European Journal of Human Genetics (2013), 1–8& 2013 Macmillan Publishers Limited All rights reserved 1018-4813/13
www.nature.com/ejhg

ARTICLEA novel intermediate mucolipidosis II/IIIab caused by
GNPTAB mutation in the cytosolic N-terminal domain
Jules G Leroy1, David Sillence2, Tim Wood1, Jarrod Barnes3, Robert Roger Lebel4, Michael J Friez1,
Roger E Stevenson1, Richard Steet3 and Sara S Cathey*,1Mucolipidosis (ML) II and ML IIIa/b are allelic autosomal recessive metabolic disorders due to mutations in GNPTAB. The geneencodes the enzyme UDP-GlcNAc-1-phosphotransferase (GNPT), which is critical to proper trafficking of lysosomal acidhydrolases. The ML phenotypic spectrum is dichotomous. Criteria set for defining ML II and ML IIIa/b are inclusive for all but
the few patients with phenotypes that span the archetypes. Clinical and biochemical findings of the ‘intermediate’ ML in eightpatients with the c.10A4C missense mutation in GNPTAB are presented to define this intermediate ML and provide a broader
insight into ML pathogenesis. Extensive clinical information, including radiographic examinations at various ages, was obtained
from a detailed study of all patients. GNPTAB was sequenced in probands and parents. GNPT activity was measured andcathepsin D sorting assays were performed in fibroblasts. Intermediate ML patients who share the c.10A4C/p.K4Q mutationin GNPTAB demonstrate a distinct, consistent phenotype similar to ML II in physical and radiographic features and to ML IIIa/b
in psychomotor development and life expectancy. GNPT activity is reduced to 7–12% but the majority of newly synthesizedcathepsin D remains intracellular. The GNPTAB c.10A4C/p.K4Q missense allele results in an intermediate ML II/III with
distinct clinical and biochemical characteristics. This delineation strengthens the utility of the discontinuous genotype–phenotype correlation in ML II and ML IIIa/b and prompts additional studies on the tissue-specific pathogenesis in
GNPT-deficient ML.
European Journal of Human Genetics (2013) 0, 000–000. doi:10.1038/ejhg.2013.207
Keywords: mucolipidosis; phosphotransferase; storage disease; GNPTABQ1INTRODUCTION
Mucolipidosis II (ML II; I–cell disease: MIM 252500) and mucolipidosis
IIIa/b (ML III; pseudo-Hurler-polydystrophy; MIM 252600) areallelic autosomal recessive metabolic disorders directly correlated withhomozygous or compound heterozygous mutant GNPTAB genotypes.
The GNPTAB gene encodes the a and b polypeptides in the hexameric
enzyme UDP-GlcNAc-1-phosphotransferase (GNPT: IUBMB no.
2.7.8.17). The g polypeptide is encoded by the GNPTG gene.
The enzyme complex catalyzes the first step in the biosynthesis of
the mannose-6-phosphate (M6P) recognition marker in theoligomannosyl-type N-linked glycans in lysosomal acid hydrolases.
Without the M6P marker most soluble hydrolases are not targetedto the lysosomal compartment in connective tissues, resulting inimpaired lysosomal degradation.
GNPTAB genotypes, either homozygous or compound heterozygous for mutant alleles with ‘amorph’ or ‘null’ effect (frameshift andnonsense mutations), result in the early-onset, severe ML II. The
later-onset ML IIIa/b with prolonged course is the morbid result inpatients with at least one ‘hypomorph effect’ allele (missense andmost splice mutations) in the GNPTAB mutant genotype. The
phenotypic spectrum of patients with GNPT-deficiency disorders is
dichotomous rather than continuously variable. ML II and ML IIIa/b
are phenotypically distinguishable. Within the largest publishedcohort of ML patients, the phenotype of only a few subjects straddled
the criteria set for defining ML II and ML IIIa/b.1
1Department

