Supplementary Information
1. Patient phenotype
The proband is the second of three children born to non-consanguineous Caucasian
parents following an uneventful pregnancy. Birth weight was 3.9 kg. She presented in infancy
with mild dysmorphic features and global developmental delay.
On review at age 16 her height is 157.7cm (9-25th centile), weight is 54.8kg (50th
centile) and OFC 57cm (91st-98th centile). She has pronounced facial hypotonia, with an
everted lower lip and protruding tongue (Figure 1A), with drooling. She has a high-arched
palate, and her ears are low-set and protruding with over-folded helices. She has an occipital
cystic scalp lesion with an underlying scalp defect associated depigmentation of the
surrounding hair, as a result of a congenital naevus. She has small hands with short, tapering
fingers which have mild skin syndactyly, and small feet with friable nails. She sustained a
pathological fracture of her right tibia at age 5, which revealed an underlying mono-osteotic
fibrous dysplastic lesion. Ophthalmology examination was normal apart from an astigmatism
in the right eye.
She is globally developmentally delayed and was statemented for educational needs
from the age of 3, and at the age of 16 has severe non-progressive learning difficulties. In
terms of her motor development she walked at 21 months, although was subsequently noted
to fall frequently and has poor fine motor skills, remaining clumsy and uncoordinated. She
has a protuberant abdomen, suggesting a degree of postural muscle weakness, however there
is no clinically detectable limb hypotonia and she has good muscle power and exercise
tolerance. At age 16 she is able to walk long distances, run and ride a bicycle. Her speech
development has been more markedly delayed. Her first words were at the age of 2.5 years,
she was using single words and sign language at age 5 years and short sentences by age 10
years. Her cognitive speech difficulties have been compounded by motor difficulties and she
has been assessed as having severe dysarthria and velo-pharyngeal insufficiency with
significant oral-motor dyspraxia. She required grommets and adenoidectomy as a child but
her hearing is normal. She has poor concentration with a diagnosis of attention deficit
hyperactivity disorder (ADHD), with some improvement on medical treatment.
Investigations have included serial serum creatine kinase measurements which have
been persistently elevated (600-1200 u/l). Whilst initially attributed to her tibial lesion, her
ALP and ALT have remained in the normal range, suggesting a mild myopathy as the more
likely cause of the elevated creatine kinase. A comprehensive metabolic screen was entirely
normal. Magnetic resonance imaging (MRI) of the brain revealed extensive, symmetrical,
non-progressive signal abnormalities in the subcortical cerebral white matter, slightly more
marked in frontal regions (Figure 1B); basal ganglia were normal. A computed tomography
(CT) scan showed no evidence of white matter calcification. Electroencephalography (EEG)
and electroretinography (ERG) were normal however visual evoked response (VER) testing
demonstrated a slightly delayed flash response in the left eye.
All the studies described were carried out as part of fully consented routine diagnostic
testing, and ethical approval was not therefore required.
2. Immunofluorescence
Immunofluorescent microscopy images from a biopsy of the patient’s vastus lateralis muscle.
Neonatal myosin
Fast myosin
Slow myosin
Supplementary Figure 1.
Staining for myosins shows type 1 fibre predominance; occasional staining for
neonatal myosin suggests a small amount of fibre regeneration.
Dystrophin (Dys3)
α-Dystroglycan (core)
α-Dystroglycan (IIH6)
Laminin β2
Supplementary Figure 2.
Staining for dystrophin, α-dystroglycan (both core and IIH6) and laminin β2 shows
essentially normal staining patterns, though the laminin β2 staining was noted as reduced. The
limited quantitative resolution of immunofluorescence means that we cannot reliably
distinguish between 50% and 100% of wild-type levels. Staining was performed as previously
reported1.
