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Original Studies |
Service d’Endocrinologie Pédiatrique (M.N., M.D., P.C.), Laboratoire de Biochimie Pédiatrique (T.F., B.M.), and INSERM U 329 (M.F.) Hôpital Debrousse, 69322 Lyon, France; INSERM CRI 9401 (H.C.), Faculté de Médecine Alexis Carrel, 69372 Lyon, France; and CNRS UMR 5534 (C. G.), Université Claude Bernard Lyon I, 69622 Villeurbanne, France
Address all correspondence and requests for reprints to: Marc Nicolino, M.D., Service d’Endocrinologie Pédiatrique, Hôpital Debrousse, 29, rue Soeur Bouvier, 69322 Lyon Cedex 05, France.
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Abstract |
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The presence of the same mutation in different patients with various endocrine conditions supports the view that the 7.4 kb mtDNA deletion should be considered as one of the candidate causes for phenotypically uncommon cases of endocrinopathies, specially in children with deafness. This is the first report of a mitochondrial disease with primary adrenocortical insufficiency as the clinical onset.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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In 1992, an A to G transition at nucleotide (nt) position 3243 in the mitochondrial tRNALeu(UUR) gene was reported in large pedigrees with maternally inherited diabetes and deafness (3, 4). Phenotypically, this distinct subtype of mitochondrial diabetes is associated with deafness in more than 60% of the cases and is observed mostly after age 40, accounting for about 1% of noninsulin-dependent diabetes mellitus (NIDDM) (5). On the other hand, sporadic cases with Kearns-Sayre syndrome (KSS) or chronic progressive external ophthalmoplegia (CPEO), two particular forms of mitochondrial myopathy with large-scale deletions in mtDNA, are often associated with endocrine dysfunctions, in particular short stature, hypogonadism, diabetes, hypoparathyroidism, and hypothyroidism (6, 7).
Thus, endocrine dysfunctions are not the main clinical features in the majority of patients affected by a large mtDNA defect, in contrast to predominant encephalomyopathy (8). Consequently, the correct etiological diagnosis is often missed when the disease first manifests as an endocrinopathy with normal neuromuscular function and no particular family history. We report two unrelated patients presenting with endocrinopathy and deafness in childhood, in whom investigations revealed that both had an identical heteroplasmic 7.4 kb mtDNA deletion. These observations suggest that mtDNA deletions should be screened in children with endocrinopathies followed by typical manifestations of mitochondrial encephalomyopathies.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Patient 1 was the only child of unrelated healthy parents. At age 4, a salt-losing adrenal crisis, in the course of bronchitis, was the first manifestation of the disease. Weight loss, hypotension, and increased pigmentation were noted. Serum electrolytes revealed sodium 118 mmol/L, potassium 5.0 mmol/L, glucose 2.10 mmol/L, and CO2 13 mmol/L. Daily excretion of urinary 17-hydroxy-corticosteroids was 0.27 mmol/day (normal: 1.0–5.6). Plasma hormone investigations demonstrated a severe adrenal insufficiency: ACTH = 6600 pmol/L (normal < 100), 17-hydroxy-progesterone = 0.45 nmol/L (normal: 0.49–1.10), PRA = 33 ng/hr/mL (normal: 3.54–8.82), and aldosterone = 8.31 pmol/L (normal: 7.31–14.91). Adrenal antibodies were negative, and there was no evidence for any causative process such as steroid biosynthesis deficiency, congenital ACTH insensitivity, congenital adrenal hypoplasia, adrenal hemorrhage, or tuberculosis. A substitutive treatment with hydrocortisone, fludrocortisone, and salt was instituted. At age 6, mild sensorineural hearing loss was diagnosed. At that time, she had progressive growth failure with height at -2.5 SD. A complete evaluation for growth retardation (-3.8 SD) was performed when she was 10 yr old. A skeletal survey showed slightly advanced bone age (+1 yr), decreased bone density, marked bradymetacarpia, and metaphyseal dysplasia consistent with unexplained osteochondropathy. Peak responses of GH to two consecutive provocative tests with glucagon-propranolol were 8.2 and 10.1 ng/mL (normal > 10). She had a normal sexual maturation and first menses occurred at age 12. During follow-up, a mild hypercalciuria (7 mg/kg·day; normal < 4) and lithiasis on ultrasound of the right kidney were observed.