The purpose of this report is to present and discuss the clinical andbiochemical findings of the ‘intermediate’ ML in eight patients withthe c.10A4C missense mutation in exon 1 in their compound
heterozygous GNPTAB genotype. The amino acid lysine is replacedwith glutamine, p.K4Q (designated K4Q hereafter). The name ‘Webb
type ML’ is proposed following the explicit permission for use of thesurname of one of the families and honoring all participating families
and patients in this study. The findings corroborate the overallgenotype–phenotype correlation, underscore the predictive power ofmutation screening and illustrate the potential for generating heuristic
hypotheses on the pathogenesis of ML.
SUBJECTS AND METHODS
Probands 1, 2 and 3 are isolated patients in three unrelated families. Only
patient 3, who died at the age of 23 6/12 years, was never examined by at least
one of the authors. However, the wealth of clinical data made available by themother prompted inclusion in the core group of subjects reported earlier andin this study.1 The postmortem study of patient 3 was recently published as thereport of autopsy findings in a patient with ML IIIa/b.2 Patients 4–8 are
siblings. The earlier part of the disorder’s course in the older siblings wasreported by Kozlowski et al.3 All sibs were included in the recent report byDavid-Vizcarra et al,4 who recognized the affected as examples of ML
intermediate between ML II and ML IIIab. The parents in each of the fourfamilies were non-consanguineous.
In addition to plasma samples and fibroblast cell lines representing the eightsubjects, cultured fibroblasts from two additional patients, one homozygousof Clinical Genetics, Greenwood Genetic Center, Charleston Office, North Charleston, SC, USA; 2The Children’s Hospital at Westmead, Westmead,
New South Wales, Australia; 3Complex Carbohydrate Research Center, University of Georgia, Athens, GA, USA and 4State University of New York Upstate Medical School,
Syracuse, New York, USA*Correspondence: Dr SS Cathey, Department of Clinical Genetics, Greenwood Genetic Center, Charleston Office, 3520 W. Montague Avenue, Suite 104, North Charleston,SC 29418, USA. Tel: þ 1 843 746 1001; Fax: þ 1 843 746 1002; E-mail: scathey@ggc.org
Received 31 January 2013; revised 5 July 2013; accepted 11 July 2013

A novel intermediate ML II/IIIab
JG Leroy et al2
and one compound heterozygous for the K4Q missense mutation, wereobtained from NIGMS Human Genetic Cell Repository at Coriell Institute(Camden, NJ, USA) and included in the biochemical studies. No clinicalinformation except the diagnosis of ML III was available. The mutant
genotypes in patients and cell lines are presented in Table 1.
The activity of selected lysosomal acid hydrolases was assayed in plasma andcultured fibroblasts according to the methods described by Thomas et al.5
Metabolic labeling and sorting of the lysosomal hydrolase cathepsin D incontrol and patient fibroblasts were performed as described previously.6,7
Briefly, cell monolayers at about 60–80% confluency were washed twice withphosphate-buffered saline and methionine starved using cysteine/methioninefree Dulbecco’s modified Eagle’s medium for 30 min, followed by a pulse labelfor 1 h with 1 ml of 1 mCi/ml Tran35S-label (MP Biomedicals) in the same
media. Excess unlabeled methionine (10 mM final concentration) was added toinitiate a 4-h chase. The amount of cathepsin D sorted was determined byovernight immunoprecipitation of equivalent aliquots of the lysed cell andmedia high-speed supernatants at 4 1C with rabbit anti-human cathepsin D
antiserum and protein A-Sepharose beads. After five washes with buffer
containing 1 M KCl and 1% Triton X-100, immunoprecipitates were subjected
to 10% SDS-PAGE under nonreducing conditions. Gels were dried andexposed on an autoradiography film. Quantification of the various bands
was performed using Image J software. The percentage of cathepsin D thatremains intracellular was calculated as the densitometric ratio of theintracellular forms (both intermediate and mature) to the sum of theintracellular and secreted (precursor) forms.
GlcNAc-1-phosphotransferase activity was measured in microsomal preparations from fibroblasts as described earlier.8,9 Briefly, cells were lysed bysonication in Tris buffer pH 7.5 containing 0.25 M sucrose and microsomes
prepared by ultracentrifugation. Reactions using 10 mM a-methylmannoside as
an acceptor and 3H-UDP-GlcNAc (along with 200 mM ‘cold’ UDP-GlcNAc) as
substrate were conducted in the following reaction buffer: 50 mM Tris pH 7.5,
0.3% Triton X-100, 50 mM N-acetylglucosamine, 1 mM DTT, 20 mg/ml bovine
serum albumin, 10 mM sodium molybdate, 1 mM MgCl2, and 1 mM MnCl2.
Activity units are calculated as pmoles of 3H GlcNAc transferred per hour.
Protein concentration was measured using the micro BCA protein assay kit(Pierce, Rockford, IL, USA).
Screening for the GNPTAB mutations by PCR and sequencing protocols was
performed according to the methods reported earlier.1

Q2

Q3

Q4

RESULTSClinical phenotype
Patient 1. Patient 1 was delivered at term after an uncomplicatedpregnancy (panels A1 and A2 in Figure 1). Size was appropriate for
gestational age (Table 2). Early features included plagiocephaly treatedwith helmeting, kyphosis noted at approximately 9 months andregurgitant cardiac valves confirmed by echocardiogram at 12
months. Skeletal survey was performed at 20 months when adecreased range of motion in the shoulders prohibited lifting theTable 1 GNPTAB mutant genotype in GNPTAB c.10A4C/p.K4Q ML
patients and in mutant fibroblast lines
Patient

