Biochemical, clinical and molecular findings in LCHAD and general mitochondrial trifunctional protein deficiency

Biochemical, clinical and molecular findings in defects of mitochondrial
trifunctional protein

S. E. OLPIN 1*, S. CLARK 1, B. S. ANDRESEN 2, C. BISCHOFF 2, R. K. J.
OLSEN2, N. GREGERSEN 2, A. CHAKRAPANI 7, M. DOWNING 1, N. J. MANNING 1, M.
SHARRARD 3, J. R. BONHAM 1, F. MUNTONI 4, D. N. TURNBULL 5, M. POURFARZAM
6.

1Dept. of Clinical Chemistry and 3Dept. of Paediatrics, Sheffield
Children's Hospital, UK 2Research Unit for Molecular Medicine, Aarhus
University, Denmark, 4Deptartment of Paediatrics and Neonatal Medicine,
Hammersmith Hospital, London, UK, 5School of Clinical Neurosciences,
University of Newcastle, UK, 6James Spence Institute of Child Health, Royal
Victoria Hospital, Newcastle, UK, 7 Department of Paediatrics, Birmingham
Children's Hospital, Birmingham, UK

*Correspondence: Department of Clinical Chemistry, Sheffield Children's
Hospital, Sheffield S10 2TH, UK


Summary: General mitochondrial trifunctional protein TFP deficiency leads
to a wide clinical spectrum of disease ranging from severe
neonatal/infantile cardiomyopathy and early death to mild chronic
progressive sensorimotor polyneuropathy with episodic rhabdomyolysis.
Isolated long chain 3-hydroxyacyl-CoA dehydrogenase LCHAD deficiency
resulting from the common E474Q mutation usually gives rise to a moderately
severe phenotype with multi-organ involvement with a high morbidity and
mortality. However, isolated LCHAD deficiency can also be consistent with
long term survival in patients identified and treated from an early age. We
present biochemical, clinical and mutation data in 9 patients spanning the
full spectrum of TFP disease.
Fibroblast acylcarnitine profiling shows good correlation with clinical
phenotype using the ratio C18(OH) / C12(OH) + C14(OH). This ratio shows a
gradation of values, from high in four patients with severe neonatal
disease (2.5 ( 0.8), to low in two neuromyopathic patients (0.35, 0.2).
Fibroblast fatty acid oxidation flux assays also show correlation with the
patient phenotype, either when expressed as percentage residual activity
with palmitate or as a ratio of percentage activity of myristate / oleate
(M/O ratio). Fibroblasts from four patients with severe neonatal disease
giving an M/O ratio of 4.0 ( 0.6 compared to 1.97 and 1.62 in two
neuromyopathic patients. Specific enzyme assay of LCHAD and long-chain 3-
ketothiolase LKAT activity in patient cells shows lack of correlation with
phenotype, probably due to instability of the mutant proteins in disrupted
cells. These results show that measurements in intact cells, which allows
all determinative and modifying cellular factors to be present, better
reflects patient phenotype. Mutation analysis reveals a number of ( and (-
subunit mutations.
A notable feature of TFP deficiency is respiratory distress, either in
the neonatal period or in older surviving infants. Patients who are
affected with viral bronchiolitis may have a particularly poor outcome.
This probably reflects disrupted surfactant synthesis as a consequence of
interference from hydroxyacyl metabolites.
Peripheral sensorimotor polyneuropathy often as the initial major
presenting feature but usually later accompanied by episodic
rhabdomyolysis, is a manifestation of mild TFP protein deficiency. The mild
clinical presentation and relative difficulty in diagnosis suggests that
this form of TFP is probably under-diagnosed.


