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Biochemical and genetic analysis of 3-methylglutaconic aciduria type IV: a diagnostic strategy

Saskia B. Wortmann, Richard J. T. Rodenburg, An Jonckheere, Maaike C. de Vries, Marjan Huizing, Katrin Heldt, Lambert P. van den Heuvel, Udo Wendel, Leo A. Kluijtmans, Udo F. Engelke, Ron A. Wevers, Jan A. M. Smeitink, Eva Morava
DOI: http://dx.doi.org/10.1093/brain/awn296 136-146 First published online: 16 November 2008


The heterogeneous group of 3-methylglutaconic aciduria type IV consists of patients with various organ involvement and mostly progressive neurological impairment in combination with 3-methylglutaconic aciduria and biochemical features of dysfunctional oxidative phosphorylation. Here we describe the clinical and biochemical phenotype in 18 children and define 4 clinical subgroups (encephalomyopathic, hepatocerebral, cardiomyopathic, myopathic). In the encephalomyopathic group with neurodegenerative symptoms and respiratory chain complex I deficiency, two of the children, presenting with mild Methylmalonic aciduria, Leigh-like encephalomyopathy, dystonia and deafness, harboured SUCLA2 mutations. In children with a hepatocerebral phenotype most patients presented with complex I deficiency and mtDNA-depletion, three of which carried POLG1-mutations. In the cardiomyopathic subgroup most patients had complex V deficiency and an overlapping phenotype with that previously described in isolated complex V deficiency, in three patients a TMEM70 mutation was confirmed. In one male with a pure myopathic form and severe combined respiratory chain disorder, based on the pathogenomic histology of central core disease, RYR1 mutations were detected. In our patient group the presence of the biochemical marker 3-methylglutaconic acid was indicative for nuclear coded respiratory chain disorders. By delineating patient-groups we elucidated the genetic defect in 10 out of 18 children. Depending on the clinical and biochemical phenotype we suggest POLG1, SUCLA2, TMEM70 and RYR1 sequence analysis and mtDNA-depletion studies in children with 3-methylglutaconic aciduria type IV.

  • 3-methylglutaconic aciduria
  • deafness
  • cardiomyopathy
  • cataract
  • mtDNA depletion
  • mitochondrial encephalomyopathy
  • TMEM70


The heterogeneous group of 3-methylglutaconic aciduria (3-MGA-uria) syndromes includes several different inborn errors biochemically characterized by increased urinary excretion of 3-methylglutaconic acid (3-MGA) and 3-methylglutaric acid. At the present time five distinct types have been recognized. 3-MGA-uria type I (MIM 250950) is an inborn error of leucine catabolism with either an acute, life threatening presentation or a late onset form, caused by the deficiency of 3-methylglutaconyl-CoA hydratase (3MGH; EC The additional four types of 3-MGA-uria all affect mitochondrial function through different pathomechanisms. Type II (Barth syndrome; MIM 302060) occurs due to mutations of the TAZ gene and abnormal cardiolipin metabolism, presenting in males with cardiomyopathy (CM), cyclic neutropenia, muscle hypotonia, and (in the classical form) with normal cognitive function. In type III (Costeff syndrome, MIM 258501) patients present with bilateral optic atrophy and progressive extrapyramidal symptoms. The underlying mutations are in the mitochondrial OPA3 gene, whose function is not fully understood.

3-MGA-uria type V (MIM 610198; Davey et al., 2006) is a novel autosomal recessive condition with early onset dilated CM with conduction defects and non-progressive cerebellar ataxia (DCMA) in the Canadian Dariusleut Hutterite population, further characterized by testicular dysgenesis and growth failure. It is speculated that the underlying mutation in the DNAJC19 gene leads to a defective mitochondrial protein import.

The diagnosis of 3-MGA-uria type IV (‘unspecified’ MIM 250951) is based on the exclusion of the other, well-defined clinical subtypes. The underlying aetiology is not yet elucidated but certainly heterogeneous (Gunay Aygun et al., 2005). 3-MGA-uria can occur secondarily in patients with Smith-Lemli-Opitz syndrome, a defect of cholesterol biosynthesis, because of abnormal isoprenoid/cholesterol biosynthesis (Kelley et al., 1995) as well as in patients with glycogen storage disease Ib (Law et al., 2003), where it is speculated that imbalanced gluconeogenesis and de novo cholesterol synthesis result in secondarily increased 3-MGA excretion.

However, 3-MGA-uria type IV is frequently associated with progressive neurological impairment, variable organ dysfunction and the common finding of biochemical features of a dysfunctional oxidative phosphorylation (OXPHOS). In some 3-MGA type IV patients the underlying genetic defect has been elucidated, mostly in patients with multiple complex deficiencies, like in mitochondrial DNA depletion syndromes (Figarella-Branger et al., 1992; Scaglia et al. 2001; de Vries et al., 2007), mitochondrial DNA deletion syndromes (Jakobs et al., 1991; Gibson et al., 1992; Lichter-Konecki et al., 1993) or MELAS syndrome (De Kremer et al., 2001).