Paternal allele

Maternal allele

1

Exon 1: c.10A4C/p.K4Q

Exon 13: c.1964delC

2
3

Exon 11: c.1399delG
Exon 13: c.2190delT

Exon 1: c.10A4C/p.K4Q
Exon 1: c.10A4C/p.K4Q

4–8

Exon 1: c.10A4C/p.K4Q

Exon 11: c.1399delG

Exon 1: c.10A4C/p.K4Q
Exon 19: 3665_3666delTC

Exon 1: c.10A4C/p.K4Q
Exon 19: 3665_3666delTC

Linea
GM00113
GM01586

aAdopted from Human Genetic Cell Repository, Coriell Institute for Medical Research, Camden,

NJ, USA; genotypes.

European Journal of Human Genetics

arms above the head. The radiographs showed features of dysostosis
multiplex (DM) that included 331 kyphosis, varus angulation of theproximal humeri, bowed, dysplastic radii and ulnae, proximal anddistal tapering of the widened metacarpals, and marked coxa valga
and subluxation of the hips. The diagnosis of ML II was assigned at 22
months. While anthropometric measurements were normal in thefirst year of life, growth failure was apparent from his second year. At
1 year, length was 76 cm (50th percentile). The maximal height of
80.5 cm (mean for a 16-month-old male) was recorded at 7.5 years.
Speech and expressive language development were normal but
unaided walking was not achieved until 20 months. Cognitive
development was delayed. Formal assessment at 12 11/12 years
showed a global intellectual ability score of 39, comparable to anage equivalent of 5 5/12 years. Obstructive sleep apnea was treatedwith continuous positive airway pressure at night, beginning before 5
years. Recurrent infections led to one hospitalization for pneumonia
and three sets of myringotomy tubes by 8 years. Several surgicalattempts were made to stabilize his progressive thoracolumbar
kyphosis, with operations at 3, 6, and 10 years. Surgery for relief ofbilateral carpal tunnel syndrome was performed at 7 years. Postoperative extubation was consistently difficult. Prolonged ventilator
support with slow weaning was required. When examined at 12 7/12
years progressive joint contractures had decreased measured height to78.7 cm (mean for a 15-month-old male), weight was 15 kg (mean fora 3-year-old male), and head circumference 51.5 cm (mean for a
6-year-old male). He wore glasses for nearsightedness. Dysmorphic
features included flat and coarse facies, ridged palate, thickened gums,
broad and short hands held in ulnar deviation, broad and camptomelic fingers. There was a small umbilical hernia and diastasis of thestraight abdominal muscles without organomegaly. Despite joint
restriction, the boy was mobile, interactive and actively playful. He
conversed appropriately with hoarse voice.
Patient 2. Patient 2 was delivered by cesarean section for transverse
lie following a pregnancy complicated by hyperemesis in the firsttrimester (panel B in Figure 1). Ultrasound examination at 18 weeks
gestation was normal. Neonatal anthropometrics were normal(Table 2). Features noticed on the first day of life included unusual
facies with micrognathia, contractures at the knees and hips but
hypermobile wrists, slender fingers and narrow feet. Slow weight gain
in infancy was improved temporarily when breastfeeding was
supplemented with bottle-feeding. Impaired growth was apparent
by 22 months. Speech developed normally. He had significant grossmotor delay, persistent truncal hypotonia and mild thoracolumbar
kyphoscoliosis noticed at 12 months. He did not sit unassisted until
18 months and walked at 30 months. At 33 months, he did not haveorganomegaly, corneal clouding or gingival swelling, but lysosomal
storage disease was suspected based on his coarse facies, short stature,and skeletal changes. Testing revealed increased activity of multiple
lysosomal enzymes in plasma. The initial diagnosis of ML II was
assigned, but by age 40 months the overall clinical phenotype wasmore in support of intermediate ML and called ‘type II/III’.
Examination at 10 2/12 years showed an alert and conversant,ambulatory boy with stature 86.5 cm (mean for a 23-month-oldmale), weight 16.4 kg (mean for a 12-month-old male), and headcircumference 51.5 cm (mean for a 6-year-old male). Relevant
findings included normocephaly, coarse facies, gingival hypertrophy,
narrow thorax, soft ejection murmur, mildly prominent abdomen
without organomegaly, umbilical hernia, thoracolumbar kyphoscoliosis, severely limited range of motion in all joints, short claw-likehands in ulnar deviation, and broad camptomelic fingers.

