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Case Series Vinca alkaloid induced cranial neuropathy

Neluwa-Liyanage Ruwan Indika1,2, Arndt Rolfs3,4, 5, Christian Beetz5, Sabine Schröder5, Catarina Pereira5, Volha 
Skrahina4,5, Mihika Fernando2, Dinesha Maduri Vidanapathirana2,6, Subhashinie Jayasena2, Eresha Jasinge2

1Department of Biochemistry, Faculty of Medical Sciences, University of Sri Jayewardenepura, Nugegoda, Sri Lanka.
2Department of Chemical Pathology, Lady Ridgeway Hospital for Children, Colombo 8, Sri Lanka.
3University Rostock, Medical Faculty, Rostock/Germany.
4Arcensus GmbH, Goethestrasse 20, 18055 Rostock, Germany.
5CENTOGENE GmbH, Am Strande 7, 18055 Rostock, Germany.
6Department of Pathology, Faculty of Medical Sciences, University of Sri Jayewardenepura,  Nugegoda, Sri Lanka.  

Genotypes and phenotypes of Sri Lankan Patients with 
Mucopolysaccharidosis type IVA

Mucopolysaccharidosis type IVA is a rare autosomal recessive lysosomal 
storage disorder occurring worldwide in all ethnic groups. It is caused by  bi-
allelic variants in the GALNS gene (OMIM 612222). We report five cases of 
mucopolysaccharidosis type IVA with short stature and severe skeletal dysplasia. 
An optimized diagnostic strategy that combined enzymatic testing and genetic 
screening was applied. All the tested urine samples showed increased urinary 
glycosaminoglycan / creatinine ratios. In all five cases, the enzyme activity of 
galactosamine-6-sulfate sulfatase was pathologically decreased. Gene-targeted 
sequencing revealed a previously unreported homozygous c.139-12T<C variant 
of the GALNS gene in one patient and three previously reported missense 
variants in four patients; c.253T<C (p.Cys85Arg), c.626C<T (p.Ala209Val) 
and c.878C<T (p.Ser293Leu). Genetic studies not only confirm the diagnosis 
of mucopolysaccharidosis IVA, but also enable predicting the prognosis and 
facilitate genetic counseling. Enzyme replacement therapy is not available in 
Sri Lanka to date. However, the quality of life in these patients can be improved 
by a multidisciplinary approach.

Abstract

*Corresponding Author
Neluwa-Liyanage Ruwan Indika
Department of Biochemistry, 
Faculty of Medical Sciences,
University of Sri Jayewardenepura,
Nugegoda, Sri Lanka.
Email: ind.liyanage@sjp.ac.lk

Article History 
Received On : 06 Apr, 2022
Accepted On : 15 Dec, 2022

Funding sources: None 

Conflict of Interest: None

Keywords: Mucopolysaccharidosis 
type IVA, GALNS, phenotype, variant, 
glycosaminoglycans

Online Access

DOI: 
https://doi.org/10.3126/jnps.v42i2.41954

Introduction
Mucopolysaccharidosis type IV (MPS IV), also known as Morquio syndrome, is a rare 
autosomal recessive lysosomal storage disorder (LSD) caused by deficiency of two 
enzymes catalyzing degradation of glycosaminoglycans; N-acetylgalactosamine-6-
sulfate-sulfatase (GALNS) deficiency in Morquio A (MPS IVA) and beta-galactosidase 
(GLB1) deficiency in Morquio B (MPS IVB). Accumulation of keratan sulfate (KS) and 
chondroitin-6-sulfate in tissues is responsible for the characteristic clinical manifestations 
of MPS IV that include, short-trunk dwarfism, skeletal dysplasia, fine corneal deposits, 
hearing impairment (conductive or sensorineural), dental abnormalities and cardiac 
valve abnormalities.1

Case Presentation 
The clinical characteristics of the probands are listed below and are in accordance with 
those of patients previously diagnosed with MPS IVA, as described in the literature. 

Case 1

This female child had, kyphoscoliosis, genu valgum, ulnar deviation of the wrist and 
joint hypermobility. She also had Mongolian blue spots over the posterior aspect of the 
trunk. She was clinically diagnosed to have MPS IV at the age of two years. 

Case Series

DOI: 103126/JNPS.V4113

Copyrights & Licensing © 2022 by author(s). This is an Open Access article distribut-
ed under Creative Commons Attribution License (CC BY NC )



J Nepal Paediatr Soc | VOL 42 | ISSUE 02 |MAY-AUG,  2022 81

Case SeriesSri Lankan patientS with mucopoLySaccharidoSiS type iVaiVa

Case 2

Among the clinical features of this female child were short neck, 
lumbar lordosis, pectus carinatum, genu valgum, abnormal gait, 
joint hypermobility and dental abnormalities. The diagnosis of 
MPS IV was made at the age of 10 years.

