Ataxia and CoQ10 deficiency: progress report the following is an update on research funded by NAF in 2001.

1. Identification of new patients with primary CoQ deficiency

To better define the "ataxic" syndrome due to primary CoQ deficiency described by our group last year in six patients (Musumeci et al, Neurology 2001;56:849-855) we have received over 100 muscle specimens from patients with unexplained ataxia and cerebellar atrophy through a rich network of collaborators both in the US and in Europe. We have analyzed 112 muscle specimens and found 12 new patients with abnormally low levels of CoQ (defined by us as below 15 [micro]mol/g tissue; our control value [+ or -] SD is 27.5+2.5) and childhood onset of ataxia. The clinical features of the 12 patients are summarized in the Table. There were six male and six female patients, ranging in age from 3 to 35 years. The first symptoms were hypotonia and motor delay in six individuals, apparent before two years of age; ataxia in four, manifesting between two and nine years of age; and seizures in two, at ages two and nine years. Ataxia became apparent in all patients by 10 years of age and affected trunk, limbs, and speech. In fact, patient 2 never learned to walk independently, patient 11 requires a walker at age 9; and patients 1 and 5 never developed intelligible speech, although they interact with signs. Although intellectual development may well be affected by motor and language limitations, most of the patients are described as friendly and interactive, with good verbal understanding. Patients 2 and 3 attend regular school, and patient 4 has a normal IQ. On the other hand, patients 7 and 10 are considered mentally retarded, while patient 11 attends a special school and is two years behind her grade level. Four patients had seizures, which were generalized tonic-clonic in two, psychomotor in one, and febrile in another. Pyramidal signs were described in four patients, ranging from hyperreflexia with bilateral Babinski signs in patients 9 and 11 to spastic paraparesis (patient 6) or tetraparesis (patient 10). Proximal more than distal limb weakness was described in patients 6 and 11. Unusual neurological signs included both spontaneous and action myoclonus (patient 4), recurrent right hemiplegia with EEG evidence of left hemispheric slowing (patient 5), and bilateral ophthalmoparesis for both horizontal and vertical gaze (patient 11).

Laboratory abnormalities were mostly confined to cerebellar atrophy, documented by MRI in all patients and affecting both the hemispheres and the vermis. In patient 5, MRI of the brain was normal at age 10 months, but showed severe cerebellar atrophy at 34 months. Patient 6 had increased lactate in blood and CSF, and patient 7 had mevalonic aciduria. Amino acid and organic acid profiles and liver function tests were normal in all patients. Nerve conduction studies and electromyograms were also normal. Muscle biopsy was normal in all patients but showed increased lipid droplets in the child with mevalonic aciduria. Axonal neuropathy was documented by nerve biopsy in patient 10.

Family history was negative: there was no consanguinity and no history of neurological disorders, except for patient 8, whose only brother had severe behavioral problems. The patients belonged to diverse ethnic groups, including Anglo-Saxon American, African American, Ashkenazi Jewish, Italian, Albanian, and East Indian. Genetic testing for all known causes of SCA was negative.

We measured the activities of the mitochondrial respiratory chain enzymes in muscle extracts from eight patients: in only two (patients 1 and 3) the activities of complex I+III and II+III were markedly decreased, suggesting a block at the COQ10 level (data not shown). Similar data were found in another laboratory (Dr. Brian Robinson, Toronto, Canada) for patient 11. COQ10 concentrations were, by definition, below 15 [micro]mol/g tissue in all patients, and below 10 [micro]mol/g in seven (Table).

All patients have been started on high doses (600-1,000 mg/d) oral COQ10 supplementation. Although formal quantitative assessments of their clinical response are not yet available, anecdotal feedback from physicians, physical therapists, and family members are uniformly positive, including improved standing balance, shorter episodes of marching clonus, better speech articulation.

A paper describing these patients is in preparation and will be submitted to Neurology.