At age 16 yr, 6 months, she was readmitted because the course of the disease, which involved unrelated symptoms, led to consider a possible mitochondrial disease. She had a severe short stature with final height 134 cm (-4.2 SD). Psychomotor development and school performance were normal. She was given hydrocortisone 17.5 mg, fludrocortisone 50 µg, and sodium chloride 1 g daily. She presented with painful carpopedal spasm in stressing conditions, and Chvostek’s sign was positive. Serum electrolytes were normal except for calcium, which oscillated around 2.0 mmol/L (normal: 2.25–2.60), with low PTH level 6 pg/mL (normal: 10–55). Horizontal and vertical eye movements were restricted, and mild ptosis was noticed. These results were consistent with mild ocular myopathy. Serum CK were slightly elevated 235 IU/L (normal: 60–200), and electromyography was normal. Metabolic investigations demonstrated an elevated lactate level ranging 2.45–3.75 mmol/L 1 h after carbohydrate rich meals (normal: 0.30–2.20). iv glucose loading test (1 g/kg) confirmed elevated lactate level 2.60 mmol/L, suggesting an abnormal oxido-reduction in plasma. However, under all these conditions, lactate/pyruvate (L/P) and 3-hydroxy-butyrate/aceto-acetate (3-OH-B/AA) molar ratios were normal. A cerebrospinal fluid (CSF) examination revealed elevated protein concentration (1.28 g/L; normal < 0.4), with normal lactate and pyruvate. A cerebral magnetic resonance imaging (MRI) scan showed symmetrical degeneration of the white matter in cerebral peduncles and bulb, with hyperintense areas on T2-weighted sequences. Ophthalmoscopy, electroretinography, and visual-evoked potentials showed no abnormalities. Cardiac function was normal on electrocardiography and ultrasonography. She was noticed to have elevated (11.8 mmol/L) plasma glucose, 90 min after meals. Impaired glucose tolerance was confirmed by oral glycaemia tolerance test (OGTT) with 2-h value at 8.5 mmol/L (normal < 6.7). HbA1c was 5.5% (normal: 5.0–6.5); insulin secretion was not evaluated.
At age 17 yr, 2 months, she was referred to the clinic for having proximal muscle weakness and ataxia. Serum electrolytes showed no abnormalities. She rapidly developed neurological deterioration with bilateral pyramidal track signs, difficulties in swallowing, and progressive cognitive dysfunctions. Sudden death occurred by cardiorespiratory arrest 72 h after admission. Autopsy with collection of adrenal tissue was not feasible.
Patient 2 was admitted at age 15 yr, 2 months, for diabetic ketoacidosis. She presented with a 1-week history of polyuro-polydipsia and asthenia. She had lost 13% of her weight and complained of abdominal pain. Laboratory results showed plasma glucose 21.7 mmol/L, sodium 132 mmol/L, potassium 3.7 mmol/L, CO2 11 mmol/L, and heavy ketonuria. Her history was unremarkable, apart from a sensorineural hearing loss since age 6. Antibodies against insulin, islet cells, and GAD were negative. HLA alleles were HLA-DRW11/DRW52. There was neither family history of endocrine or neuromuscular disorders, nor consanguinity. A younger brother was normal.
She had been previously investigated at age 13 for progressive growth retardation. At that time, her height was 140 cm (-2.5 SD) with bone age 13. Physical examination showed poorly developed secondary sex characteristics (A2 P2 S3, according to Tanner classification). She had marked coxa vara associated with genu valgum, and X-rays indicated poorly mineralized bones. GH peak responses of GH to propranolol-glucagon and GHRH were 19.1 and 24.0 ng/mL, respectively. GnRH stimulation test revealed plasma levels of LH and FSH consistent with P2-P3 pubertal stage. A pelvic ultrasonography showed a 29-mm-long uterus and normal sized ovaries with follicles. Menarche subsequently occurred at age 15. Because of the presence of growth retardation associated with deafness, a defect of oxidative metabolism was considered but metabolic investigations (L/P and 3-OH-B/AA molar ratios) were normal. Brain MRI scan was normal. She complained of photophobia with blepharospasm, and an ophthalmological examination revealed a severe oedematous keratitis of unknown cause, and normal lens. Ophthalmoscopy, electroretinography, and visual-evoked potentials were normal.