Q5

A novel intermediate ML II/IIIab
JG Leroy et al
3

Figure 1 GNPTAB c.10A4C/p.K4Q ML clinical features in three unrelated patients: short stature, normocephaly, coarse facies, limited mobility in all joints,
short and broad hands with fingers postured in ulnar deviation. A1 and A2: Patient 1 at 8 and 12 years; B: Patient 2 at 6 years; C1 and C2: Patient 3 at 8
and 20 years.

Q6

Patient 3. Patient 3 was the third of three sons born to a Japanesefather and Caucasian mother (panels C1 and C2 in Figure 1).
Pregnancy was uncomplicated until 4 weeks prior to the expected
delivery date when rupture of membranes and labor led to vacuumassisted vaginal delivery. Size was appropriate for gestational age(Table 2). By 9 months, parents regarded his development to be
slower than that in his healthy siblings and noted differing physicalfeatures, including proptotic eyes, short neck, relatively large head,narrow shoulders, and protruding abdomen. Speech was late (firstwords at 15 months) and difficult to understand. He achieved
unaided walking at 18 months despite stiff hips and knees. By 23
months, he had limited range of motion at the shoulders, wrists andfingers, and thoracolumbar kyphoscoliosis. ML II was first suspected
at 26 months; ML III was his diagnosis at 29 months. Minimal
statural growth occurred after age 3 years. He had speech therapy andwas in special education classes throughout the years of schooling.
Testing of academic achievement at 14 years revealed a school
performance at the third to fourth grade level. At 11 years, he had
mild thickening of aortic and mitral valves, mitral regurgitation andleft ventricular hypertrophy. Cardiac and pulmonary function

worsened in the second decade. At 21 years, the left ventricularejection fraction was below 60% and associated with mild tomoderate regurgitation at all cardiac valves. He had severe sleep
apnea by mid-teens and required continuous supplemental oxygen by
22 years. He died at 23 years. Autopsy confirmed severe dilated
hypertrophic cardiomyopathy.2
Patients 4–8: common features in sibling patients. Five of sevenchildren born to healthy parents were affected (Figure 2; Table 2).
The two surviving affected siblings (patients 5 and 8) are ages 28 and15 years. Patients 4, 6, and 7 died at 21, 24, and 14 years, respectively.
Placental abruption at term complicated one pregnancy. The third
affected child, a male, was diagnosed prenatally and had an unaffected
female twin. No ultrasound abnormalities had been detected in either
fetus. Kyphosis and dislocated hips were diagnosed in the firstborn
child (patient 4) when she was 9 months old. Dysmorphic facial
features and joint contractures were subsequently recognized ininfancy in each of the affected siblings. The diagnosis of ML II was
made simultaneously in the two oldest children at 26 months and7 months. All of the children were of appropriate size for gestational
European Journal of Human Genetics

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A novel intermediate ML II/IIIab
JG Leroy et al
4

Table 2 Clinical features in eight patients with GNPTAB c.10A4C/p.K4Q ML
Patient

1

2

3

4

5

6

7

8

Gender

Male

Male

Male

FemaleFemaleMale

FemaleFemalePregnancy term (weeks)
Birth weight (g)

39
3200

39
3430

36
2390

39
3060

41
3290

40
2520

40
3290

42 þ
3970

Birth length (cm)Birth OFC (cm)

53
33

53
34

49

54
35

54
34

1 day
6 months

1 day
1 day

9 months
9 months

1 day
8 months

1 day
5 months

1 day
1 day

1 day
1 day

1 day
1 day

Age at ML diagnosis

22 months

33 months

23 months

26 months

7 months

1 month

2 weeks

2 weeks

Age at motor milestones
Sitting

12 months

18 months

12 months

18 months

9 months

9 months

9 months

9 months

14 months
20 months

18 months
30 months

18 months

36 months
7 years

12 months
21 months

11 months
42 months

12 months
42 months

12 months
42 months

80.5
7.5

87.0
12

88.0
22

84.5
13

87.8
15

Age at first signs
Craniofacial
Skeletal

Crawling
Walking
Max height (cm)Age at max height (years)
Age at death

23 years

90
26
21 years

24 years

14 years

Patients are from four unrelated families. Patients 4–8 are siblings.