Furthermore there are several reports about patients with CM, mitochondrial dysfunction and 3-MGA-uria type IV (Ibel et al., 1993; Besley et al., 1995, Ruesch et al., 1996; Morava et al., 2004; Di Rosa et al., 2006; Sperl et al., 2006). Di Rosa et al. described five patients presenting with a severe early-onset phenotype with hypertrophic cardiomyopathy (HCM), cataract, hypotonia/developmental delay and lactic acidosis (Di Rosa et al., 2006). Another distinct phenotype has been defined in 14 cases with an isolated deficiency of the mitochondrial complex V of nuclear genetic origin presenting with HCM, hypotonia, hepatomegaly, facial dysmorphism and microcephaly. Half of the patients died, mostly within the first weeks of live; the survivors showed psychomotor and various degrees of mental retardation (Sperl et al., 2006).

Recently we performed biochemical and genetic investigations in four children with 3-MGA-uria presenting with neuro-radiological evidence of Leigh disease, hearing loss, recurrent lactic acidemia and hypoglycemia, and defined a new mitochondrial phenotype within 3-MGA-uria type IV (MEGDEL association: 3-MGA-uria with sensori-neural deafness, encephalopathy and Leigh-like syndrome; Wortmann et al., 2006).

In this study we evaluated 18 children sequentially diagnosed with 3-MGA-uria type IV to assess the occurrence of associated symptoms, malformations, minor anomalies and biochemical features in order to define distinct phenotypes and elucidate the underlying genetic etiology in 3-MGA-uria type IV syndrome.


We evaluated the clinical data, collected by the same clinical investigator, of 17 out of 262 children evaluated by muscle biopsy in the period of 2002–06 at our centre who were consecutively diagnosed with a mitochondrial disorder and 3-MGA-uria. We additionally included one child related to the index patients with overlapping clinical features, who underwent a skin biopsy. All children were referred under suspicion of a OXPHOS disorder and underwent a standard diagnostic protocol including organ function, imaging and laboratory studies (Morava et al., 2006).

Urine organic acid analysis/in vitro NMR spectroscopy

Organic acids in urine were analysed by gas chromatography/mass spectrometry (GC-MS) after extraction of the urine sample with ethylacetate and derivatization with N,N-bis(trimethylsilyl)trifluoracetamide containing 1% trimethylchlorosilane. The concentration of 3-MGA was quantified by comparing the signals obtained with calibration curves of the pure compound, using a CP-Sil 8 CB column (Varian, Middelburg, The Netherlands) on a HP 6890 Gas Chromatograph (Agilent, Amstelveen, The Netherlands). An 8000 Top Gas Chromatograph and Trace Mass Spectrometer Plus (InterSciences, Breda, The Netherlands) was used to verify the identity of both 3-methylglutaconic isoforms. Body fluid NMR spectroscopy and quantification of the cis and trans forms of 3-MGA were performed essentially as described elsewhere (Engelke et al., 2004).

Biochemical and histological analysis in muscle and fibroblast samples

A surgical muscle biopsy was performed as well as a skin biopsy in all cases using standard methods. The samples were used for histological, histochemical and detailed biochemical investigations. Measurement of the 3-methylglutaconyl-CoA hydratase activity and Cardiolipin analysis were performed in blood or fibroblasts by standard methods (Valianpour et al., 2002). Oxidative phosphorylation was evaluated in muscle and fibroblasts, including ATP production measurement from pyruvate oxidation in muscle, and complex I–V activity in both tissues (Janssen et al., 2006).

Complex V assembly and activity/ANT1 evaluation

BN-PAGE, blotting and complex V in-gel activity was performed as described (Nijtmans et al., 2002), loading 60 µg of protein of OXPHOS complexes isolated from a mitoplast fraction of the patient and control cybrids and muscle homogenates. For Western blotting, monoclonal antibodies against mitochondrial ATP synthase subunit alpha and CoII-70 kDa (complex II) (Invitrogen, Breda, The Netherlands) were used. For the complex V in-gel activity assay the gel was incubated overnight at room temperature with the following solution: 35 mM Tris, 270 mM glycine, 14 mM MgSO4, 0.2% Pb(NO3)2 and 8 mM ATP, pH 7.8 (Nijtmans et al., 2002). ANT1 evaluation was performed by Western blot analysis (Morava et al., 2004).

Genetic analysis

PCR amplification of both exons and their flanking intronic DNA sequences of the OPA1 and OPA3 genes were performed as described (Anikster et al., 2001) in all patients. Mitochondrial depletion studies were performed in Patients 1, 3, 7–9, 11, 12 and 19 as described (Carrrozo et al., 2007). Mitochondrial DNA mutations were evaluated using a standard mitochip analysis (GeneChip® Human Mitochondrial Resequencing Array 2.0, Affymetrix, Inc.). Deletions were analysed by long template PCR. Sequencing of the nuclear coded structural complex I genes and RYR1 mutation analysis was performed on an ABI 3730 DNA analyser using BigDye terminator chemistry (Applied biosystems, Lekkerkerk a/d IJssel, The Netherlands).