At age 16 yr, 6 months, she was admitted for further investigations including muscle biopsy. Her final height was 152 cm (-2 SD), and her weight was 43 kg. Her mental development was normal. Neurological examination revealed mild bilateral ptosis but no external ophthalmoplegia. No muscle weakness was noted. CK enzymes were slightly elevated 311 IU/L. Determination of L/P and 3-OH-B/AA molar ratios, before and after meals, still showed normal values. CSF examination revealed elevated protein 1.52 g/L and normal L/P. MRI of brain showed spotty and symmetrical hyperintense areas on T2-weighted sequences in periventricular, cerebral, and cerebellar peduncle white matter. Additional investigations (plasma calcium and phosphate, PTH, liver enzymes, urine analysis, ACTH, electrocardiography, electromyography, cardiac ultrasonography) were normal. The A3243G mtDNA mutation was negative in muscle. Glycaemia was controlled under atypically low doses of insulin (0.4–0.5 U/kg/day) and in absence of diabetic diet, with HbA1c 6% (normal, 4.3–6.1). Peak serum level of C-peptide was 2.58 ng/mL (mean value in subjects with IDDM = 1.80), showing significant residual ß-cell function. Insulin therapy was temporarily stopped; then oral treatment by glibenclamide (5 mg/day) was given alternatively with insulin. Until now, at age 19 yr, 6 months, she remains partially insulin dependent, with HbA1c 7.2%. Her neurological status is unchanged.
Mitochondrial investigations
Muscle biopsy of the quadriceps was performed by an open surgical technique under local anesthesia. Peripheral blood was obtained from both patients and their mothers. Informed consent for the current studies was obtained from patients and parents.
Histochemistry. The stains included modified Gomori’s trichrome, periodic acid Schiff, Sudan black, myosin ATPase at pH 9.4 and 4.65, succinate dehydrogenase (SDH), and cytochrome c oxidase, according to standard procedures.
Respiratory and enzymatic measurements. Oxypolarographic and spectrophotometric analysis of the respiratory chain were performed on isolated mitochondria from muscle of both patients as described previously (9, 10).
Molecular analysis of the mtDNA.
Total DNA derived from
muscle or whole blood was submitted to PCR amplification using eight
combinations of primers (Table 1
) (Fig. 1A). For each combination, the template
DNA (0.25 µg) was added to a final volume at 50 µl of reaction
mixture containing 0.2 mM of each
deoxyribonucleotide, 200 pM of each primer, 2.5 U
Taq polymerase (Eurobio, France). PCR conditions for 25
cycles were 30 sec at 94 C, 30 sec at 55–61 C, and 90 sec at 72 C. PCR
products were further analyzed using appropriate restriction enzymes to
define the approximate breakpoints of the deletions. The restriction
enzymes were selected according to the restriction sites, which are
close to the forward (L) and reverse (H) primers: DraI,
KpnI, MscI, StuI, and XbaI.
For characterization of the nucleotide sequences at the breakpoints of
deleted mtDNA, direct sequence analysis of amplified fragments was
performed by the Sanger method (Thermo Sequenase cycle sequencing kit,
Amersham, France). Samples were electrophoresed through 7
M urea 6% polyacrylamide gels for 2–4 h at 50 W
and autoradiographed for 24–72 h. For Southern-blotting analysis (11),
DNA (5 µg) was digested with restriction enzymes PvuII and
SnaBI, which linearize the mtDNA molecules (cleavage site at
nt 2652 and 10736, respectively).
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Results |
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In the muscle biopsies from both patients, there was a significant number of typical ragged-red fibers (3%). The histoenzymatic reaction of SDH was reinforced for the two muscle biopsies. In addition, black sudan revealed lipid storage in type 1 fibers. For the two patients, ragged-red fibers were cytochrome c oxidase-negative.