3. Quantitative RT-PCR.
Patient muscle RNA was prepared from ~100 mg of vastus lateralis biopsy material by
homogenising in 1 ml of QIAzol (RNA extraction kit, QIAGEN) and subjecting to RNA
extraction according to the manufacturers’ instructions. Human muscle RNA purchased from
Ambion was used as a normal control sample. RNA was quantified using an RNA Nano Chip
on an Agilent 2100 Bioanalyser (Agilent Technologies). Single-strand cDNA was synthesized
from approximately 3 μg of total muscle RNA using a cocktail of gene-specific primers
(dystroglycan, dystrophin and α-dystrobrevin) and M-MLV Reverse Transcriptase (Applied
Biosystems). 2 μl cDNA was used for the first round of nested PCR using gene-specific outer
primer pairs. Second-round nested RT-PCR products were cloned into pCR4-TOPO
(Invitrogen), linearised using NcoI or PmeI (NEB), quantitated using Nanodrop ND-100
(Labtech International), serially diluted and used as standards to control for differing
efficiencies of amplification. Diluted first-round RT-PCR products were used as templates in
a quantitative second round PCR using SYBR green detection in an ABI Prism 7000®
Sequence Detection System. For each amplimer, measurements of threshold cycle (Ct) were
used to infer initial template copy number when compared to Ct measurements of the
respective serially diluted standard. Each datum plotted is the mean of three measurements
(±SD) normalised to α-dystrobrevin and with the normal muscle expression level set to 100%.
Primer sequences are given in Supplementary Tables 1 and 2.
Supplementary Tables 1 & 2. Primer sequences.
Outer
Forward
Reverse
Gene
Name
Sequence
Name
Sequence
Dystroglycan
HumDGFO
cagaggctgtcagggactggg
HumDGRO
ccagccgtgtagcgctcactg
Dystrophin
DysSBSFO
gggctacctgccagtgcagac
DysSBSRO
ggaaatcaagatctgggcagg
Dystrobrevin
ADybE8FO
ggaaatcacctgctaagaagctg
ADybE15RO
ctagctcagcaatcagctgcc
Inner
Forward
Reverse
Gene
Name
Sequence
Name
Sequence
Dystroglycan
HumDGFI
ccatgcactcagtgctctcag
HumDGRI
gtaatgcacacccttatcagtg
Dystrophin
DysSBSFI
ccatggaaactcccgttactctgatcaac
DysSBSRI
ggatcctcactgggcaggactacgaggctg
Dystrobrevin
HumADybE9-F
gctcacatcgtgcctcccag
HumADybE10-E14R
ctcaagcatgctcctggtaa
4. Dystroglycan Amplification and Sequencing
Patient and control DAG1 transcripts were amplified using nested RT-PCR. A single
outer PCR reaction was used, which then served as a template for two overlapping inner PCR
reactions of ~1.5 kb each. Nested RT-PCR was carried out as described above for generating
QRT-PCR standards. Instead of cloning, the resulting PCR products were sequenced directly
using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Inc.) and
BetterBuffer (Microzone Ltd) according to manufacturers’ instructions, followed by
electrophoresis on an Applied Biosystems 3730 DNA Analyser. Inner primers plus two
additional “Seq” primers gave complete sequence coverage of the coding region. All
oligonucleotides were synthesised by MWG-Biotech. The sequence of the patient’s intact
DAG1 transcript was found to be identical to that of nucleotides 726-3506 of GenBank
accession number NM_001165928. Primers sequences are given in Supplementary Table 3.
Supplementary Table 3. Primer sequences.
Forward
Reverse
Reaction
Name
Sequence
Name
Sequence
Outer
Inner 1
Inner 2
Seq
Seq
HumDAG1FO
HumDAG1FI(1)
HumDAG1FI(2)
HumDAG1FI(3)
HumDAG1FI(4)
cctacagttcaggcggatggag
gaacccagctctgggaccaag
cagaagctggcaccacagttcc
ggatgccgacctcaccaagatg
cgagtatttcatgcatgcc
HumDAG1RO
HumDAG1RI(1)
HumDAG1RI(2)
cccaaagtctctccagctgc
ggcagtttccaatctggtgatgg
gacaacaccatgtcgtctccag
1.
Jimenez-Mallebrera C, Torelli S, Feng L et al: A comparative study of alphadystroglycan glycosylation in dystroglycanopathies suggests that the
hypoglycosylation of alpha-dystroglycan does not consistently correlate with clinical
severity. Brain Pathol 2009; 19: 596-611.