Molecular investigations included sequence analysis of the structural genes of the oxidative phosphorylation complex I (NDUFS1, NDUFS2, NDUFS4, NDUFS7, NDUFS8, NDUFV1), and of the POLG1, DGUOK and SUCLA2 genes depending on the biochemical results from the muscle biopsy and fibroblasts (Morava et al., 2006). In the three patients with complex V deficiency, highly overlapping clinical phenotype and consanguinity we performed genome-wide homozygosity mapping using the 10K and 250K Affymetrix Single Nucleotide Polymorphisms arrays (Woods et al., 2007). Homozygous areas were found on the chromosomes 2, 4, 8, 10 and 14. Upon the overlapping clinical and biochemical features with the patients described by Cizkova et al. (2008) we checked the TMEM70 gene for disease causing mutations.


Clinical patient characteristics

Based on the clinical phenotype we divided the patients into the following four distinctive sub-groups: encephalomyopathic form (CNS and muscle involvement without liver involvement), hepatocerebral form (encephalomyopathy and liver dysfunction), cardiomyopathic form (motor developmental delay with CM, no or mild encephalopathy) and myopathic form (skeletal muscle involvement, no central nervous system involvement). We describe 8 new patients, the other 10 patients (1–6, 8, 15, 16, 18) have already been published as case reports, we therefore refer to the original papers. The essential clinical findings are summarized in Table 1.

View this table:
Table 1

Clinical and biochemical features in 18 patients with 3-MGA-uria type IV

Age at onset (years)000000.8160.30.390.
Present age (years)1393a16a121.5a16a1a3a113a395117410
Hearing loss+++++NA
Brain atrophy+++++++, DM++, DMNANANA+, DMNANA
Leigh-like syndrome+++++NANANANANA
3-MGA excretionb20–2224–2531–7616–6837–141282625384–2316–338012144–6429–6013–3818–2009–38
Other metabolites in urineMMAMMAEMAEMA
Complex I deficiency in muscle or fibroblasts+++++++++++++++++
Complex V deficiency in muscle or fibroblasts++++++NANA+
  • a Age deceased (years).

  • b Normal <20 μmol/mmol creat.

  • CCH = corpus callosum hypoplasia; DM = delayed myelinization; EM = encephalomyopathic type; EMA = ethylmalonic acid; HC = hepatocerebral type; M = myopathic type; MDC = mitochondrial disease criteria; MMA = methylmalonic acid; NA = not available; S = suspected; 3-MAG = 3-methylglutaconic Acid; +, mild; ++, moderate; +++, severe.

Encephalomyopathic form

Patients 1 and 2: (Patients 11 and 12; Carozzo et al., 2007). Both patients initially presented with muscular hypotonia, poor suck and failure to thrive (FTT). In Patient 1 later hearing loss, kyphoscoliosis and developmental delay as well as severe regression occurred. Patient 2 developed extrapyramidal symptoms and myoclonic seizures in the first year of life. Hearing evaluation revealed a sensorineural deafness followed by chochlear implant.

Patients 3–5: These patients were recently reported as MEGDEL-association by our group (Patients 1, 2 and 4; Wortmann et al., 2006). The three children presented with severe infections in the neonatal period, hypoglycemia and lactic acidemia were seen. Feeding problems and FTT made tube feeding necessary in two cases. All but one child had a delayed motor development and muscle hypotonia. One patient did not develop at all, the other patient showed psychomotor regression, one had epilepsy and all were mentally retarded. The patients developed severe spasticity, combined with extrapyramidal symptoms. One patient showed behavioural problems with constant laughing. Two patients died at the age of 3 and 16 years, respectively. No cardiologic alteration was noted. The MRI of the brain revealed characteristic bilateral hyper-dense lesions of the basal ganglia and diffuse cerebellar and/or cerebral atrophy in all patients. Visual evoked potential analysis was bilaterally delayed in two patients, however, no optic atrophy was noted. The BAEP studies showed severe sensori-neural hearing loss in all patients, making hearing devices necessary.

Hepatocerebral form

Patient 6: (Patient 3; de Vries et al., 2007). The male patient had an older brother with a similar clinical phenotype. At the age of 8 months FTT, hypotonia, mental retardation and myoclonic seizures became obvious. He died at the age of 17 months following intractable seizures and a progressive synthetic and cytotoxic liver failure.

Patient 7: The female patient presented at the age of 16 years with a progressive neurological dysfunction of the left hemisphere (headache, hemi-anopsia, latent left-sided paresis and aphasia). Up to then, the medical history was negative, as was the family history. She was diagnosed with, and treated for acute disseminated encephalomyelitis. She suffered from an organic psychosyndrome and epilepsy, treated with valproic acid. After a brief period of clinical improvement she presented with a relapse in combination with gastrointestinal symptoms, and showed a neurological regression with apathy, agitation, anorexia and liver failure (synthetic/cytotoxic) and deceased in a multi-organ failure due to a gram-negative sepsis. Autopsy revealed spongiosis in thalamus and nucleus caudatus, suggestive of Morbus Alpers-Hüttenlocher.