Case 3 

Lumbar lordosis, pectus carinatum, genu valgum, platyspondyly, 
hip dysplasia, and dental abnormalities were the presenting 
clinical features of this two-and-a-half-year-old female child. 
On slit lamp examination there were stromal opacities in the 
cornea. Ultrasound scan of abdomen revealed a liver with coarse 
echotexture. The 2D-echocardiogram was normal.

Case 4

This two-year-old male child was born to consanguineous 
parents who are first cousins. He presented with short neck, 
pectus carinatum, joint hypermobility and umbilical hernia. The 
2D-echocardiogram of the child showed mildly myxomatous 
mitral valve and trivial mitral regurgitation.

Case 5 

This three-and-a-half-year-old male child was born to 
consanguineous parents. He had pectus carinatum, genu valgum, 
platyspondyly, dental abnormalities, corneal opacity and inguinal 
hernia. Dysostosis multiplex and short stature were common to all 
the cases. 

Quantification of total glycosaminoglycans (GAGs) 
in urine: Urine total GAGs were analyzed at Institute of 
Human Genetics, Foundation for Research in Genetics and 
Endocrinology (FRIGE) House, Gujarat, India. GAGs were 
determined by dimethylmethylene blue (DMMB) dye-binding 
assay with slight modifications to the method reported by De Jong 
et al.2 Absorbance of the GAG-DMMB complex was measured 
at 520 nm immediately after mixing 20 µl of standard / sample 
and 100 µl of distilled water with 100 µl of DMMB, in Cobas Bio 
centrifugal analyzer. The urine creatinine levels were measured 
and results were expressed in terms of mg GAG / mmol creatinine. 
All four patients whose total GAG levels were quantified showed 
increased excretion of total GAGs (Table 1).

Quantification of enzyme activities: The enzyme studies 
were carried out again at CENTOGENE GmbH, Rostock, 
Germany. GALNS activity was measured in dried blood spots 
by LC / MRM-MS (liquid-chromatography / multiple-reaction-
monitoring mass-spectrometry) based method.3 In all five cases 
enzyme activity of GALNS was pathologically decreased (table 
1).

Table 1. Urine glycosaminoglycan levels and blood GALNS 
activity in patients with MPS IVA.

 Case Age as on the 
date of urine 
sample collection

Urine GAG / 
creatinine ratio
(mg GAG / mmol 
creatinine)

GALNS   activity 
(µmol / L / h)

Case 1 2.7 years 9.2 (4.4 – 8.0) < 0.3 (≥ 2.0) 

 Case 2 10.5 years 53.5 (1.9 – 4.3) < 0.1 (≥ 2.0) 

Case 3 2.5 years 10.9 (4.4 – 8.0) < 0.3 (≥ 2.0) 

Case 4 2.2 years 14.75 (4.4 – 8.0) < 0.1 (≥ 2.0) 

Case 5 3.2 years Not documented < 0.1 (≥ 2.0) 

N.B: Lower limit of detection [LOD] and lower limit of quantification 
[LOQ] of the GALNS activity at CENTOGENE are 0.1 µmol/L/h 
and 0.3 µmol/L/h respectively. Reference intervals are given in 
parenthesis. 

Genetic diagnostic testing: The GALNS and GLB1 genes 
were analyzed by sequencing PCR products that cover the 
entire coding region and the highly conserved exon-intron 
splice junctions; GALNS by bi-directional Sanger Sequence 
Analysis while GLB1 by NGS-Illumina as the laboratory started 
updating the platform for sequencing the genes to NGS-
Illumina from 2017. Our patients were investigated during the 
transition period. The reference sequence of the GALNS gene is 
NM_001323544.1. The reference sequence of the GLB1 gene 
was NM_001317040.1. Deletion/duplication analysis for case 
5 was carried out by quantitative PCR assay (qPCR) by using 
gene-specific amplicons targeting every coding exon of the 
GALNS gene. No pathogenic variants were detected in GLB1 
gene. Pathogenic variants in GALNS gene were detected in all 
five cases resulting in a diagnosis of MPS IVA. The genotypes of 
the cases are summarized in table 2.

Table 2. Genotypes observed in patients with MPS IVA. 