INTRODUCTION


Mitochondrial trifunctional protein TFP is a heterooctamer of four (- and
four (- subunits and this multi-enzyme complex catalyses the last three
steps in (-oxidation of long chain fatty acids. The (-subunit contains the
long-chain enoyl-CoA hydratase and LCHAD activities and the (-subunit
contains the LKAT activity.
Isolated LCHAD deficiency (McKusick 600890) is associated with the
common (-subunit mutation E474Q that is located at the catalytic site of
the LCHAD domain. This mutation results in intact mutant protein but with
significantly reduced LCHAD activity, the activity of the other two enzymes
remains at >60% of normal. Patients with LCHAD deficiency usually exhibit
moderate/severe multi-organ involvement, often with cardiomyopathy. There
is generally a high mortality and morbidity but the disorder can also be
consistent with long term survival in patients identified and treated from
an early age (Tyni et al 1997; Den Boer et al 2002).
General mitochondrial trifunctional protein TFP deficiency was first
described by two groups independently in 1992 (Wanders et al ; Jackson et
al). This disorder is defined as a reduction in the activity of all three
enzymes, and is due to heterogeneous mutations in either one of the TFP
genes encoding the (-subunit (McKusick 600890) and the (-subunit (McKusick
143450) (Ushikubo et al 1996; Schaefer et al 1996; Orii et al 1997; Ibdah
et al 1998; Matern et al 1999; Hintz et al 2002; Spiekerkoetter et al 2003,
2004a).
Patients with TFP deficiency exhibit a wide clinical spectrum of
disease with severe neonatal manifestation including cardiomyopathy and
death, through moderate/severe infantile presentation with hepatic
manifestations to mild peripheral neuropathy with episodic rhabdomyolysis
(Spiekerkoetter et al 2003, 2004a, 2004b). We have investigated 9 patients
spanning the full clinical spectrum of disease and demonstrate correlation
of biochemical indices with patient phenotype.


MATERIALS AND METHODS


Fibroblast culture and measurement of (-oxidation flux in cultured
fibroblasts using [9,10-3H]myristate, [9,10-3H]palmitate and [9,10-
3H]oleate was by the methods of Manning and colleagues (1990) and Olpin and
colleagues (1992, 1997). Specific enzyme analysis in fibroblast sonicates
was by the method of Wanders et al (1990). Mutation analysis was
…………………………..
Acylcarnitine profiling of cultured fibroblasts was essentially by the
method of Pourfarzam et al (1994) with minor modification. Unlabelled
palmitic acid was used as substrate and incubations were performed using
intact fibroblasts monolayer culture. Quantitation of long-chain (ie C12-
C18), saturated, unsaturated and hydroxyacylcarnitines was achieved by
reference to [methyl-2H9]palmitoylcarnitine internal standard .

MUTATION METHODOLOGY PLEASE

RESULTS

Fibroblast acylcarnitine profile analysis shows good correlation with the
phenotype using the ratio C 18(OH) / C14(OH) + C12(OH) (see tables 1 & 3).
Four patients with severe neonatal disease having ratios of 2.5 ( 0.8
(range 1.7 – 3.6). Three patients with moderate/severe phenotypes having a
mean acylcarnitine ratio of 1.1 ( 0.12 significantly different to the
values of 0.20, 0.35 in two neuromyopathic patients. Fibroblast fatty acid
oxidation flux expressed as % palmitate oxidation and the ratio of %
residual activity of myristate / oleate (M/O ratio) also show correlation
with clinical phenotype (table 1). Plotting the acylcarnitine ratio against
% palmitate flux shows good separation of the three phenotypes (figure 1).
Fibroblasts from four patients with severe neonatal disease gave a mean %
palmitate residual activity and M/O ratio of 26.0 ( 3.5 and 4.0 ( 0.6
respectively as compared to three moderate/severe phenotypes with
corresponding values of 50 ( 11 and 2.5 ( 1.0 respectively. However patient
5 gives an M/O ratio that overlaps with the severe infantile group. Both
palmitate % flux and M/O ratios fail to discriminate between the
moderate/severe patients and the 2 mild neuromyopathic patients (table 1).
Specific enzyme assay in patient cells shows lack of correlation with
phenotype (table 1) see discussion.
Clinical and biochemical information for each patient is shown in
table 2. Patients 1-4 & 6 have previously been described (Chakrapani et al
2000). These results demonstrate that urine organic acid analysis is often
unreliable as a means of detecting TFP deficiency in both severe neonatal
and neuromyopathic patients. Blood acylcarnitine profiling however reliably
detects severe and moderate/severe TFP deficient phenotypes but is
unreliable in neuromyopathic patients. Mutation analysis in the MTF
patients revealed a number of (-subunit and (-subunit mutations (table 1).