Patient 8: (Patient 5; de Vries et al., 2007). From the age of 4 months the male patient was followed for FTT, muscular hypotonia and delayed psychomotor development, later also regression. The EEG showed a burst suppression pattern and he had severe epilepsy. Hepatomegaly and elevated liver-function tests were noted as well. At the age of 13 months he died due to cardiorespiratory insufficiency after multi-drug treatment (without Valproic acid) during status epilepticus.

Patient 9: The female patient was born prematurely by 31 weeks gestation after a pregnancy complicated by severe hyperemesis. Two other pregnancies, in which solely intrauterine growth retardation (IUGR) was noted, ended with a stillbirth. The neonatal period was uncomplicated, the cerebral ultrasound showed a right sided subepidermal hemorrhage and bilateral mild periventricular leucomalacia. At the age of 3.5 months she was diagnosed with epilepsy, muscle hypertonia and psychomotor development delay. From the age of 2 years on she suffered from unexplained encephalopathic periods with atonia, apneas and stupor. These episodes of deep coma (Glasgow coma scale 3), lasted for ∼72 h and then she became conscious again spontaneously and quickly. Continuous EEG-registration then showed a bizarre pattern of burst suppression changing with hours nearly without any cortical activity. MRI showed mild periventricular leucomalacia, irregularly widened ventricles, gracile corpus callosum and a partial pachygyria. Repetitive drug-screening in blood and urine was negative. She received a gastrostomy due to severe FTT and gastro-esophageal reflux after malrotation correction. Transient elevations of transaminases were noted. At age 3 years she died due to cardio-respiratory failure in an encephalopathic state.

Patient 10: The male patient was born at 26 weeks of gestation, with intrauterine growth retardation. No family history is available. He had an infantile encephalopathy with epilepsy and a delayed psychomotor development. He was noted to have episodes with hypo- and hyperthermia, extraordinary resistance to pain and severe FTT. At the age of 9 years he was admitted with a stroke-like episode with hemiplegia after valproic acid use for seizures. The MRI suggested a hemi-encephalitis. After a multi-organ failure with liver dysfunction and synthesis-failure, pancreatitis and a long-time depressed consciousness, his residual hemiparesis and speech problems are improving over time.

Patient 11: The pregnancy with this female patient was complicated by an early growth retardation and microcephaly, and she was born at term by caesarean section. Genetic and metabolic analysis was initiated at the age of 3 months due to severe microcephaly, dysmorphic features comparable to Brachmann-de Lange syndrome, psychomotor retardation, no development (could not sit or roll over, and made no contact), muscle hypotonia, epilepsy, severe FTT, feeding difficulties and tracheomalacia. She received tube feeding. She suffered from recurrent episodes of hyperthermia, liver dysfunction and intractable seizures and died at the age of 2.5 years.

Cardiomyopathic form

Patient 12–14: These three female patients from a large Roma family descent from a possible inbred community. There are several additional siblings known with an overlapping phenotype. Patient 12, the index patient presented with severe FTT, arterial hypertension, CM and Wolf-Parkinson-White syndrome at the age of 3 years. Patient 13, a maternal cousin of Patient 12 had HCM in combination with a aorta stenosis, pulmonary valve stenosis and occasional extrasystoles. Patient 14 presented with feeding problems leading to FTT and was evaluated from the age of 8 months onwards four times for periods of encephalopathy with extremely elevated lactate (11.0 mmol/l, N < 2.1 mmol/l), alanine and glutamine/glutamate levels by normal liver enzyme and intermittently high ammonia concentrations. She had a normal motor development, only slight mental retardation and suffered from myoclonic episodes. Besides a HCM, an aortic valve stenosis was detected with rhythm disturbances (occasional extrasystoles). None of the patients had hypotonia. At the age of 3, 5 and 9 years, respectively, they have a nearly normal psychomotor development, short stature, FTT, but no neurological or other organ involvement. They all shared dysmorphic facial features including high forehead, curved eyebrows, flat midface, long philtrum, low implanted ears and thin lips.

Patient 15 and 16: (Cases 1, 2; Morava et al., 2004). Patient 15 was evaluated for severe muscle hypotonia with hyporeflexia, delayed motor development and a severe, stable HCM with mild left ventricular outflow obstruction. His sister, Patient 16, had a lactic acidosis at birth, no hypotonia. An echocardiogram detected HCM without an obstructive component. Both children developed early bilateral cataracts. Cranial MRI showed delayed myelinization for Patient 15, in Patient 16 it was normal. At the age of 11 and 7 years, respectively, they have a normal psychomotor development and severe exercise intolerance, both receive speech therapy.