Figure 2 GNPTAB c.10A4C/p.K4Q ML clinical features in five siblings: Similar findings as in Figure 1 are shown early and late in disorder’s natural
course. D: Patient 4 at 21.5 years; E: Patient 5 at 20 years; F: Patient 6 at 17.5 years; G: Patient 7 at 7.5 years; H: Patient 8 at 6 years.

age, but statural growth slowed from infancy and ceased in earlychildhood. Receptive and expressive language development occurred
at a near normal pace but neuromotor development was delayed.
Ambulation was not achieved until age 7 years in patient 4, who had
severe congenital flexion contractures at the knees. Patient 7 maintained independent ambulation only briefly. Cognitive development
was impaired, and all participated in special education schooling.
None truly mastered reading but could copy letters and words.
Recurrent bouts of otitis contributed to conductive hearing loss andEuropean Journal of Human Genetics

led to repeated sets of myringotomy tubes in all. All patients had
surgery for carpal tunnel syndrome. Systolic heart murmurs were
present from early childhood. Echocardiography revealed slow
thickening and gradually worsening incompetence of the mitral andaortic valves. Later in the clinical course, enlargement of the left
ventricular wall and dilatation of the left atrium and ventricle were
noticed. From infancy, breathing was louder, more superficial andfaster than normal. Adenoidectomy was performed in some. Sleep
apnea, obstructive and central, was treated with continuous positiveA novel intermediate ML II/IIIab
JG Leroy et al5airway pressure. There was limited development of secondary sexual
characteristics; only one of the females ever had menses. Diseaseprogression was characterized by severe bone and joint pain evenwhen at rest. Periodic intravenous infusion of biphosphonate
provided transient pain relief. All patients developed radiographic
and neurologic signs of narrowing of the spinal canal. Althoughdisease progression was apparent in every system, the morbid
consequences of cord compression dominated end-of-life issues.
The clinical diagnosis of intermediate mucolipidosis (ML II/III)
was reached in the two prior reports that included clinical andradiological data from affected members of this family.3,4
The radiographic phenotype
The osteochondrodysplasia (OCD) documented radiographically inthis group of ML patients fits within the concept of DM defined andillustrated by Spranger et al.10 The skeletal radiographs in this groupof patients at various ages show the remarkably consistent DM that iscongruent with ML II and significantly different from the ML IIIa/b
radiologic phenotype (Table 3; Figures 3 and 4).1,3,4,10,11
In the lateral skull films available from late childhood only the sella
turcica has an elongated shape, whereas in age-matched and older ML
IIIa/b patients it maintains the normal configuration. The thoracolumbar vertebrae demonstrate a much foreshortened and near ovoid
shape with convex upper and lower borders, and concave anterior andposterior irregular rims. At least one vertebra, either T12 and/or L1 at
the top of the kyphotic spine deformation appears more hypoplastic,

Table 3 Distinctive phenotypic findings in GNPTAB c.10A4C/p.K4Q
ML
Finding

ML II

K4Q MLML III

Coarsening of facies
Impaired growth

þþ
þþ

þ
þþ

±

Short hands, fingers
Limited motion range in joints

þþ
þþ

þþ
þ

(early childhood)
Unaided ambulation

a

þb

þb

Speech (normal, near normal)
Intellectual disability


þþ

þ
þ

þ
±

-

þ

þ

þþþ

þþþ

þ

c

Clinicalþ±±Biochemical and molecular results
Acid glycosidase levels in serum and cells. The activity of total
b-D-hexosaminidase, a-L-fucosidase and b-glucuronidase wassignificantly increased in plasma and decreased in cultured fibroblasts
derived from patients. These results overlap with the ranges ofhydrolase activity observed in ML II and ML IIIa/b. These findings
do not contribute to distinguishing the GNPTAB c.10A4C/p.K4Q
ML patients from either of the archetype GNPT-deficientdisorders.1,12,13
Assay of UDP-GlcNAc 1-phosphotransferase (GNPT). The results of
the GNPT assay are presented in Table 4. The activity of thephosphotransferase towards the substrate a-methylmannoside is
reduced to a range of 7–12% in the GNPTAB c.10A4C/p.K4Q ML
patients, about half of that in the K4Q homozygous patient fibroblasts(GM00113), which is to be expected.14 The significantly higher GNPT
activity in the K4Q samples compared with classic ML II supports thedistinct nature of the GNPTAB c.10A4C/p.K4Q ML.

