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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_2984_Библиотеки_им_академика_М_И_Перельмана
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Fedah E. Mohamed, Jozef Hertecant, Fatma Al-Yahyai et al.
12
premature codon. At the protein level, the frame shift starts from 1167
amino acid for up to four amino acids before the premature stop codon that
results in the loss of 86 amino acids compared to the wild type protein
sequence (Figure 2). Enzymatic analysis using patient’s sample confirmed
ML II which was consistent with the patient clinical presentation and the
diseases progression.
4.2. Family 2
A 23-month-old male Pakistani child born to a consanguineous
marriage was clinically diagnosed with type II ML. He presented with
coarse facial features and developmental delay. Parents are first cousin,
had three miscarriages due to intrauterine death of unknown reason (Figure
3). Paternal uncle died at two years as result of high fever, suspected to
have ML. The proband has a five years old female sibling who is clinically
healthy but living with hearing aid.
Figure 3. Family 2 pedigree.
4.2.1. Clinical Presentation and Phenotype
The proband is 23-month-old with confirmed diagnosis of ML II with
initial clinical presentation to metabolic service at 18 months of age. He
presented with coarse facial features and developmental delay. Prenatal
history revealed decreased Fetal movement at 7 months of gestation.
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Mucolipidosis Disease in the United Arab Emirates
13
abnormal bone structure of fetus was noted in the last trimester and mother
informed that he may die in utero. At term, he arrived with spontaneous
vaginal delivery and directly admitted to NNU for 21 days. He required
phototherapy for a week due to jaundice secondary to ABO
incompatibility. Developmental history; fix and follow at four months of
age but no head control. At 10 months of age, started to roll over “half a
turn” and sits only with support while he started to hold his hands together
at the age of 12 months. He was noticed to have a pincer and palmar grasps
at 14 months of age and started babbling around 17 months of age. No
history of developmental regression. He presented with recurrent URTI,
snoring all night and sometimes sleep apnea. On physical examination:
coarse facial with thick eyebrows, no corneal clouding long eye lash,
depressed nasal bridge, gum hypertrophy, high arched palate, claw hands,
short neck, and contracture of multiple joints (shoulders, elbows, wrists,
hips and knees). He had nail clubbing, thick pale skin with papular rash
over the lower back, trunk and between scapula. Hepatosplenomegaly and
heart murmur were noted on examination. He had several hospitalizations
because of recurrent chest infections. There was no history of recurrent
otitis media. Echo showed thickened aortic valve, causing mild aortic
insufficiency.
4.2.2. Biochemical and Molecular Analysis
Clinically patient was suspected to have mucopolysaccharidosis type II
but iduronate-2-sulphatase activity was within the normal range.
Lysosomal enzymes activities were below the normal range indicating that
the proband is affected by mucolipidosis type II/III. The full sequencing of
GNPTAB sequencing detected a dinucleotide deletion in exon 19 of the
gene which the similar variant reported in Family 1. The detected deletion
interferes with the normal reading frame resulting in a frame shift mutation
that lead to the generation of a premature codon. At the protein level, the
frame shift starts from 1167 amino acid for up to four amino acids before
the premature stop codon that results in the loss of 86 amino acids
compared to the wild type protein sequence (Figure 2).
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Fedah E. Mohamed, Jozef Hertecant, Fatma Al-Yahyai et al.
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4.3. Family 3
The proband is a four-year-old Egyptian female born to non-
consanguineous parents (Figure 4A). She was presented with coarse facial
features, short stature, and severe skeletal deformities with joint
restrictions. The clinical examination predicted ML II/III which was
confirmed by GNPTAB/GNPTG genes sequencing and biochemical
analysis of lysosomal enzymatic analysis.
4.3.1. Clinical Phenotype and Symptoms
The proband presented to our metabolic service at 28 months of age
with coarse facial features, short stature, severe skeletal deformities with
joint restrictions which affected her range of motion. It was noted by her
parent at 15 months of age, mother also reported a change in lower back
since birth with bulging deformity. Family sought medical attention and
seen by several physicians in Egypt and UAE. Suspected to have MPS
based on physical and radiological findings of dysostosis multiplex but
Urine MPS screening was negative.
She had history recurrent URTI with adenoid hypertrophy and sleep
disturbance (snoring). Developmental assessment at 28 months of age;
gross motor: sitting with support at 7 months, roll over at 12 months,
walking without support at 18 months, at 28 months could not walk up or
downstairs, can throw and kick a ball (but weak as per parent), fine motor:
good pencil grasp, scribbles but cannot draw a line or other shapes. Speech
and language: cannot say a sentence of more than 2 words, follow simple
command. On physical examination: coarse fascial features, no corneal
clouding, macrocephaly, frontal bossing, protruding eyeballs, low set ears,
long philtrum, gingival hypertrophy, wide spaced teeth, poor dentition,
high arch palate, short neck, chest wall deformity. Systolic heart murmur
grade II-III. hepatomegaly on abdominal examination, contracture of
elbow, hands, knee and shoulders with limitation of range of movement.
