² Department of Biochemistry, Children’s Hospital, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

16.2 Multiple Sulfatase Deficiency

Graciana Diez-Roux and Andrea Ballabio

Telethon Institute of Genetics and Medicine , Naples , Italy

16.1 Defects in Transport: Mucolipidosis II alpha/beta, Mucolipidosis III alpha/beta and Mucolipidosis III gamma

Mucolipidosis type II (ML II) and mucolipidosis type III (ML III) are autosomal recessive disorders caused by defects in the N-acetylglucosamine-1-phosphotransferase (GlcNAc-1-phosphotransferase) complex, which is composed of three subunits, α, β and γ. Mutations in the gene encoding the alpha/beta subunits (GNPTAB) lead to ML II alpha/beta, which was also called I-cell disease (OMIM #252500), or to the clinically milder condition, ML III alpha/beta (OMIM #252600), which was formerly called pseudo-Hurler polydystrophy (ML IIIA – Table 16.1). ML III gamma (OMIM #252605), which was formerly called variant ML IIIC, arises from mutations in the gene encoding the γ subunit of GlcNAc-1-phosphotransferase (GNPTG) [1]. The GlcNAc-1-phosphotransferase complex catalyses the first step in the generation of the mannose 6-phosphate (Man6P) recognition marker required for efficient targeting of soluble lysosomal proteins (acid hydrolases and activator proteins) to lysosomes [2].

Case histories

Case history for ML II alpha/beta

Ultrasound examination of a foetus at week 24 of pregnancy showed a short femur with increased echogenicity and polyhydramnion. The child was born at week 37 and presented soon after birth with bowed legs, gingival hypertrophy, coarse face and respiratory difficulties (achydyspnea). X-ray examination of the bones showed osteopenia, wide ribs and other additional findings consistent with dysostosis multiplex. At 5 months she had growth deficiency with short stature (55 cm [–3SD]) and failure to gain weight (4.5 kg [–3SD]) (Figure 16.1). Echocardiogram revealed a thickened aortic and mitral valve. No hepatosplenomegaly was observed.

The activities of the serum lysosomal enzymes, arylsulfatase A, β-galactosidase and β-glucuronidase were elevated 10-fold. Mutation analysis of the GNPTAB gene showed a homozygous deletion (c.3503_4delTC) leading to a frameshift and a premature termination (p.L1168QfsX5). The constellation of the clinical, biochemical and molecular findings permitted confirmation of the diagnosis of ML II.

Case history for ML III alpha/beta

A 3-year-old girl presented with severe claw hands, genu valgum, a severe kyphosis, and contractures of the hips and knees but without facial dysmorphism. The contractures of the fingers and large joints had been noticed as first symptoms by her parents at the time of consultation. Her psychomotor development was normal for her age. Her height was 80 cm (–3SD). She had no hepatosplenomegaly or gingival hypertrophy. Echocardiography revealed mitral valve thickening. X-rays of the spine showed kyphosis, platyspondyly, anterior–inferior beaking of L2; and the iliac wings were small. The girl presented with mild coxa valga of the femoral heads. The activities of the serum lysosomal enzymes arylsulfatase A, β-galactosidase and β-glucuronidase were elevated 20-fold. The combination of clinical findings and the elevated lysosomal serum enzyme activities led to the suspicion that the patient was affected with ML III because of the less severe phenotype than ML II. The patient was compound heterozygous for the mutations c.1120 T > C (p.F374L) and c.6565 C > T (p.R1189X) in the GNPTAB gene, which confirmed the diagnosis of ML III alpha/beta.

Case history for ML III gamma

A 12-year-old boy presented with claw hands, genu valgum, progressive stiffness of the fingers and reduced joint mobility of the knees and hips and shoulders. The claw hands had been observed as first symptoms at the age of 6 years. He had normal psychomotor development and went to a regular school. He had no facial dysmorphism. In addition he presented with a mild kyphosis and genu valgum (Figure 16.2). His height was 130 cm (–2SD). He had neither hepatosplenomegaly nor gingival hypertrophy. Echocardiography revealed mild mitral valve thickening. He had been followed up in the rheumatologic clinic for suspected rheumatoid arthritis since the age of 10 years. X-ray examination of the spine showed mild kyphosis, and mild platyspondyly. He had mild coxa valga of the hips and dystrophic changes of the femoral heads.