bIndependent ambulation consistently lost from adolescence because of hip dysplasia andSorting efficiency of cathepsin D. The phenomenon of missorting ofimmature lysosomal enzymes and their appearance outside the cell
was studied in nascent cathepsin D by pulse-chase immunoprecipitation experiments. Cathepsin D is a highly mannose phosphorylated
lysosomal protease that is hypersecreted from the cell when mannose
phosphorylation is impaired. The pulse/chase assays thus provide an
opportunity to gauge the amount of newly synthesized cathepsin Dthat remains intracellular versus that secreted into the media. Whereas
the level of intracellular cathepsin D in fibroblasts from either controls
or obligate heterozygous parents exceeds the normal 90% (Figure 5),
ML II-type ‘I-cells’ (GM01586) retain only about 20% of newly
synthesized cathepsin D. The fibroblasts from our patient 2 and thehomozygous K4Q cells (GM0113) exhibit surprising levels of intracellular cathepsin D (81% and 65%, respectively). The comparatively
reduced percentage in the GM00113 cells is attributed to the latepassage number and consequent slow-growing nature of this culture.
Greater heterogeneity in all forms of cathepsin D was detected inpatient 2 and GM00113, which may reflect altered trafficking and/or
processing of this hydrolase within the secretory pathway.

painful osteopenic dystrophy.
cShort life span may preclude elongation of sella turcica in ML II.
dDocuments for proof or exclusion not available.
eSonographic finding in fetal ML II; inconsistent in ML II infants.

GNPTAB mutation screening. The GNPTAB mutation analysis datain patients 1–3 and 8 were previously reported.1 Table 1 also lists theSurvival beyond childhood
Radiographic
Overall dysostosis multiplex before
age 3 years (skeletal survey)
Sella turcica elongated (lateral skull)Thoracolumbar vertebrae foreshortened

þþ

þ
þþ




(lateral spine)
Deformation of carpal, metacarpal,

þþ

þþ

±

d



d



phalangeal bones (hands, wrists)
Transient phenomena in the perinatal, infantile periods
Periosteal ‘cloaking’ alongside diaphysesþþof large long bones
Periarticular punctate calcificationsþ þe(prenatal ultrasound)Q9

insufficiently ossified and often triangular (Figure 3). The latterfeature is consistently present in ML II, but absent in some ML IIIa/bpatients. In the anteroposterior thoracic films, the ribs are considerably widened in the lateral and anterior segments, but abnormallynarrow near the vertebral insertion (not shown). The pelvis is mostobviously osteopenic with severely slanted acetabular roofs, narrowbasilar portions of the ilia and relatively large and elongated ischial
and pubic bones, features consistently present in ML II and also ML
IIIa/b. All long bones are short and poorly tubulated with thickcortices and small, coarsely trabeculated epiphyses. The knee
epiphyses are small and almost rectangular. Ossification of the lateral
portion of the tibial submetaphyseal zone is overmodeled. Changes in
the hands and wrists (Figure 4) fit into the severe side of the DM
spectrum. All epiphyses in the tubular hand bones and the carpal
bones remain small and irregular. The metacarpal diaphyses areexcessively widened and ends are constricted. While nearly congruent
with the changes in ML II, the hand skeleton in these intermediate
ML patients differs significantly from the much milder OCD in ML
IIIa/b hand bones (Figure 4). In the longest surviving patients, theradiographic findings are altered by the worsening osteopenia.

aIndependent walking rarely and very transiently achieved.

European Journal of Human Genetics

A novel intermediate ML II/IIIab
JG Leroy et al
6

Figure 3 Lateral radiographs of spinal columns in eight patients with GNPTAB c.10A4C/p.K4Q ML compared with one ML II and two ML III a/b patients.
Thoracolumbar vertebral bodies remain significantly foreshortened with concave frontal and convex upper/lower margins; delayed and deficient ossification.
Similar findings are seen in ML II. In ML IIIa/b vertebral bodies are oblong, and show mild platyspondyly and irregular margins higher anteriorly thanposteriorly. In all types of ML ossification is most deficient in either T12 or L1 or in both, often at the top of thoracolumbar kyphoscoliosis. A. Patient 1 at
8 years; B: Patient 2 at 6 years; C: Patient 3 at 9 years; D: Patient 4 at 19 years; E: Patient 5 at 19 years; F: Patient 6 at 18 years; G: Patient 7 at
7 years; H: Patient 8 at 4 years; ML II: female ML II patient at 4 years; ML III-1: male with ML III a/b at 18 years; ML-III-2: female with ML III a/b
at 13 years.

mutant GNPTAB genotypes for the two fibroblast lines obtained fromthe Coriell Institute.
DISCUSSION
Of the 61 probands included in the report by Cathey et al,1 theclinical features in only a few patients with GNPT-deficient ML
straddled the boundaries of the variability spectra set for delineationof ML II and of ML IIIa/b.1 Upon referral for GNPTAB mutation
screening, all had been diagnosed as ‘intermediate ML’ or ‘ML II/III’.
The majority of these ‘intermediate’ patients was found to have acompound heterozygous mutant genotype that consistently included
c.10A4C/p.K4Q missense mutation in exon 1 and a frameshift
mutation in a downstream exon (Table 1). This group of ML patientshas a distinct and remarkably consistent phenotype that shares
physical (Figures 1 and 2) and radiographic (Figures 3 and 4) featureswith ML II, but more closely resembles the ML IIIa/b phenotype
regarding life expectancy, psychomotor development and degree ofintellectual disability.
European Journal of Human Genetics