DISCUSSION

Fibroblast acylcarnitine profiling and expression of the results as C
18(OH) / C14(OH) + C12(OH) provides good correlation with patient phenotype
and can be usefully employed as a measure of prognosis in TFP deficiency.
Plotting the fibroblast acylcarnitine ratio against % palmitate oxidation
shows separation of these three phenotypic groups (figure 1). Patient 5
(E747Q homozygote) with isolated LCHAD deficiency was included in this
study for comparison with general TFP deficiency. Although LCHAD patients
may exhibit multi-organ involvement with moderate/severe disease that may
result in early death, the disorder can also be consistent with a good
clinical outcome in well managed patients treated from an early age. This
is exemplified by patient 5 in this series. Despite at least two episodes
of severe decompensation with cardiomyopathy and rhabdomyolysis as an
infant, he is now a well 14 year old and remains on a strict dietary
regimen. At 14 years of age this patient has a normal IQ, height and
weight, and actively participates in sport, having swum a kilometer for
charity at the age of 11 years. He does however have a mild but non-
progressive pigmentary retinopathy. Fibroblast acylcarnitine profiling in
patient 5 gave a C 18(OH) / C14(OH) + C12(OH) ratio of 1.2. This was
similar to the two patients with general TFP deficiency who presented
outside the neonatal period and who did not have cardiomyopathy on
presentation, see table 3. Although both these moderate/severe patients
died during an infection of viral bronchiolitis, they had previously been
either completely asymptomatic for the first nine months of life (patient
7), or had initially responded well to dietary intervention and had not
shown any evidence of cardiomyopathy (patient 6) see table 2. This group of
moderate/severe patients which includes isolated LCHAD deficiency is almost
certainly a clinically distinct group as compared to the severe neonatal
patients. This latter group generally develop cardiomyopathy at an early
age (3 of 4 patients in this series) and the prognosis for medium term
survival, even with early treatment is extremely poor (den Boer et al 2003;
Spierkerkoetter et al 2004b). The fibroblast acylcarnitine ratio in these 4
severe neonatal patients was 2.5 ( 0.8 vs. 1.1 ( 0.12 in the 3
moderate/severe patients as compared to 0.20 and 0.35 in two neuromyopathic
patients.
The comparison of fatty acid oxidation using % palmitate flux activity
against phenotype shows a degree of correlation. Percentage palmitate
activity in severe neonatal patients having lower values than the other two
groups. The M/O ratio is also higher in all the severe infantile patients
with the exception of patient 5 which shows overlap with this group.
Specific enzyme assay in fibroblast sonicates of both LCHAD and LKAT
activity shows lack of correlation with the patient phenotype (table1).
This has been previously reported (Spiekerkoetter et al 2003). As with the
study of Spiekerkoetter and colleagues, these specific enzyme assays were
performed over an extended period of time, and therefore may suffer from
increased inter assay variability. It is also known that significant
residual activity of the LCHAD enzyme using 3-keto-palmitoyl-CoA as
substrate is due to matrix short-chain hydroxyacyl-CoA dehydrogenase
activity which has overlapping substrate specificity with TFP (Jackson et
al 1991). However, it seems most probable that the major reason that
specific enzyme activity does not correlate with phenotype is that mutant
proteins show variable instability in disrupted cells. The activities of
both LCHAD and LKAT were significantly reduced in all patients with the
exception of patient 5, where only the LCHAD activity was reduced. This
patient has the common E474Q mutation and would therefore be predicted to
have LKAT activity of >60%. The low activity of both LCHAD and LKAT in the
other 8 patients clearly defines them as suffering from general TFP
deficiency.
In conclusion these fibroblast studies indicates that measurements in
intact cells, which allows all determinative and modifying cellular factors
to be present, better reflects patient phenotype and may provide a useful
prognostic indicator, in this very variable disorder.
Severe TFP disease is generally characterized by multi-organ
involvement including cardiomyopathy, while in the moderate/severe
infantile form of TFP deficiency, cardiomyopathy is usually not a
presenting feature (see table 2) (Spiekerkoetter et al 2004b; 2004c).
A notable feature of severe and moderate/severe TFP deficiency in this
series of patients is acute respiratory distress syndrome ARDS, being
recoded in 4 of 7 patients. This has been previously reported in both LCHAD
and TFP deficiency (Lundy et al 2003). Acute respiratory distress may occur
in the neonatal period or in older surviving infants. Patients who are
affected with viral bronchiolitis may have a particularly poor outcome as
with patients 6 and 7, both succumbing during such an illness. This
susceptibility to develop poor lung function probably reflects disrupted