Clubbing of nails and toes, back deformity with gibbus. shuffling gait.
Head CT: mildly dilated ventricular system, mild dilated subarchnoid CSF
spaces, thinning of periventricular white matter on both sides particularly
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Mucolipidosis Disease in the United Arab Emirates
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in peritrigonal region. Echo of heart showed intraventricular septum
hypertrophy with mild Left Ventricular obstruction. Proband died in Egypt
at 4 years of age due to respiratory illness.
4.3.2. Biochemical and Molecular Analysis
For diagnostic purposes, both GNPTAB and GNPTG genes coding
regions and ~50 bases of flanking noncoding regions were sequenced and
aligned to reference sequences. GNPTG gene sequencing was negative. In
contrast, GNPTAB gene sequencing showed a homozygous point mutation
at position 1567 (c.1567T > C) that is predicted to result in an amino acid
substitution at the protein level at position 523 (p. Cys523Arg) (Figure
4B). The wild type cysteine at the stated position was found to be highly
conserved among multiple eukaryotic species indicating its importance to
the underlying protein function and/or structure (Figure 4C).
Table 2. Lysosomal enzymes activities in patient’s fibroblast cell lysate
Nmol/min/mg protein
Enzyme
Activity
Normal range
Effect
Beta-glucuronidase
0.4
(2.0 – 20.0)
↓
Alpha-fucosidase
0.1
(0.8 – 6.0)
↓
Alpha-mannosidase
0.2
(0.9 – 6.0)
↓
Beta-hexosaminidase
1.7
(6.3 – 50.0)
↓
Arylsulphatase A
< 0.1
(5.0 – 25.0)
↓
Alpha-N-acetylgalactosaminidase
1.0
(0.9 – 6.0)
↓
Acid phosphatase (Normal control)
49
(14.0 – 50.0)
Normal
For further confirmation of clinical diagnosis, patient fibroblasts were
subjected for lysosomal enzymatic activity analysis of a group of different
hydrolases. The analysis showed that the level of enzyme activities of all
the tested lysosomal enzymes in fibroblast are either at or below the
bottom of the respective normal range (Table 2). In contrast, the level of
the control enzyme (acid phosphatase) is clearly within the normal range.
In addition, urine analysis of the proband revealed oligosaccharide pattern
that is consistent with ML II/III.
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Fedah E. Mohamed, Jozef Hertecant, Fatma Al-Yahyai et al.
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Figure 4. ML III family 3 characterization. A) Family 3 pedigree. Affected individuals’
symbols are filled with black. Proband is marked by an arrow. B) Wild type and
mutated GNPTAB gene and N-acetylglucosamine-1-phosphate transferase subunits
alpha and beta protein sequences. Nucleotides and amino acids at the mutation
positions are highlighted in yellow. C) Conservational analysis via multiple sequencing
analysis performed via multiple sequencing alignment.
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Mucolipidosis Disease in the United Arab Emirates
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4.4. Family 4
The proband is a two-year-old female born to first cousin Emirati
couple (Figure 5). She was presented with claw hand and restriction of
joint mobility especially the hip joint. The rest of her examination was
unremarkable. No corneal clouding, or organomegaly. Sanger sequencing
and clinical presentation confirmed that the patient is affected with ML III.
Despite the milder form of ML, the proband was found to carry the same
GNPTAB variant detected in Family 3. Detailed family history revealed
three of her paternal aunts and one uncle are affected (Figure 5).
4.4.1. Clinical Symptoms/Phenotype
Decreased range of motion of joints was noted by grandmother earlier
around one to two years of age as she became more observant for the
subtle features. Contractures of the hands were evident between 2 and 4
years of age. The patient with the late onset of skeletal symptoms began
complaining of joint stiffness and loss of flexibility at 7 years. Regarding
the developmental milestone, fine motor delay was noted in the male
patient. The oldest patient had bilateral hip replacement in her late teens.
The rest of clinical findings of the patients studied are presented in Table 3.
The time of diagnosis for this family ranged from 2 year to 26 years.
4.4.2. Biochemical and Molecular Analysis
In addition to the clinical examination, complete sequencing of the
GNPTAB gene in the affected individual revealed novel homozygous point
mutation at position 1567 (c.1567T > C) which at the protein level results
in a the substitution of the wild type cysteine to arginine at position 523
(p.Cys523Arg) (Figure 4B). The mutated amino acid at this position was
found to be highly conserved among multiple eukaryotic species as
indicated previously via multiple sequencing alignment (Figure 4C).
Serum lysosomal enzymatic analysis of probands serum samples were
10-30 folds higher than normal control (Table 2). While, in leukocytes
enzymatic activities were in borderline low/normal.
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Fedah E. Mohamed, Jozef Hertecant, Fatma Al-Yahyai et al.
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Figure 5. Family 4 pedigree. Affected individuals’ symbols are filled with black.
