Tubular aggregates (TAs) are cytoplasmic aggregates of membranous tubules derived
from the sarcoplasmic reticulum and usually 50–70 nm in diameter [1]. They may be
seen in a range of genetic myopathies, including gyrate atrophy caused by ornithine
aminotransferase deficiency, periodic paralysis and two disorders of glycosylation
caused by mutations in the GFPT1 and DPAGT1 genes [2, 3]. In addition, they may occur
as a minor or inconsistent feature in a wide range of myopathies or be the predominant
feature in idiopathic or congenital myopathies [4]. However, the mechanisms underlying
TA formation remain poorly understood. We now report the development of TAs in a patient
with a lipid remodelling disorder, providing new insight into the formation of TAs.
We report the case of a 13‐year‐old boy with a progressive mitochondrial encephalopathy
in the Leigh syndrome spectrum. He is the first child of healthy unrelated Bengali
parents and has an unaffected younger sister. He initially presented on day one of
life with transient neonatal lactic acidosis and hyperammonaemia, which resolved with
conservative management. Muscle biopsy in the neonatal period revealed undetectable
activity of succinate‐cytochrome c reductase [respiratory chain (RC) complexes II + III],
with normal activities of complexes I and IV (Table 1). By 6 months, poor feeding
and growth were apparent, and at 18 months, he was treated with hearing aids for bilateral
sensorineural hearing loss (SNHL). At 2 years, he developed challenging behaviour,
associated with developmental regression. From 3 years, he had recurrent chest infections,
progressing to bronchiectasis. At 6.5 years, he had an episode of encephalopathy with
status dystonicus requiring invasive ventilation. At 9 years, Nissen fundoplication
was performed to treat severe gastro‐oesophageal reflux, and a Percutaneous Endoscopic
Gastro‐Jejunostomy (PEG‐J) was inserted. Muscle biopsy was repeated at 12 years at
the time of a gastrostomy revision and revealed that RC activities were now essentially
normal, other than borderline reduction of cytochrome c oxidase (COX, complex IV)
activity (Table 1). RC activities were normal in cultured skin fibroblasts (Table 1).
Now 13 years, he has microcephaly, optic atrophy, severe dystonia and self‐harm involving
biting and scratching. He receives prophylactic antibiotics to prevent further respiratory
infections and requires regular suction and intermittent face mask oxygen. The results
of metabolic investigations are summarised in Table 1. Neuro‐imaging demonstrated
abnormal signal in the striatum bilaterally and associated atrophy in the caudate
heads.
Table 1
Metabolic investigations in a patient with SERAC1 deficiency
Investigation
Results
Blood lactate
Ranged from 5.7–7.4 mmol/L (reference range 0.7–2.1)
CSF lactate
2.0 mmol/L (normal <2)
Urine organic acids
Grossly raised lactate, pyruvate and 2‐hydroxybutyrate with moderately raised 2‐hydroxyisovalerate
and mildly raised 2‐oxo‐isocaproate.
Strongly raised 3‐methylglutaconate with moderately raised 3‐methylglutarate
Plasma amino acids
Increased levels of
Alanine 587 μmol/L (150–450)
Glutamine 1143 μmol/L (480–800)
Tyrosine 394 μmol/L (30–120)
Proline 569 μmol/L (85–290)
Fibroblast enzyme activities
Pyruvate dehydrogenase activity 0.85 nmoles/mg
Protein/min (reference range of 0.7–1.1)
Complex II + III 0.146 (0.07–0.243)
Complex IV 0.016 (0.007–0.036)
Muscle respiratory chain enzyme activities
At age 15 days
Complex I 0.101 (0.104–0.268)
Complex II and III undetectable (0.040–0.204)
Complex IV 0.015 (0.014–0.034)
At age 12 years
Complex I 0.149 (0.104–0.268)
Complex II + III 0.103 (0.040–0.204)
Complex IV 0.013 (0.014–0.034)
CSF, Cerebrospinal fluid.
2014 British Neuropathological Society
A biopsy taken at 15 days of age from the left quadriceps did not show significant
diagnostic abnormalities on routine histochemical stains including oxidative stains
[Nicotinamide adenine dinucleotide tetrazolium reductase (NADH‐TR), COX and succinate
dehydrogenase (SDH)] or fibre typing stains (Figure 1
a–d). Furthermore, ultrastructural examination only revealed prominent nonmembrane‐bound
sarcoplasmic glycogen but no abnormalities of mitochondrial ultrastructure (Figure 1
e).
Figure 1
Muscle biopsies taken at 15 days (a–e) and 12 years of age (f–i). At 15 days, the
biopsy did not show significant diagnostic abnormalities (a, H&E; b, Gömöri trichrome;
c, Oil red O; d, NADH‐TR). Ultrastructural examination revealed prominent nonmembrane‐bound
glycogen in the sarcoplasm (e). Specifically, there were no tubular aggregates. In
contrast, at 12 years of age, there were small, predominantly angular, fibres and
scattered fine vacuolation and occasional deposits highlighted by the Gömöri stain
(f,
H&E; g, Gömöri trichrome). There was excess lipid for the age (h, Oil red O). There
was a little granularity on NADH‐TR staining (i) but without well‐defined aggregates.
Histochemical staining for adenylate deaminase (j) showed scattered dense staining
structures (arrow). Electron microscopy revealed very frequent collections of tubular
aggregates, many of which are relatively small (indicated by the circles) (k). These
showed a typical pattern of concentric double‐lumen tubules (l). Scale bars, a, b,
c, d, f, g, h, i, j: 50 μm; e: 10 μm; k: 4 μm; l: 100 nm.
figure
2014 British Neuropathological Society
In contrast, a right quadriceps biopsy taken at 12 years showed a number of abnormalities
including some small, predominantly angular, fibres (down to approximately 5 μm) (Figure 1
f). There were occasional internal nuclei, but these were not a prominent feature.