Patient 17: The male patient was noted to have a severe muscle hypotonia from the age of 5 months, swallowing problems, recurrent infections and loss of motor skills. By 10 months of age a non-obstructive CM was detected with mitral valve insufficiency, a tricuspidal aortic valve and pericardial effusion. Due to severe FTT and swallowing problems he got a gastrostomy. He received a lipid enriched diet. Physical examination revealed mild hepatomegaly, with transient elevation of the liver function tests. At the age of 4 years he is developing impressively, walks independently and communicates on an approximate age appropriate level.

Myopathic form

Patient 18: (Patient 5; Monnier et al., 2008). In this male patient generalized hypotonia, multiple contractures, swallowing problems and a lack of facial expression were apparent at birth. In spite of several severe infections he never needed artificial ventilation. He had FTT and his multiple episodes of hypoglycaemia were successfully treated with raw cornstarch. Besides a severe generalized muscle disease, he is unable to crawl or walk and therefore wheelchair dependant, he has no other organ involvement. By the age of 10 years the patient attends a regular school.

Biochemical patient characteristics

As shown in Table 1, urine organic acid analysis with GC-MS and in vitro NMR spectroscopy showed an increased urinary excretion of 3-MGA in all patients in at least one sample. The excretion of 3-MGA in the children ranged between 4 and 200 μmol/mmol creat (control <20), with a strict 1 : 1 ratio of the cis and trans isoforms of 3-MGA (Fig. 1). The excretion of 3-hydroxyisovaleric acid (3-HIVA) in the samples was within the normal range (data not shown). Hence, we conclude that our patients show 3-MGA-uria.

Fig. 1

NMR spectroscopy demonstrating two resonances of the cis and trans forms of 3-methylglutaconic acid (urine sample of Patient 5).

3-MGA-uria types I, II (Barth syndrome) and III (Costeff syndrome) were excluded in all patients as the activity of 3-methylglutaconyl-CoA hydratase, the cardiolipin levels and molecular analysis of the OPA3 gene, respectively, showed no abnormalities (data not shown). Smith-Lemli-Opitz-syndrome and glycogen storage disorder were ruled out as well.

As all our patients scored above three points on the clinical diagnostic scoring system for mitochondrial disorders (MDC, see Table 1; Morava et al., 2006) the possibility of a respiratory chain defect was further explored. Indeed, as shown in Table 2, the biochemical analysis of mitochondrial function in the fresh muscle biopsy and/or fibroblast cell lines showed disturbed oxidative phosphorylation in all 18 patients with an ATP production from pyruvate oxidation ranging between 0.9 and 37 nmol ATP/h/mU CS (control range 42–81 nmol/h/mU/CS).

View this table:
Table 2

Biochemical analysis of the mitochondrial function in the muscle biopsy and fibroblast lines of 18 patients with 3-MGA-uria type IV

ATP productionbNANA372426303411NAa31130.9NANA4.
Complex I activityc856993NN54N7754876763NA8221261383
Complex III activitycNANNNAN90N82NNANNNAN27268024
Complex IV activitycN43NNANNN99NNNNNAN3520NN
Enzyme complex deficiencies in fibroblastsNACI 84%CI 80%, CV 20%CI 63%, CV 5%CI 97%, CV 37%
Abnormal muscle histology/histochemistry+++NA+NANA++++
Abnormal Electron microscopy+NANANA+NANANANANANA++NA+
  • Abnormal findings in bold.

  • a Frozen muscle biopsy.

  • b ATP production from pyruvate oxidation (control range 42–81 nmol/h/mU/CS).

  • c Enzyme activities as percentage of lowest controls.

  • HC = hepatocerebral type; M = myopathic type; LM = lipid myopathy; N = normal; NA = not available.

In the subgroup of children with the encephalomyopathic form (Patients 1–5), including the Patients 3–5 with the MEGDEL association, all children had (when measured) a decreased ATP production from pyruvate oxidation (24–37 nmol ATP/h/mU CS) in fresh muscle biopsy. Four patients demonstrated complex I deficiency (either in muscle or fibroblasts), and one patient with SUCLA2 defect had additional complex IV deficiency. The activities in fibroblasts were normal, with exception of a complex I deficiency in Patient 5. The 3-MGA excretion levels were mildly to moderately and intermittently increased in the MEGDEL patients (16–141 μmol/mmol creat) and mildly elevated (20–25 μmol/mmol/creat) in the other patients.

In the subgroup of children with the hepatocerebral form (Patients 6–11) all children had moderately to severe decreased ATP-production (13–34 nmol ATP/h/mU CS). All but Patient 7 had a decreased activity of complex I, two had additional complex III deficiency, Patient 8 demonstrated multiple enzyme deficiencies. The enzyme measurements in fibroblasts were normal in all children. Patient 11 additionally underwent a liver biopsy which revealed a complex I and IV deficiency. The 3-MGA-uria was intermittent in most of the children with normal results alternating with mildly elevated excretion (23–38 μmol/mmol creat).