2. Developing sensitive HPLC-EC assay protocol to measure CoQ level in brain and CSF

Although there are several methods to measure the level of CoQ in blood, no reliable and well tested methodology has yet been established to measure CoQ in different regions of the brain and in the cerebrospinal fluid (CSF). We sought to develop such a sensitive methodology. Since the level of CoQ in the CSF is extremely low (about 500 to 1000 times lower than in blood) the standard technique of HPLC with UV detection system could not be employed. Instead, we have adopted a methodology based on HPLC and electrochemical detection. Application of this technique enabled us to measure simultaneously oxidized and reduced form of CoQ in the brain. In order to establish reference range for CoO level in brain and CSF, we need to measure CoQ in a statistically acceptable number of specimens. To date, we have obtained brain biopsies and CSF specimens from five patients, but we have sensitized our colleagues both in neurology and in neuropathology, and we expect to have no problem in accumulating large numbers of specimens. We will also compare CoQ values in brain biopsies and in postmortem brain specimens.

3. Studying CoQ biosynthesis in vitro We reasoned that if low level of CoQ in muscle from ataxic patients were due to a defect in the biosynthetic pathway of CoQ, this might well result in accumulation of intermediary metabolites upstream of the block. To examine this hypothesis, we grew skin fibroblasts from control subjects and from CoQ-deficient patients in the presence of radiolabeled CoQ precursors, either mevalonolactone (a polyprenyl precursor) or p-hydroxy benzoate (a benzoquinone ring precursor). However, data from two experiments failed to demonstrate any detectable accumulation of intermediary metabolites in cultured cells from patients whose CoQ concentration in fibroblasts was about 60% of normal. This observation can be explained by a partial biosynthetic defect in fibroblasts, not severe enough to cause substantial accumulation of intermediary metabolites. Measuring the activity of the individual enzymes involved in CoQ synthesis is not an option both because of the large number of assays required and because the levels of these enzymes in the human cells are extremely low and very large amounts of cells would be required to obtain any measurable activity.

To circumvent these obstacles we are growing myoblasts from a patient of ours with the "myopathic" variant of primary CoQ deficiency (Sobreira et al, Neurology 1997;48:1238- 1243). Although we had not measured CoQ in this patient's muscle cultures, we noticed that myoblasts grew very sluggishly and were "rescued" by addition of CoQ to the medium of culture. These observations suggest that myoblasts may have very low levels of CoQ (we are in the process of verifying this) and may, therefore, be ideal cells for the biosynthetic experiments. Of course, a definition of the metabolic block in this patient would help us understand the pathogenesis of the myopathic, not the ataxic variant. However, this would be an important first step towards a better understanding of primary CoQ deficiency in general.

TABLE

Clinical characteristics of patients with primary CoQ deficiency

Patients    Age (yr)/     Age     Ataxia/CA (1)
               sex       Onset

    1         24/F       2 mo           +
    2          8/F       birth          +
    3          9/F       6 yr           +
    4         11/M       9 yr           +
    5          9/M       10 mo          +
    6         12/F       2 yr           +
    7          5/M       1 yr           +
    8          6/M       2 mo           +
    9         10/M       5 yr           +
   10         35/F       3 yr           +
   11         12/F       birth          +
   12         11/M       9 yr           +

Patients    Seizure             DMD (2)    MR

    1       generalized,           +       -
            tonic- clonic
    2       -                      +       -
    3       Psychomotor/           -       -
            pseudoabsences
    4       -                      -       -
    5       -                      +       +
    6       generalized,           +       +
            tonic- clonic
    7       -                      -       +
    8       Febrile seizures       +       -
    9       -                      -       -
   10       -                      +       +
   11       -                      +       +
   12       -                      -

Patients    Other Symptoms         COQ10
                                   Muscle
                                   (ug/g)

    1                              12.18
    2       Muscle weakness         9.94
    3                               2.93
    4       myoclonus               5.94
    5       hemiplegia             12.12
    6       spastic paraparesis    11.01
            muscle weakness
    7       headache                2.92
    8                               8.16
    9       pyramidal signs        12.60
   10       Spastic paresis         9.20
   11       Muscle weakness        12.20
            ophtalmoparesis
   12                               2.90

(1) Cerebellar Atrophy; (2) Developmental Motor Delay; (3) Mental
Retardation

Salvator DiMauro, MD
Columbia University