surfactant synthesis as a consequence of interference from hydroxyacyl
metabolites (Lundy et al 2003).
Patient 9 with the neuromyopathic phenotype had a history of
respirtaory failure and upper respiratory tract infections. As a young
adult he required ventilatory support for respiratory failure following an
acute episode of decompensation & rhabdomyolysis followed by acute renal
failure. Such episodes of transient respiratory failure due to severe
muscle weakness, frequently precipitated by viral infection or exercise,
were previously reported in 5 of 11 neuromyopathic patients (Spiekerkoetter
et al 2004a).
Patients 8 & 9 were reported as having a mild pigmentary retinopathy.
Retinal changes were only observed in 1 of 11neuromyopathic patients
reported by Spiekerkoetter and colleagues 2004a. However pigmentary
retinopathy has been frequently observed in LCHAD deficiency (Tyni et al
1998) and has been causally linked to the high requirement for long-chain
fat oxidation in retinal pigment epithelium (Tyni et al 2004).
Peripheral sensorimotor polyneuropathy often as the initial presenting
feature but usually later accompanied by episodic rhabdomyolysis, are the
typical manifestations of mild neuromyopathic TFP deficiency (Schaefer et
al 1996; Ibdah et al 1998; Spiekerkoetter et al 2003, 2004a). Patients 8 &
9 both experienced delayed early milestones with gait abnormalities,
restricted mobility and exercise intolerance during childhood. Patient 8
had Achilles tendon contractures at 15 months and muscle weakness appeared
to affect predominantly the lower limbs both proximally and distally.
Predominance of lower limb involvement with Achilles tendon contractures,
bilateral foot drop and gait abnormalities were also frequently reported in
a study of 11 neuromyopathic TFP patients (Speikerkoetter et al 2004a).
Patient 9 experienced episodic rhabdomyolysis from early childhood but
patient 8 experienced his first episode of rhabdomyolysis at 10 years of
age (CK 26,000) with three further episodes during the following year. This
first episode of myoglobinuria at 10 years old is somewhat later than in 7
of 8 patients reported by Spiekerkoetter and colleagues. Detailed
investigations in patient 8 at this time showed a motor neuropathy,
predominantly axonal, affecting the legs more than the arms. There was
evidence of some demyelination. The muscle biopsy showed a type 1 fibre
predominance but no ragged red fibres, although there was some
subsarcolemmal accumulation of mitochondria. These findings are consistent
with previous reports (Spiekerkoetter et al 2004a).
Biochemical abnormalities in both patients 8 & 9 were subtle, with
generally no specific abnormalities on urine organic acid analysis. Both
patients showed an increase in blood long-chain 3-hydroxyacylcarnitine
species but this was typically only a mild increase (see table 2 for values
in patient 8). This general absence or only mild picture of biochemical
abnormalities is consistent with previous reports (Spiekerkoetter et al
2004a). This group of neuromyopathic patients is therefore not easy to
diagnose. The major clinical abnormality is a slowly progressive
sensorimotor polyneuropathy with muscle weakness that has many features in
common with hereditary motor-sensory neuropathies HMSN and spinal muscular
atrophy SMA. Patient 8 was indeed diagnosed initially with HMSN (Charcot-
Marie-Tooth Type 2). Patient 9 was for many years also given the diagnosis
of HMSN and was not correctly diagnosed until he was 29 years old. Episodic
rhabdomyolysis may often be the feature which suggests an underlying
metabolic disease but this, as in patient 8, may not appear for many years
after the onset of the neurological symptoms (Spiekerkoetter et al 2003;
2004a). The mild clinical presentation and relative difficulty in diagnosis
suggests that neuromyopathic TFP deficiency is probably under-diagnosed and
should be included in the differential diagnosis of HMSN and SMA.
It is nevertheless, important to make an early diagnosis in this
patient group, as acute episodes of rhabdomyolysis can be effectively
controlled with a low long-chain fat diet with high carbohydrate feeds,
avoidance of fasting and avoidance of prolonged exercise (Schaefer et al
1996; Spiekerkoetter et al 2004a). Limb-girdle myopathy has shown sustained
clinical improvement on treatment with prednisone (Tein et al 1995). Medium
chain triglyceride MCT with cod liver oil / docosahexaenoic acid DHA
supplementation has proved effective in improving exercise tolerance and
has been objectively demonstrated by nerve conduction studies to improve
nerve function (Tein et al 1999; Spiekerkoetter et al 2004a).
Patient 9 is still able to continue his occupation as a manual worker
at 37 years of age. His rhabdomyolysis is controlled on a low long-chain
fat, high carbohydrate diet. Control of episodic rhabdomyolysis has been
achieved in patient 8 by similar dietary measures but he has nevertheless
shown further deterioration of his neuropathy / myopathy and is at 12
years, largely wheelchair dependent. Further dietary intervention is being
undertaken in this patient.