The Patients 12–17 with the cardiomyopathic form show very low ATP production from pyruvate oxidation; 0.9–5.5 nmol ATP/h/mU CS. Patients 12–14 have similar biochemical features as described by (Sperl et al., 2006) with severe complex V deficiency (0.4–4.9% of lowest controls). In Patient 13, due to a positive family history, only a skin biopsy was performed, showing mild complex I (63%) and severe complex V deficiency (5%). In Patients 12 and 14 mild complex I (80–97%) and severe complex V deficiency (20–37%) were found in fibroblasts. The two patients with Sengers-like syndrome (Patients 15 and 16) had severe combined multi-complex deficiency (complexes I, III and IV). The 3-MGA-uria was moderate in patients 12, 13 and 14 (44–121 μmol/mmol creat), intermittently very high in patient 17 (up to 200 μmol/mmol creat) and mildly to moderately elevated (13–60 μmol/mmol creat) in the other patients.

Patient 18 with the myopathic form had a multiple complex deficiency (complex I activity 83%, complex II 93%, complex III 33% and complex IV activity 24% of lowest controls, respectively) and a very low ATP production from pyruvate oxidation of 4.0 mmol ATP h/mU CS. No abnormalities were detected in fibroblasts. 3-MGA-excretion in urine was mild (9–38 μmol/mmol creat).

Histology/electron microscopy

Alterations were found on histology by light microscopy in the majority of patients, ranging from fibre type disproportion in Patients 3, 6, 11, 15 and 16, critical illness myopathy in Patient 7 to increased percentage of lipid vacuoles, suggesting lipid myopathy in Patient 17 (Table 2). In Patient 15 necrotizing regions were detected and severely decreased COX-activity, SDH-staining was diffusely increased in several type I fibres (Fig. 2). The histology and histochemistry of Patient 16, with the identical clinical phenotype, was normal. Significant histological alterations were also found in Patient 18 (Fig. 2) with the myopathic subtype. Areas devoid of oxidative activity (cores) were observed in the skeletal muscle biopsies, as well as predominance of type I fibres with the presence of centrally located nuclei or of rods comparable to central core disease.

Fig. 2

(A) One of the patients with SUCLA2 mutation with myopathic face by rigid dystonia. (B) Patient 8 with a POLG1 mutation demonstrating the mask-like face due to facial dystonia, note the tube feeding. (C) Patient 16 at the age of 8 months with extreme hypotonia. (D) Patient 18, note the myopathic face. (E) Cerebral MRI of a patient with SUCLA2 mutation: Leigh disease and hyperintensities on T2-weighed images in both thalami. (F) Cerebral MRI of Patient 7 with a POLG1 mutation: hyperintensities in all basal ganglia with sparing of the putamina in T2-weighed images. (G) Transverse light microscopic serial sections of quadriceps muscle of Patient 15, note the myosin ATP-ase staining (bar 50 µm) and fibre type dysproportion. (H) Transverse light microscopic serial sections of quadriceps muscle of Patient 18, Areas devoid of oxidative activity (cores), as well as predominance of type I fibers with the presence of centrally located nuclei or of rods comparable to central core disease (NADH TR staining).

Electron microscopy showed abnormal structure of mitochondria in Patient 2, absent cristae in Patient 8 and zones with absent mitochondria in Patient 18. In Patient 15 many mitochondrial aggregations and an increased number of lipid droplets were found in many fibres. The mitochondria were abnormal with a severe loss of christae, also ring-shaped mitochondrial were seen. These alterations were also seen in Patient 16, but less impressive.

Genetic studies

Molecular analysis of the mitochondrial DNA detected no mutation in any of the patients. Additional sequencing of the nuclear coded structural genes of the oxidative phosphorylation complex I (NDUFS1, NDUFS2, NDUFS4, NDUFS7, NDUFS8, NDUFV1) showed no disease causing mutations.

Mitochondrial depletion analysis was performed in eight children, and mitochondrial depletion was confirmed in Patients 1, 2, 6, 7, 8, 11, but not in Patients 10 and 18 (Carrozzo et al., 2007; de Vries et al., 2007). No associated mitochondrial DNA deletions were found. Mutations in the DGUOK and POLG1 genes were evaluated in all patients, TK2, SUCLA2, P53R2, MPV17 were analysed appropriate to the phenotype. Patients 1 and 2 (Patients 13 and 11, respectively, Carrozzo et al., 2007) harboured homozygous SUCLA2 mutations (c.534 + 1G > A). POLG1 mutations were detected in the Patients 6 (G2869C/G1399A; Patients 3 in de Vries et al., 2007), 7 (G1399A/G1399A) and 8 (G680C/G1399A, Patient 5 in de Vries et al., 2007). In the three Patients 12–14 with complex V deficiency we performed genome-wide homozygosity mapping revealing several homozygous regions that were shared by all three patients tested. No homozygosity was found in any of the previously described nuclear encoded structural assembly genes. Upon the finding of a homozygous region on chromosome eight and the overlapping clinical and biochemical phenotype with (Ciskova et al., 2008), we performed mutation analysis of the TMEM70 gene. This revealed a homozygous substitution c.317-2A > G in Patients 12–14 identical with those recently described (Cizkova et al., 2008). Western blotting with monoclonal antibodies against mitochondrial ATP synthase subunit alpha and CoII-70 kDa (complex II) showed a severely reduced amount of holo-complex V in the fibroblasts of these patients (Fig. 3). In Patient 18 RYR1 mutations (c14804-1G > T/c.10616G > A; Patient 5 in Monnier et al., 2008) were detected.