The GNPTAB missense (MS) mutation c.10A4C/p.K4Q causes aunique intermediate type ML. The two cytosolic domains of GNPTAB
at the N- and C-termini contain motifs that mediate the export ortrafficking of the enzyme through the endoplasmic reticulum and
Golgi. The importance of the N-terminal domain has been highlighted by recent work from Franke et al15 showing that the lysine
residue at position 4—the same residue mutated in the patients
described in this report—affects a neighboring dileucine sequence
responsible for the export of the GlcNAc-1-phosphotransferase
ab precursor from the endoplasmic reticulum.15 The possibility that
other mutations may be found to result in an identical phenotype
cannot be excluded. It remains to be seen whether other mutations
within the N-terminal domain, or perhaps the C-terminal cytosolic
domain, are associated with the unique clinical and biochemical
features described in the patients reported here. This distinct ML
subtype may be associated with other mutations that directly impact
the cytosolic sorting motifs, a unique alteration of GNPTAB.
Differences within the organization of the secretory pathway andA novel intermediate ML II/IIIab
JG Leroy et al
7

Figure 4 Hand radiographs in GNPTAB c.10A4C/p.K4Q ML patients: comparison with ML II and ML IIIa/b. DM in the hands is similar to that in ML II anddistinctly more severe than in ML IIIa/b. Figures show shortening and diaphyseal widening in all tubular hand bones; severely delayed and deficientossification of epiphyses, carpal bones and the distal ulnar and radial epiphyses; and apparent regression of mid-diaphyseal widening in older patientsconcomitant with progressive generalized osteopenia. A: Patient 1 at 8 years; B: Patient 2 at 4 years; C: Patient 3 at 9 years; D: Patient 4 at 9 years;
E: Patient 5 at 19 years; H: Patient 8 at 6 years; ML II: female with ML II at 4 years; ML III-2: female with ML III a/b at 13 years.

Table 4 GNPT activity for GNPTAB c.10A4C/p.K4Q ML patientsand other patient fibroblast lines
Patient/
cell line

GNPT activity% of averageID(pmol/mg/h)

control activity

Genotype

ClinicalPatient 2

1.79

11.1

p.K4Q/c.1399delG

Intermediate

Patient 5
Patient 8

1.86
1.14

11.6
7.1

p.K4Q/c.1399delG
p.K4Q/c.1399delG

Intermediate
Intermediate

GM00113ML III

3.14
2.13

19.5
13.2

p.K4Q/p.K4Q
p.S399F/‘ML III’a
ML III

affected
GM015860.31

1.9c.3335 þ 6T4G
c.3665_3666delTC/

ML II
Control
Control
Control

Control 1

13.8—

c.3665_3666delTC—

Control 2Control 3

18.5
16.0

—
—

—
—

diagnosis

The three control activities were averaged and used to compare the relative activity in patient
samples. The GNPT activity for GM01586 and Patient 2 represents the average of two
independent experiments. All other activities were single determinations.
aThe GNPT activity result for GM00113 is in good agreement with earlier data from two groups
who analyzed these cells.16,17 Unfortunately, no clinical information could be obtained for thispatient.

the trafficking machinery within certain cell types may lead todifferences in GlcNAc-1-phosphotransferase trafficking. We believe
such differences may help explain the tissue-specific phenotypes seen
within the GNPTAB c.10A4C/p.K4Q ML patients.

Figure 5 Sorting of newly synthesized cathepsin D in control and ML
fibroblasts GM01586 (ML II), patient 2 (K4Q compound heterozygote), and
35S-labeled
GM00113(K4Q
homozygote).
Autoradiographsofimmunoprecipitates of cell and secreted cathepsin D are shown. The
percentage of intracellular enzyme is determined by dividing the amount ofcell-associated cathepsin D by the total cathepsin D (cell-associated andsecreted).