Respiratory and enzymatic measurements. The oxidation rates of NADH-linked substrates and succinate were slightly decreased on isolated mitochondria from patient 1. Enzymatic activities of the respiratory chain complexes were normal. For patient 2, oxidation rates of all the substrates were decreased (49–75% of the control means). The complex I (NADH ubiquinone reductase) activity was decreased (62% of the mean control value). The decreased complex I/complex III and complex I/complex IV ratios supported a partial complex I deficiency.
Molecular analysis of the mtDNA.
A large-scale mtDNA deletion
of 7.4 kb-long was detected in both patients by the PCR procedure using
seven combinations of primers. The distance between two primers varies
from 4.5–10.8 kb. In these conditions, a selective amplification of
deleted mtDNA was obtained because the distance between the two primers
flanking the deletion is significantly reduced. For both patients, the
0.6 kb and 3.3 kb PCR fragments obtained with the primer pairs nos. 2
and 7, respectively revealed the 7.4 kb deletion (Fig. 2). A more precise mapping of deletion
breakpoints was performed using restriction enzyme digestion. For both
patients, the 5' breakpoint was between position 8571 (presence of
restriction site StuI) and position 8837 (absence of
restriction site MscI). The 3' breakpoint was included
between position 16008 (absence of restriction site DraI)
and 16115 (hybridization of the reverse primer of pairs nos. 2 and 7)
(data not shown). Following restriction enzyme digestion, sequence
analysis showed that the 7.4 kb deletion was the second common deletion
spanning from nt 8648 in the ATPase 6 gene to nt 16085 in the
D-loop (7, 436 nt deleted). The deletion encompassed genes encoding for
a part of ATPase 6, the whole COIII, ND3, ND4L/4, ND5, ND6,
cytochrome b, a part of the noncoding D-loop and 8 tRNAs
(Gly, Arg, His, SerAGY, LeuCUN, Glu, Thr, Pro). The deleted region is
flanked by two perfect 12 nt direct repeats in the native sequence (1),
one of which being lost during the deletion process (Fig. 1B
).
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). The total relative amount of deleted
mtDNA was 25% and 15% for patients 1 and 2, respectively. No complex
mtDNA rearrangement such as partial duplication was identified. In
addition, the 9.1 kb deleted mtDNA was not detected in more than 100
muscle controls.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The 7.4 kb mtDNA deletion would be expected to mainly result in a rapidly progressive neuromuscular disease, as seen in most patients with KSS or CPEO, which are also characterized by mtDNA deletions between 2 and 7 kb (2). It is conceivable that the relatively low proportion of deleted mtDNA found in the muscles of our patients (25% and 15%) was insufficient to clinically impair the respiratory chain in muscle, possibly explaining the absence of severe myopathy in both cases. This should also explain that routine metabolic and enzyme histochemical investigations were not strongly conclusive. For unclear reasons, the 7.4 kb mtDNA deletion produced predominant endocrine manifestations for a long period in both cases. We suggest that the accumulation of deleted mtDNA may be higher in adrenal cortex and pancreatic ß-cells rather than in muscle or the central nervous system, resulting in a more rapidly progressive decline in oxidative phosphorylation in these endocrine tissues.
In fact, the 7.4 kb deletion is the second most common among those that have been previously described, encompassing 8/13 genes encoding for the mitochondrial OXPHOS subunits and 8/22 tRNAs (2). The proteins encoded by the mitochondrial genes, which are eliminated through the 7.4 kb mtDNA deletion could be rate-limiting in the affected tissues. Furthermore, it is likely that the elimination of 8/22 tRNAs results in a generalized translational defect. This deletion was previously reported in the cardiac muscle (9% deleted mtDNA) of elderly individuals as a probable consequence of normal ageing (13) and in cirrhotic liver surrounding hepatic tumors (14). More recently, it was also reported in leukocytes of a case with Pearson marrow-pancreas syndrome (60% deleted mtDNA) (15). However, none of these patients had any endocrinopathy. In the present study, it is noteworthy that both patients presented as children. The mode of inheritance was probably sporadic as it is described in most diseases caused by mtDNA deletions, namely KKS, CPEO, Pearson syndrome, or mitochondrial disorders with multiorgan involvement (2, 16). The lack of deleted mtDNA in the blood of the two unaffected mothers and the absence of affected relatives suggested a de novo deletion. In addition, the presence of perfect direct repeats at the boundaries of the deletion might have easily promoted this de novo mtDNA recombination via a slipped mispairing during mtDNA replication (17, 18). However, the presence of deleted mtDNA in the germ-line cells of the two mothers cannot be excluded (19). DNA studies in other tissues of the mothers were not available for providing definitive proof that the deletion was not maternally transmitted.