Fig. 3

One-dimensional Blue Native-PAGE showing severely reduced expression of holo-complex V in fibroblasts of two patients (8836 = Patient 13, 8719 = Patient 14) from the cardiomyopathic subgroup. Complex V was detected by an antibody against ATP-synthase subunit alpha. As a loading control complex II levels were visualized using an antibody against the 70 kDa FP subunit.


Until now, several distinct metabolic disorders have been described demonstrating 3-MGA-uria, affecting the leucine degradation pathway or mitochondrial function in variable degree and severity. Besides these clinically and biochemically well-defined forms, the highly heterogeneous diagnostic category of 3-MGA-uria type IV encompasses patients with progressive neurologic impairment, variable organ involvement and OXPHOS dysfunction (Gibson et al., 1991). During our study within a 5-year period in the group of patients undergoing muscle biopsy based on standard clinical criteria, we detected a high number of children (18 out of 262) with 3-MGA-uria and oxidative phosphorylation complex deficiencies; almost 7% of the patients were diagnosed with 3-MGA-uria type IV. In nine children we confirmed a nuclear coded mutation underlying the disease, in one patient only mitochondrial depletion was found. One child, who is not included in the current study was additionally diagnosed with Barth syndrome. No OPA1 mutations were elucidated in any of the patients. In spite of the presence of mitochondrial depletion in a high percentage of the mutation positive patients none of the children were diagnosed with mitochondrial mutations and no mitochondrial DNA deletion was confirmed in muscle tissue. Based on our findings we confirm the previous observation that 3-MGA-uria is an important diagnostic marker and could be used for screening in the diagnostic workup of a suspected dysfunction in the oxidative phosphorylation.

3-MGA-uria has been described as an associated finding in various mitochondrial DNA depletion syndromes (Figarella-Branger et al., 1992; Scaglia et al., 2001), however the finding of this organic aciduria is highly variable and frequently intermittent. In our patient group diagnosed with POLG1 mutations, 3-MGA-uria was present in only 3 out of 10 patients and in a large cohort of children with SUCLA2 defect only 5 out of 16 showed 3-MGA-uria during their disease course (U. Steuerwald 2008, personal communications). The majority of these patients have normal concentration of 3-MGA in the urine (Carrozzo et al., 2007). This suggests that the metabolic marker could be indicative for mitochondrial depletion, but not directly related to the disease, even in the presence of the same mutation.

One should interpret the presence of mild 3-MGA-uria very cautiously, the same level of excretion can be detected in children with primary and secondary liver disease, including glycogen storage disorder type I (Law et al., 2003) and type IX (E. Morava: personal communication) or cholesterol synthesis defects (Kelley et al., 1995) as a marker of lipid mobilization and upregulated cholesterol synthesis. These patients might present with elevated lactate levels and hypoglycaemia, still the clinical picture is distinct enough to be able to find the correct diagnosis.

In nearly all of our patients the finding of 3-MGA-uria was intermittent. Even the children carrying POLG1 mutations had periods during the course of their disease without 3-MGA-uria. No correlation was found with either possible episodes of infection or the nutritional state in our patients. Since the underlying mechanism leading to 3-MGA-uria is still undiscovered, we have no appropriate explanation for this phenomenon. Interestingly variable excretion, even within 24 h (Cantlay et al., 1999), of 3-MGA was reported in several patients with Barth and Costeff syndrome (own experience; Elpeleg et al., 1994, Christodoulou et al., 1994) seemingly unrelated to the clinical course or the severity of metabolic derangement (Fig. 4).

Fig. 4

Excretion levels of 3-MGA in urine in the different subtypes of 3-MGA-uria.

By far the most patients described so far with 3-MGA-uria type IV dysfunction present with HCM (Ibel et al., 1993; Besley et al., 1995; Ruesch et al., 1996; Morava et al., 2004; Di Rosa et al., 2006; Sperl et al., 2006). The most striking biochemical feature in the patients described by Sperl et al., is a severe complex V deficiency, measured in muscle in fibroblasts and associated with a mild complex I deficiency (Sperl et al., 2006). This characteristic pattern has been described so far only in children with gypsy ethnicity originating from highly consanguineous families, in combination with normal mental development and characteristic facial features. These patients were recently diagnosed with a novel nuclear genetic defect leading to abnormal biogenesis of the respiratory chain complex V (Cizkova et al., 2008). We have identified three patients (Patients 12–14) with the same TMEM70 mutation, sharing the same ethnic background.