The reduced intracellular and much enhanced plasma activity ofseveral acid glycosidases in this cohort does not differ significantly
from the corresponding data obtained in the reference phenotypes,
ML II and ML IIIab. The GNPT assays (Table 4) confirm earlier
reports in leukocytes and fibroblasts that activity of the causative
enzyme is nearly completely absent in ML II and between 2 and 20%
of normal in ML IIIab.1,8,9,16 The residual GNPT activity in each of
the K4Q compound heterozygous ML lines is higher than that in ML
II-derived fibroblasts and almost matches that in the ML IIIab line.
Remarkably, but not unexpectedly, GNPT activity in the homozygous
European Journal of Human Genetics

A novel intermediate ML II/IIIab
JG Leroy et al
8

K4Q mutant cell line is 19.5% of that in controls and confirms theresults previously obtained by others.17 This observation proves that
the K4Q GNPTAB allele is indeed pathogenic but fairly leaky at leastin cultured fibroblasts. The molecular characterization and traffickingof the enzyme may be further elucidated by additional studies of themechanism by which this mutation alters GNPT activity.
The subnormal yet still effective maintenance of intracellularcathepsin D levels demonstrates the utility of this assay in distinguishing this specific intermediate type ML at least from the ML II
reference phenotype. The fact that the majority of newly synthesizedcathepsin D remains inside the cell in the GNPTAB c.10A4C/p.K4Q
ML patients is surprising, and inconsistent with both the severity ofthe phenotype and the elevated plasma levels of lysosomal enzymes.
However, these patient cells do retain substantial activity towards thesimple sugar substrate, a-methylmannoside. We cannot rule thepossibility that this mutation results in hydrolase-specific effects onlysosomal sorting. Further research on cathepsin-sorting efficiency
may provide insight into the early failure of statural growth, nearly as
severe as in ML II. The robust lysosomal enzyme missorting observedin ML II cells may extend to other cathepsins (for example, cathepsin K)
known to play central roles in skeletal development. The relatively
high efficiency of cathepsin D sorting may explain the moderatecognitive disability associated with this genotype. Roles for cathepsin
D have been suggested in neural development and degeneration, andmaintenance of near-normal intracellular levels of this protease may
confer a protective effect within the brain of these patients.18–20
Delineation of this c.10A4C/p.K4Q GNPT-dependent intermediate ML has more than merely clinical and prognostic significance. The
partial ‘uncoupling’ of physical and cognitive disability represents theapparently rare exception to the rule that nearly complete absence of
GNPT activity yields the ML II phenotype and more residual activity
results in ML IIIab.1 The severe DM, including the transient skeletal
changes in infancy, is compatible with the ML II archetype. However,the specific intermediate type ML we describe is associated with
GNPT activity and extension of life expectancy compatible with the
MLIIIab archetype, and thus illustrates the progressively morbid
clinical course not granted to the ML II patient. Manifestations
include progressive and painful osteopenia, areas of frank osteolysis,
and severe sclerosis in fasciae with contractures in tendons and otherperiarticular structures. Progressive destruction of the weight-bearing
skeleton occurs both in the lower third of the ilia and particularly thefemoral necks, occasionally resulting in pathological fracture. Clinicalmanifestations of osteoporosis are the late consequences of abnormalbone formation and excessive regional overmodeling, compatible withthe observation of increased osteoclast recruitment to bone surfaces insome areas.21 Cyclic infusion of bisphosphonate may offer partial
relief of pain as it is a potent analgesic in painful bone diseases andimproves bone mineral density. The use of bisphosphonates is limitedby the increasing risks for rare complications such as bisphosphonaterelated atypical fractures. The gradual disappearance of the diaphyseal
widening in the metacarpals in long-surviving patient 5 exemplifies
the effects of late progressive overmodeling of qualitatively abnormal
cortical and trabecular bone bone. The qualitative degrading anddysfunction of soft connective tissue adversely affects not only
periarticular and peri-osseous structures, but also bronchopulmonary
elasticity and cardiac valve anatomy and function, ultimately
contributing to lethal outcome.2 Elucidation of the mechanisms bywhich this mutation leads to partial ‘uncoupling’ of the severe physicalimpact and the rather moderate cognitive disability in this specificintermediate ML will expand understanding of pathogenesis of ML
beyond inadequately phosphorylated glycoproteins.
European Journal of Human Genetics

CONFLICT OF INTEREST
The authors declare no conflict of interest.
ACKNOWLEDGEMENTS
Funding has been provided by Award Number U54NS065768 from the
National Institute of Neurological Disorders And Stroke and the Office of Rare
Diseases Research (ORDR) through the Lysosomal Disease Network
Consortium, a part of the NIH Rare Diseases Clinical Research Network(RDCRN), and by the National Institute of General Medical Sciences(GM086524). Additional support has been provided by ISMRD, theInternational Advocate for Glycoprotein Storage Diseases, the National MPS
Society, and by the Genetics Endowment of South Carolina.

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