 (116)

Severe

Uttarilli et al., 2015

One Novel and other Known Known

No

No

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Exon 1

Prenatal diagnosis in the family

Novel

Parent al Mutati on Analys is No

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Family

No

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Exon 5

12.4

Severe

Yes

13

M

3 Yrs

14

M

15

No

Exon 5

16.7 (213)

Severe

Novel

Yes

Intermediate

Uttarilli et al., 2015

Novel

Yes

16.3 (126)

Severe

Novel

No

15.6 (126)

Severe

Kantaputr a et al., 2014A Kantaputr a et al., 2014A

Yes Heterozygous mutation in fetus (MPS VI carrier) Yes Heterozygous mutation in fetus (MPS VI carrier) No

Yes

[p.His393Arg]/[p. His393Arg]

Exon 6

57.4 (215)

16 Yrs

Yes

[p.Ser403Tyrfs*]/ [p.Ser403Tyrfs*]

Exon 6

F

6Yrs

Yes

Exon 7; Exon 8

16

M

3Yrs

No

17

F

7 Yrs

Yes

18

F

10 Yrs

Yes

19a

F

11 Yrs

Yes

19b

M

6 Yrs 6 mths

Yes

[p.Pro445Leu]/[p. Pro445Leu]; [p.Trp450Cys]/[p. Trp450Cys] [p.Trp450Cys]/[p. Trp450Cys] [p.Trp450Cys]/[p. Trp450Cys] [p.Trp450Cys]/[p. Trp450Cys] [p.Trp450Leu]/[p. Trp450Leu] [p.Trp450Leu]/[p. Trp450Leu]

Both are Novel

Yes

Yes Affected fetus

Exon 8

21.2 (115)

Severe

Novel

No

No

Exon 8

15.6 (126)

Severe

Novel

Yes

No

Exon 8

10.3 (116)

Severe

Novel

No

No

Exon 8

36.2 (126)

Mild

Novel

Yes

No

Exon 8

53.5 (216)

Mild

Uttarilli et al., 2015 Uttarilli et al., 2015 Uttarilli et al., 2015 Uttarilli et al., 2015 Uttarilli et al., 2015

Novel

Yes

No

Legend: The patients 19a and 19b were siblings.

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1 Yr 6 mths

No

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M

Novel

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12

Present report Uttarilli et al., 2015

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[p.Ile350Phe]/[p.Il e350Phe] [p.Trp353*]/ p.Trp353*]

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NA

PT ED

9 Yrs

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M

AC

11

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Arylsulfatase B – ARSB Human Gene Mutation Database –HGMD Mucopolysaccharidoses –MPS Lysosomal storage disorders –LSD Dermatan sulphate- DS

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1. 2. 3. 4. 5.

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Abbreviations

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Graphical abstract

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 

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Functional characterization of novel variants in ARSB gene helped to prove the pathogenicity of the variants. The patients with p.H393R and p.W450L variants showed mild phenotype and high residual enzyme activity. ARSB protein expression was affected severely in p.A237D and p.S320R variants. The mutant, p.S320R was found to affect the mRNA stability.

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