The observations illustrate the association of a variety of endocrine symptoms with a particular mtDNA defect. Diabetes is frequently described in mitochondrial diseases with mtDNA rearrangements. Ballinger et al. (20, 21) first reported adult-onset diabetes and deafness associated with a maternally inherited complex mtDNA rearrangement; three interrelated mtDNA molecules were shown to exist within patient tissues: a deleted mtDNA that lacked 10.4 kb of the genome (present as 12 kb dimer), a partially duplicated (23 kb, containing an extra 6.1 kb) mtDNA, and the normal (16.5 kb) mtDNA. Complex mtDNA rearrangements have also been reported in diabetes mellitus associated with KSS (7), CPEO (22) or multisystem disorders (23). Some of us have previously reported a case of Wolfram syndrome (diabetes insipidus + diabetes mellitus + optic atrophy + deafness) with early onset IDDM caused by a 7.6 kb deletion of mtDNA (24). Interestingly, no patient with diabetes mellitus has been previously described with the 7.4 kb deletion. Similar to other reports (5), our findings further show that a combination of both sensorineural deafness and diabetes mellitus is not only associated with point mutations in the mt tRNALeu(UUR) gene.
Short stature and hypoparathyroidism were also obvious in both patients. However, it is the first time that a defect in mitochondrial respiratory chain is reported with a clinical onset of adrenocortical insufficiency. Hypoaldosteronism alone has been described previously in association with OXPHOS defects in two different cases (25, 26). To our knowledge, the only evidence of complete adrenal insufficiency due to a possible mitochondrial disorder is the recent report of North et al. (27) describing a 18-month-old girl with an OXPHOS defect who had a phenotype characterized by neonatal onset of chronic lactic acidosis, lipid storage myopathy, bilateral cataracts, and developed primary adrenal insufficiency at 7 months of age. Unfortunately, the molecular defect was not identified in this study. Given the fact that correct cause is not established in a number of cases with adrenal insufficiency (12), our results may be of interest by providing a potential etiology in these patients. On the other hand, it is possible that the 7.4 kb deletion is not the direct cause of the adrenal insufficiency in this study. We could not check for the presence of the 7.4 kb deletion in adrenal tissue in order to obtain definite evidence of involvement of the deleted mtDNA in the pathogenesis of the adrenal dysfunction. Nevertheless, the absence of other etiology strongly suggests the genetic disorder of OXPHOS. The possible process in which the deletion would affect adrenal function is probably a defect in the secretory capacity of adrenocortical cells, which might be due to an impaired mitochondrial ATP production, but this hypothetical mechanism remains to be established.
In conclusion, the most interesting aspects of the present study are: 1) the two patients suffer from endocrinopathies of early onset and deafness associated with a 7.4 kb mtDNA deletion; and 2) a mitochondrial disorder may account for adrenal insufficiency presenting as the onset symptom.
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Acknowledgments |
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Footnotes |
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2 Recipient of the HCL (Ministère de la Santé, Projet
National de Recherche Clinique, 1993).
Received October 14, 1996.
Accepted June 5, 1997.
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References |
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) mtDNA was
amplified with primer pairs nos. 2 and 7 for both patients. The size of
the deletion (approximately 7.4 kb) was obtained by subtracting the
sizes of amplified fragments from distances between the primers.
Wild-type (wt) mtDNA was amplified using primer pairs nos. 6 and 8 for
both patients. The amplification of the deleted mtDNA was only observed
when the two primers of each pair were located outside the deletion,
that is with primer pairs nos. 2 and 7. For both patients, the primer
pair no. 8 was used as an internal control to amplify wt mtDNA in a
region that is not usually deleted. M is a 1 kb ladder mol wt marker.