 A B C D E F

1 M No Compound
heterozygous

c.253T < C 
p.(Cys85Arg)
c.626C < T 
p.(Ala209Val)

NM_001323544.1

2 F Yes Homozygous c.878C < T 
p.(Ser293Leu)

NM_001323544.1

3 F No Homozygous c.253T < C 
p.(Cys85Arg)

NM_001323544.1

4 M Yes Homozygous c.253T < C p. 
(Cys85Arg)

NM_001323544.1

5 M Yes Homozygous c.139-12T < C
p. (?)

NM_001323544.1

Note: A = Case, B = Gender, C = Parental Consanguinity,  

D = Zygosity, E = Genotype, F = Reference sequence

Discussion
Quantification of urinary KS by LC-MS / MS (Liquid-chromatography-
tandem mass-spectrometry) is a specific and quantitative method 
for analysis of KS as opposed to spectrophotometric estimation 
of total GAG excretion or qualitative analysis using thin layer 
chromatography or electrophoresis. Measuring enzyme activity 
from a DBS is only a screening test and enzyme activity analysis in 
fibroblasts or leukocytes is recommended if screening is negative. 
Variant analysis not only confirms the results of enzyme activity 
but also facilitates genetic counseling of the family.4 In the present 
study, an optimized diagnostic strategy that combined enzymatic 
testing and genetic screening was applied. Fig. 1 illustrates how 
the diagnostic strategy in the present case study is related to the 
pathogenesis of MPS IVA. 



J Nepal Paediatr Soc | VOL 42 | ISSUE 02 |MAY-AUG,  202282

Case Series Sri Lankan patientS with mucopoLySaccharidoSiS type iVa

Figure 1. Relating pathogenesis of Mucopolysaccharidosis type 
IVA to its diagnostic approach. 

N-acetylgalactosamine-6-sulfate-sulfatase (GALNS) enzyme 
encoded by GALNS gene removes 6-sulfate groups of the 
N-acetyl-D-galactosamine 6-sulfate units of chondroitin sulfate and 
of the D-galactose 6-sulfate units of keratan sulfate. Deficiency 
of GALNS enzyme results in tissue accumulation and urinary 
excretion of the glycosaminoglycans. GALNS variants, reduced 
GALNS activity and increased urinary glycosaminoglycan 
excretion can be detected by targeted gene sequencing, enzyme 
assays and dimethylmethylene blue assay respectively. 

The c.253T < C (p.Cys85Arg) variant has been previously 
reported in Indian patients.5 The variant is reported as c.253T 
< C (p.Cys85Arg) or c.235T<C (p.Cys79Arg) depending 
on the reference sequence used; NM_001323544.1 and 
NM_000512.4 respectively. c.626C < T (p.Ala209Val) has been 
reported earlier (as c.608C < T p.Ala203Val due to the use of a 
different reference sequence) from Indian and Brazilian patients 
with severe disease phenotype of MPS IVA.5,6 Case 2 in the 
present study had significantly high urine GAG levels. The variant 
detected in this patient, c.878C < T (p.Ser293Leu) has been 
reported before using a different reference transcript as c.860C<T 
(p.Ser287Leu) in American, Australian, and Polish patients with 
severe phenotype.6 In case 5, we detected a previously unreported 
homozygous variant in intron 1 of the GALNS gene, c.139-12T < 
C. To date, this variant is not described in the Exome Aggregation 
Consortium, Exome Sequencing Project or the 1000 Genomes 
Browser. Software analyses (Alamut v.2.7.1) predicted an effect 
on splicing. All the patients reported in the present study showed 
significantly low GALNS activity.  

Enzyme replacement therapy with elosulfase alfa is not available 
in Sri Lanka to date, but can be brought down on special 
request. Nevertheless, quality of life in these patients can 
be improved also by a multidisciplinary approach, once the 
diagnosis is properly done. Caring paediatrician or physician 
may provide the patient with palliative care with nonsteroidal 
anti-inflammatory drugs (NSAIDs) for joint pain, antibiotics 
for pulmonary infections. Oxygen supplementation should be 
arranged for pulmonary compromise and obstructive sleep 
apnoea. Moreover, the coordinating physician should interact 
with other health care professionals such as orthopedic surgeons, 
otorhinolaryngologists, geneticists, dentists and physiotherapists 
to form an effective multidisciplinary team.7-9 Finally, we hope that 
our study will raise awareness for MPS IVA in Sri Lanka, eventually 
resulting in improved diagnostic and therapeutic options. 

Conclusions
The optimized diagnostic strategy that combined enzymatic testing, 
urinary glycosaminoglycan excretion, and genetic screening 
yielded clinically useful laboratory information for genotype-
phenotype correlations. All the patients showed significantly 
increased urinary glycosaminoglycan / creatinine ratios and 
significantly low GALNS activity that could be correlated with 
deleterious mutations detected in our patients.  

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