Interestingly all of our patients (Patients 12–17) with HCM had no regression and an only somewhat delayed or normal psychomotor development. Only in Patient 17 there is a moderate psychomotor retardation, but the patient is improving impressingly over time. Besides the slowly progressive HCM some of the children suffered from rhythm disturbances or congenital valve anomalies. Still one should emphasize the absence of associated neurological symptoms in this patient group. In both children diagnosed with Sengers like syndrome (Patients 15 and 16) infantile cataract was diagnosed at an early stage of the disease. Cataract seldomly occurs at such a young age in children with a respiratory chain disease, and mostly presents in association with mtDNA deletions (Bene et al., 2003), mtDNA depletion (Sarzi et al., 2007) or in the classical Sengers syndrome (OMIM 212350, Sengers et al., 1975). Cataract has been observed several times in combination with 3-MGA-uria type IV in combination with HCM (Di Rosa et al., 2006). This unique symptom therefore might lead the clinician early on to the correct diagnosis. Based on the strong correlation of CM, 3-MGA-uria, complex V deficiency and cataract we strongly advice detailed cardiological and ophthalmological evaluation in all patients with 3-MGA-uria and a suspected OXPHOS disorder.

Patients with a diagnosis of 3-MGA-uria type IV present with an extremely variable clinical phenotype. NMR-spectroscopy of the urine in 3-MGA-uria type I (Engelke et al., 2006) showed a 2 : 1 ratio for cis and trans isoforms of 3-MGA in all patients. Interestingly in the CSF only the cis stereoisomer was found. Additional to other biochemical features, these findings helped to differentiate patients with type I from the other 3-MGA types. In our study we expected to be able to delineate further patient groups upon the cis and trans ratios of 3-MGA in urine. Unfortunately the ratio was 1 : 1 in all patients and therefore NMR spectroscopy revealed no additional information compared to standard organic acid analysis.

Assigning clinical subgroups within the large patients group of 3-MGA-uria could however effectively facilitate the molecular diagnostic workup. There is a long ongoing discussion between clinical geneticists and molecular/biochemical geneticists regarding ‘splitting or lumping’ phenotypic groups in clinical syndromes; either based on biochemical and phenotypic patterns, to define relatively homogeneous groups for further genetic analysis or to be able to form larger groups for follow up and counselling. In the current study we tried to approach our patient group with ‘splitting’, leading to the discovery of the genetic aetiology in many children. Although forming smaller, but characteristic groups might lead to even more complex descriptions of an individual patient, it might also help in better prognosis assessment and optimal follow up in this unique patient group. By delineating patient groups we elucidated the genetic defect in 10 out of 18 children. Depending on the phenotype, the presence of complex V deficiency and the finding of additional biochemical markers we recommend POLG1, SUCLA2, TMEM70 and RYR1 sequence analysis and mtDNA-depletion studies in children with 3-MGA-uria type IV.

In the patient group with encephalopathic presentation we found SUCLA2 mutations in patients demonstrating an associated methylmalonic aciduria. Both patients came from the Faroer islands due to a founder effect. In the other children with progressive dystonia, Leigh like syndrome and deafness, a ‘classical mitochondrial’ presentation, no mitochondrial DNA deletion or mutation, no alteration in the OPA1, POLG1 or DGUOK genes were discovered. Homozygosity mapping might reveal the underlying suspected nuclear gene defect in these consanguinous cases.

Most patients with 3-MGA-uria type IV present with either neurodegenerative symptoms, or encephalopathy in combination with other organ involvement. In our patient cohort, but also in most of the so far published cases only a minority of children diagnosed with type IV have an isolated muscle disorder. Due to the distinct, syndromic presentation and the characteristic association of biochemical and clinical markers we suggest a practical evaluation protocol to facilitate an effective diagnostic route and optimalize counselling (Fig. 5). Solving the genetic defect and assigning a syndrome diagnosis to a child with ‘the unclassified’ type IV 3-MGA-uria, finally ‘excludes’ the patient from this unclassified group.

Fig. 5

Diagnostic flowchart. MDC = Mitochondrial disease criteria (Morava et al., 2006).

Obviously in case of a classic syndromic presentation in a child with 3-MGA-uria and a suspected mitochondrial dysfunction, invasive diagnostics, such as muscle biopsy or skin biopsy should be avoided, if possible. By using the diagnostic flowchart we elucidated the underlying genetic defect in two additional patients with 3-MGA-uria. One male patient from the Faroer islands with MMA-uria, hypotonia and deafness, in whom a SUCLA2 mutation could be confirmed, and a gipsy female with typical facial features and HCM, where we confirmed a TMEM70 mutation. Both mutations were confirmed in blood, hence we spared the children undergoing muscle biopsy.


  • *These authors contributed equally to this work.

  • Abbreviations:
    Blue native-polyacrylamide gel electrophoresis
    Dilatative cardiomyopathy and ataxia
    failure to thrive
    hypertrophic cardiomyopathy
    3-methylglutaconic aciduria
    3-methylglutaconic acid
    oxidative phosphorylation


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