There was no fibre necrosis, regeneration or splitting. There was a fine vacuolation
associated with Gömöri‐positive material but no ragged red fibres (Figure 1
g). In a few areas, larger deposits of Gömöri‐positive material were noted. There
was prominent lipid staining on Oil red O (Figure 1
h). There was some coarse staining on the NADH‐TR stain, but staining for SDH and
COX was normal (Figure 1
i). Fibre typing with immunohistochemistry for fast and slow myosin showed fast fibre
predominance. There were small fibres of both types. A few fibres stained for neonatal
myosin. We considered the possibility that the abnormality of fibre typing may have
represented either a neurogenic component or a selective loss of slow fibres. However,
apart from angular atrophic fibres, specific features to distinguish these possibilities
were not seen. Histochemical staining for adenylate deaminase showed scattered dense
aggregates of staining (Figure 1
j). Staining for SERCA1 and SERCA2 showed a fibre‐type pattern but did not reveal
significant aggregates. Electron microscopy showed frequent subsarcolemmal TAs composed
of parallel collections of concentric double‐walled tubules (Figure 1
k,l). The aggregates were very frequent but each individually was relatively small,
which may explain the difficulty of recognizing them on histochemical stains. The
excess lipid noted histochemically was confirmed on electron microscopy, but there
were no abnormalities of mitochondrial structure.
Genetic studies were performed with ethical approval from the National Research Ethics
Committee London Bloomsbury, UK. In view of the low complex II + III activities in
the first biopsy, the MT‐CYB and BCS1L genes were sequenced; no pathogenic mutations
were identified. Full mitochondrial genome sequencing was performed in muscle from
the second biopsy and was also normal. A whole exome sequencing approach was then
utilised to further investigate presumed autosomal recessive Leigh syndrome, using
the Illumina HiSeq platform. Raw fastq files were aligned to the hg19 reference genome
using Novoalign version 2.08.03 (Novocraft Technologies Sdn Bhd, Malaysia). For this
case sample, as well as a collection of 2600 in‐house control samples processed together,
we created gVCF files using the Haplotype Caller module of the Genome Analysis Tool
Kit (gatk) version 3.1.1 (Broad Institute, USA). These individual gVCF files were
combined into combined gVCF of 100 samples, which were then used for variant calling
(using the GenotypeGVCF module of gatk 3.1.1). Variants quality scores were recalibrated
according to GATK best practices separately for indels and single nucleotide polymorphisms.
Rare variants (defined as an allele frequency <0.5% in‐house control samples) which
were nonsynonymous, presumed loss of function or splicing were prioritized. Of 21 388
exonic calls, eight were rare homozygous, and 23 genes contained compound heterozygous
calls. In the mitochondrial gene SERAC1 (serine active site containing 1), encoding
a putative phosphatidylglycerol remodelling protein, we identified a known homozygous
pathogenic splice mutation c.1403 + 1G>C in exon 14. This mutation leads to a skipping
of exon 13 and nonsense mediated mRNA decay [5]. Both parents were heterozygous for
this mutation, confirming segregation with disease in this family.
SERAC1 encodes a phospholipase which has been postulated to be involved in cholesterol
trafficking and lipid remodelling at the Mitochondria‐associated endoplasmic reticulum
membranes (ER‐MAM) interface [5]. The presence of TAs in our patient's muscle at 12
years but not in his neonatal biopsy suggests that sarcotubular structure has altered
in the intervening period, which we hypothesise directly results from inefficient
lipid remodelling as a consequence of the SERAC1 mutations, leading to aggregation
of ER constituents. This hypothesis is supported by the presence of TA‐like structures
in Chinese hamster ovary cells overexpressing 3‐hydroxy‐3‐methylglutaryl‐coenzymeA
reductase, an ER enzyme involved in cholesterol biosynthesis [6]. Delayed formation
of TAs in our patient fits with previous observations that TA formation is slow in
vivo, typically months in mice [7], which may equate to years in humans. Previous
reports of EM in patients with SERAC1 mutations noted abnormal mitochondrial architecture
in liver [8] and muscle [5] but not TAs. However, all previously reported muscle biopsies
appear to have been performed in infancy or very early childhood [5, 8, 9, 10], which
likely explains the absence of TAs in previous cases. Therefore, this is the first
report of the natural course of muscle pathology in SERAC1 deficiency. SERAC1 deficiency
is one of a growing family of phospholipid biosynthesis and remodelling disorders,
which may affect the heart, skeletal muscle, brain or peripheral nerves [11].
The variable RC deficiencies observed in SERAC1 deficiency, with normal enzyme activities
in some individuals including the second biopsy in the patient reported here, mean
that abnormal RC activities cannot be used as a screening test for this disorder.
Rather, the presence of a Leigh‐like encephalopathy with SNHL and 3‐methylglutaconic
aciduria should trigger clinical suspicion of 3‐methylglutaconic aciduria with deafness,
encephalopathy and Leigh‐like syndrome (MEGDEL syndrome), and sequence analysis of
SERAC1 should be performed. Moreover, our findings suggest that energy deficiency
is not the primary pathogenic mechanism in SERAC1 deficiency and that abnormal subcellular
aggregate formation as a result of disordered lipid membrane remodelling may be more
important in the multisystemic pathogenesis of this disorder, including deafness and
progressive encephalopathy. Furthermore, based on the evidence presented here, we
suggest abnormal lipoprotein aggregation may be a common pathogenic mechanism in disorders
of phospholipid biosynthesis and remodelling.