The activities of the lysosomal enzymes arylsulfatase A, β-galactosidase, and β-glucuronidase were elevated 15-fold in serum. The combination of relatively mild symptoms compared to patients with ML III alpha/beta and the elevated lysosomal enzyme activities led to the suspicion that the patient was affected with ML III gamma. This was confirmed by mutation analysis of the GNPTG gene which showed that the patient was homozygous for the mutation c.196 C > T, leading to a premature stop codon (p.Arg66X).

Epidemiology

ML II and ML III are rare lysosomal storage disorders with a variable prevalence of 0.16 (The Netherlands), 0.22 (Czech Republic), 0.31 (Australia), 0.4 (Japan) or 0.8 (north Portugal) per 100,000 live births for ML II. The highest ML II frequency, estimated at 16.2/100,000, has been found in Sanguenay-Lac-Saint-Jean (Quebec, Canada). The prevalence of ML III was reported to be 0.08 and 1.89 per 100,000 in The Netherlands and north Portugal, respectively.

Genetic basis

The GlcNAc-1-phosphotransferase complex is composed of three different subunits α, β, and γ in a molar ratio of 2:2:2 and is encoded by two genes. The GNPTAB gene, which is located at chromosome 12q23.3, encodes a 145 kDa α/β subunit precursor [3] that is catalytically activated by proteolytic cleavage by the site-1 protease involved in the cholesterol homeostasis [4] (Figure 16.3 [5]). The GNPTG gene, which is located at chromosome 16p13.3, encodes the soluble 36 kDa γ subunit of unknown function [6]. ML II alpha/beta and ML III alpha/beta patients carry mutations in GNPTAB whereas ML III gamma patients have mutations in the GNPTG gene.

Table 16.1 Classification of mucolipidosis types II and III.

	Former nomenclature	New nomenclature
I-cell disease	ML II	ML II alpha/beta
pseudo-Hurler Polydystrophy	ML IIIA	ML III alpha/beta
ML III variant	ML IIIC	ML III gamma

Figure 16.1 Patient with ML II alpha/beta. (a) Coarse facial features, long philtrum and antevarted nares. (b) Bowed leg. (c) X-ray of the spine showing severe hip dysplasia, and dysostosis multiplex of the lower limbs bones.

Figure 16.2 12-year-old patient with ML III gamma.

Pathophysiology

Defects in GlcNAc-1-phosphotransferase lead to missorting of multiple lysosomal hydrolases in mesenchymal cells especially fibroblasts and the lysosomal accumulation of different non-degraded macromolecules, lipids, amino acids or sugars (e.g. glycosaminoglycans, GM2-gangliosides, cholesterol, cystine or sialic acid). Many other cultured cells such as hepatocytes, Kupffer cells, and leukocytes exhibit normal levels of lysosomal enzyme activities. Therefore, peripheral leukocytes are not suitable for ML II and ML III diagnostics. In addition, in liver, kidney, spleen, and brain tissue of ML II patients nearly normal activities of lysosomal enzymes are detectable. The cellular consequences of the accumulating material are still unclear and appear to depend on the composition and extent of storage and the type of storing cells.

Clinical presentation

Neonates with ML II alpha/beta might show craniofacial abnormalities with striking gingival hypertrophy. Importantly, patients with clinical phenotypes intermediate between the ML II alpha/beta and ML III alpha/beta phenotypes have been reported with slower progression than ML II alpha/beta and more severe progression than ML III alpha/beta phenotypes (Table 16.2).

Figure 16.3 Organisation of the GNPTAB and GNPTG genes and their encoded α/β subunit precursor membrane protein (1,256 amino acids) and the soluble γ subunit (305 amino acids), respectively. The α/β subunit precursor is cleaved by the Golgi-resident site-1 protease into mature and enzymatically active α and β subunits (modified from Kollmann et al. 2010 [5]).