Proband is marked by an arrow.
Table 3. Clinical findings of affected individuals in Family 4
Patient #
Sex
Age
Clinical manifestations
Development
Other features
1
F
2 y
Claw hand, restriction of
joint mobility of hip,
shoulder
Normal
-
2
F
26 y
Bilateral hip
replacement, Carpal
tunnel syndrome
College
graduate
Inguinal hernia,
hearing loss
3
F
22 y
Bilateral femoral head
necrosis, restriction of
hip movement, claw
hand, Scoliosis
College student
Inguinal hernia,
coloboma
4
F
24 y
Right hip necrosis
Restriction of elbow, hip
mobility, claw hand
College
graduate
Inguinal hernia,
umbilical hernia
mongolion spot
5
M
4y
Claw hand
Delay in fine
motor skill
Extensive mongolion
spot
4.5. Family 5
An 18 years old Emirati female from Al-Balushi tribe born to a
consanguineous marriage has been diagnosed with ML III (Figure 6A).
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Mucolipidosis Disease in the United Arab Emirates
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The family has another four daughters of which is an eight years old
female who is also affected with the same phenotype. The proband is
mainly showing multiple joints contractures since early life for which she
sought medical evaluation. The father has been diagnosed with Langerhans
cell histiocytosis.
Figure 6. Family 5 ML III characterization and in silico analysis. A) Family pedigree
where proband is marked by the black arrow. Filled shapes indicates affected
individuals. B) in silico illustration of the detected variant in intron 7 of GNPTG gene
and its effect at the protein level. Blue boxes resemble the gene exons around the
detected mutation which is highlighted in green in both DNA and protein sequences.
Wild type residues at the position of the detected change are highlighted in yellow.
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Fedah E. Mohamed, Jozef Hertecant, Fatma Al-Yahyai et al.
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4.5.1. Clinical Phenotype and Symptoms
The proband presented mainly with musculoskeletal manifestations
with major joint pain and noticeable decreased range of motion. At the
time of clinical examination, she appeared well nourished, well developed,
not dysmorphic, noted to have pectus excavatum, short neck restriction
shoulder movements in all directions, restriction of elbows extension. A
bilateral hip internal and external movements were restricted within
minimal levels. Neurological examination unremarkable.
4.5.2. Biochemical and Molecular Analysis
The full sequencing of GNPTG gene revealed a previously reported
splice site mutation in intron 7 (IVS7-10G > A) of the gene by detecting a
substitution mutation at position 11120 (g.11120G > A) [3]. This
transitional mutation occurred ten nucleotides upstream from the 30-splice
site of intron 7 generating a new cryptic acceptor splice site resulting in the
retention of eight nucleotides in intron 7 (Figure 6B). The retention of
these nucleotides leads to a change in the open reading frame and result in
a frame shift mutation and a premature truncation at the protein level
(Figure 6B). The corresponding translation product contains a premature
stop codon after 18 amino acids (p.V176GfsX18) resulting in a predicted
truncated protein of 194 amino acid which was confirmed via Pohl et al,
through western blot analysis [3].
4.6. Family 6
The proband (patient 1 of the IV generation) is a five-year-old male
born to first cousin parents of a Pakistani origin (Figure 7A). He was
clinically diagnosed with ML III at the age of two years due to an insertion
mutation in the GNPTG gene lead to a major increase in multiple serum
acid hydrolases. Screening of the rest of family members for the detected
variant showed that two of siblings are affected as well as first male cousin
(Figure 7A).
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Mucolipidosis Disease in the United Arab Emirates
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Figure 7. Family 6 MLIII characterization. A) Family pedigree where affected
individuals’ symbols are black filled and the proband is marked by an arrow. B) Image
of affected proband at the age of five years showing the restriction of shoulder
movement with no coarse facial features noted. C) Image of the proband’s affected
sibling (Patient 2) showing the contracture of inter- phalangeal joints.
4.6.1. Clinical Phenotype
He was first presented at two years of age with swelling and
progressive stiffness in his wrists and finger joints. At five years of age no
facial dysmorphism, the patient shows no signs of mental retardation. The
patient’s weight falls within the 10- 25
th
centile and his height at the 10
th
centile. Ophthalmological examinations did not show any corneal
clouding. No organomegaly on abdominal examination. Normal cardiac
echo exam. Musculoskeletal examination showed finger contracture and
reduced joint mobility of elbow, shoulder, hip and knee (Figure 7B, C).
The Skeletal survey showed dysostosis multiplex. Urine MPS was positive
with increase in dermatan and heparan sulphate. The proband was
independent in his ambulation however his affected cousin was wheelchair
dependant. The youngest sibling (one month) was asymptomatic at the
time of diagnosis with no skeletal manifestation. The hand x-ray showed
flexion contracture of interphalangeal joints more specifically the distal IP.
Carpal bones are smaller than usual. Multiplanner MRI scans of the hips
showed dysplastic changes in the form of flattening of the humeral head
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