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Table 1



Biochemical and Mutation data in LCHAD/MTF protein deficiency


"Patient"* % "* % "* % "M/O "LCHAD "Thiola"Mutation "Mutation "
"number "M "P "O "ratio "# % "se "(-subunit"(-subunit "
"and " " " " " "# % " " "
"phenoty" " " " " " " " "
"pe " " " " " " " " "
"1 (S) "47(13 "29 ( 3" 14 "3.35 "47 "23 "ND "Exon 8 insertion "
" " " "(1 " " " " "(AT cDNA 520/521) "
" " " " " " " " "Exon 15 1373C>A "
" " " " " " " " "Ala>Glu "
"2 (S) "34 "20 "7 "4.85 "35 "20 "ND "ND "
"3 (S) "46 ( 4"27( 7 "11( 2"4.18 "18 "41 "ND "ND "
"4 (S) "47 ( 8"28 ( 9"13( 3"3.61 "23 "29 "ND "Exon 7 "
" " " " " " " " "426C>A Asn>Lys "
"5 (MS) "75 ( 8"35 ( 5"19 ( "3.95 "14 "111 "1528G>C "- "
" " " "6 " " " "homozygot" "
" " " " " " " "e " "
"6 (MS) "92(18 "61(11 "47(12"1.95 "19 "16 "ND "Exon 10 "
" " " " " " " " "Gly301Asp "
"7 (MS) "66( 4 "53 ( 8"43 ( "1.53 "46 "33 "1528G>C "ND "
" " " "3 " " " "Arg676His" "
"8 (M) "94(12 "88(12 "58(11"1.62 "32 "28 "ND "Exon 6 "
" " " " " " " " "Asn114Ser "
"9 (M) "61(1.5"50(1.5"31(2 "1.97 "- "- "- "- "


Patient phenotype S = severe infantile, MS = moderate/severe, M = mild

* % percentage of simultaneous controls (n = 2-5 / assay) data is mean (
s.d. (duplicate determinations for 2-5 assays)
# % percentage activity of mean control data
LCHAD - long chain 3-hydroxyacyl-CoA dehydrogenase activity control mean
77(16 nmol/min/mg protein (n=23)
Thiolase - long-chain ketothiolase activity control mean 22.7(3.4
nmol/min/mg protein (n=15)
ND not detected





Table 2



Clinical data in 9 patients with LCHAD/TFP deficiency


"Patient"Age at "Age "Age "Urine "Blood "Cardio- "Liver "encephalopa"Acute "Peripher"Rhabdomyolys"
" "presenta"at "now "Organic"Acyl-carn"myopathy"diseas"thy "respirator"al "is "
" "tion "Death" "acids "itines " "e " "y distress"neuropat" "
" " " " " " " " " "/ "hy " "
" " " " " " " " " "bronchioli" " "
" " " " " " " " " "tis " " "
"1 (S) "day 5 "day "NA "NSA "(OH)C16-1" # "+ "+ "+ "- "- "
" " "26 " " "8 " " " " " " "
"2 (S) "day 3 "day 6"NA "lactate"(OH)C16-1"+ "+ "+ "- "- " "
" " " " "glutara"8 " " " " " " "
" " " " "te " " " " " " " "
" " " " "adipate" " " " " " " "
" " " " "ketones" " " " " " " "
"3 (S) "day 1 "day 2"NA "not "not done "+ "+ "- "- "- "- "
" " " " "done " " " " " " " "
"4 (S) "day 2 "2 m "NA "lactate"(OH)C16-1"+ "- "- "+ "- "- "
" " " " "NSA "8 on " " " " " " "
" " " " " "NNSC " " " " " " "
"5 (MS) "6 m "- "14 yrs"(OH)DCA"(OH)C16-1"+ "+ "+ "- "- "+ "
" " " " "DCA "8 " " " " " " "
"6 (MS) "3 m "11 m "NA "(OH)DCA"(OH)C16-1"- "+ "+ "+ "- "- "
" " " " "DCA "8 " " " " " " "
"7 (MS) "9 m "9 m "NA "(OH)DCA"(OH)C16-1"( "+ "+ "+ "- "+ "
" " " " "DCA "8 " " " " " " "
"8 (M) "15 m "NA "12 yrs"NSA "*mild "- "- "- "- "+ "+ "
" " " " " "(OH)C16-1" " " " " " "
" " " " " "8 " " " " " " "
"9 (M) "3 yrs "NA "37 yrs"NSA " mild "- "- "- "- "+ "+ "
" " " " " "(OH)C16-1" " " " " " "
" " " " " "8 " " " " " " "


Patient phenotype S = severe infantile, MS = moderate/severe, M = mild

( Cardiomyopathy was not detectable at initial presentation at 9 months
(previously completely well) but developed during terminal illness
# Echocardiography was normal
(OH)C16-18 elevated hydroyacylcarnitines C16 & C18
* C16:1(OH) 0.23 normal range