Table 16.2 Clinical fi ndings and symptoms of mucolipidosis types II and III.

Typical time of onset	Disorder	Signs and symptoms
Prenatal	ML II alpha/beta	Short and bowed lower limbs; hips and knees contractures; bone fracture; transient alveolomaxillary defect (TAD); osteopenia; periosteal new bone formation (“cloaking”).
Postnatal	ML II alpha/beta	Dysmorphism: coarse facies; gingival hypertrophy, short neck Neurology: Developmental delay; intellectual disabilities; hypotonia Eyes: corneal clouding Skeletal: short bowed limbs; joint contractures; pectus carinatum; thoracal asymmetry; congenital hip dislocation; osteopenia Abdomen: hepatosplenomegaly; umbilical hernia; inguinal hernia Heart: mitral valve thickening; dilated cardiomyopathy transient neonatal hyperparathyroidism
Early infancy and adulthood	ML III alpha/beta & ML III gamma^*	Skeletal: progressive joint stifness; stiffness of the small joints; claw hand; carpal tunnel syndrome; tarsal tunnel syndrome; scoliosis; kyphosis; hyperlordosis; genu valgum; shoulder, decreased mobility and/or contractures of knees and hip joints; upper limb paraesthesia; progressive growth retardation; C1-C2 instability; progressive bone dystrophy X-rays: progressive development of “J” shaped sella turcica; oar-shaped ribs; anterior inferior beaking of lower thoracic to upper lumbar vertebral bodies; flared iliac wings; progressive dysplasia of the femoral heads and necks; coxa valga; “bullet-shaped” proximal phalanges and central pointing of proximal metacarpals Eyes: hyperopic astigmatism and corneal clouding; retinal and optic nerve abnormalities Heart: aortic and /or mitral valve thickening; dilated cardiomyopathy Abdomen: umbilical hernia; inguinal hernia

*The ML III gamma patients seems to have a milder clinical picture than the ML III alpha/beta patients.

ML III alpha/beta and ML III gamma are slowly progressive disorders mainly affecting skeletal, joint and connective tissues. Clinical onset is in early childhood with restricted joint mobility, and skeletal changes that mainly affect the pelvis and spine. ML III gamma seems to be less severe than ML III alpha/beta.

Natural history

ML II alpha/beta is a severe disease with onset of signs and symptoms at birth and a fatal outcome most often in early childhood. ML III alpha/beta patients show first clinical symptoms at approximately 3 years of age whereas data on life expectancy are missing. The onset of first symptoms in ML III gamma patients is in infancy as well and they are known to survive to the fourth or fifth decades; the oldest reported patient was 86 years old [7]. ML III might be misdiagnosed as rheumatoid arthritis.

Figure 16.4 Diagnostic pathway for mucolipidosis types II and III. The activities of a set of lysosomal enzymes, e.g. β-hexosaminidase (β-hex), β-glucuronidase (β-gluc), β-galactosidase (β-gal), and arylsulfatase A (ASA) should be measured in the serum, in extracts of cultured fibroblasts and conditioned media. In comparison to controls (black column) the activities of the lysosomal enzymes are decreased in patient cells but increased in serum and conditioned cell media, demonstrating the missorting of lysosomal enzymes. (ii) Defects in the generation of the Man6P recognition marker can be analyzed by radioactive measurement of GlcNAc-1-phosphotransferase activity in extracts of cultured cells, or by Man6P-western blotting of cell extracts and aliquots of conditioned media. Both experimental approaches support the diagnosis of ML II or ML III. (iii) Since many of the known mutation in GNPTAB or GNPTG lead to frameshift and unstable transcripts due to “nonsense-mediated mRNA decay”, the mRNA expression of GNPTAB/GNPTG is performed in fibroblasts. Reduced GNPTG mRNA levels have been reported to be accompanied by compensatory up-regulation of GNPTAB mRNA. The decreased expression of GNPTG mRNA can be confirmed by western blotting for the human γ subunit. (iv) The direct sequencing and DNA analysis of the GNPTAB or GNPTG genes allow the diagnosis of ML II alpha/beta or ML III alpha/beta or gamma.