Main

KSS is mainly recognized as a neurologic disorder with variable encephalomyopathic symptoms, such as progressive external ophthalmoplegia, pigmental retinopathy, cerebellar signs, sensorineural hearing impairment, muscle weakness, and mental deterioration(1). An elevated cerebrospinal fluid protein concentration and ragged red fibers in muscle biopsy specimens are further features of this mitochondrial disease(2).

Nonneurologic manifestations may include cardiac conduction defects(3) and a wide range of endocrine disturbances(1, 4). The most frequent endocrinopathies are growth retardation, hypogonadism, and diabetes mellitus. Hypoparathyroidism, hyperaldosteronism, hypomagnesemia, and thyroid dysfunction occur in less than 10% of patients. Cases with hypoparathyroidism have been considered to constitute a distinct subgroup because they show multiple endocrine abnormalities 2-4 times more frequently(5).

More than 90% of KSS patients harbor deleted mtDNA in muscle(6) and brain(7). The deletions are heteroplasmic and span about 3-9 kb of the 16.5-kb sized mitochondrial genome. There is no clear-cut correlation between the deletion sizes and heteroplasmic proportions in muscle on the one side and the clinical and biochemical presentations on the other side(8, 9).

There is now increasing evidence that mtDNA deletions observed in KSS and in other multisystem disorders are part of complex mtDNA rearrangements(10, 11). Heteroplasmic tandem duplications of mtDNA have been detected in mitochondrial disorders with early onset diabetes mellitus(12–15). More complex mtDNA rearrangements involving partial duplications, deletion monomers, and/or deletion dimers (pleioplasmy) have been observed in KSS patients with and without diabetes mellitus but not in patients with isolated progressive external ophthalmoplegia(11, 16). To date, no molecular genetic studies are available on KSS patients with hypoparathyroidism.

We described four children (three boys and one girl) who presented with hypocalcemic tetany caused by hypoparathyroidism and with hearing impairment in the early course of KSS. All of them harbor a similar pattern of pleioplasmic mtDNA rearrangements with a detectable level of partial duplications and deletions in their blood cells. The data obtained in this study show that KSS patients with hypoparathyroidism are characterized by the same mitochondrial genotype as those with diabetes mellitus.

METHODS

Subjects. The patients, three boys and one girl, are children of four unrelated healthy parents without histories of neurologic or endocrine disorders in their families. Pregnancies and deliveries were uncomplicated, and birth sizes were normal. Patient 2 had a younger brother, and patient 4 had two older brothers, all of them healthy.

Patient 1. This boy appeared normal until his 5th y of life when loss of acquired mental skills was recognized. At the age of 12 y he experienced the first episodes of mild cramps and paresthesia in his hands. Eleven months later tetanic convulsions occurred with carpopedal spasms and generalized stiffness. Clinical examination of the mentally retarded boy showed short stature (133 cm, <P3), bilateral ptosis, external ophthalmoplegia, and hearing impairment. His teeth were carious due to enamel dysplasia. Funduscopy showed no retinal changes at this time. Cranial computed tomography demonstrated symmetric widespread calcifications of basal ganglia and central parts of cerebellum. Cerebrospinal fluid protein (0.72 g/L; normal, <0.32 g/L) and lactate (2.8 mmol/L; normal, <2.3 mmol/L) concentrations were increased. The boy was treated with 1,25-(OH)2-cholecalciferol (12.5 ng/kg/d) and calcium (0.5 g/d). During the following years his neurologic condition deteriorated progressively. At the age of 15 y he presented with almost complete external ophthalmoplegia, intention tremor, gait ataxia, and sensorineural deafness. His vision was bilaterally reduced to 0.4 caused by the now evident pigmental retinopathy with torquiated vessels. ECGs did not indicate any cardiac conduction defect. A pathologic variation in fiber size with hypotrophic type II fibers was present in muscle tissue (m. quadriceps) taken by needle biopsy. Nevertheless, neither ragged red nor COX-negative fibers were seen. At the ultrastructural level, numbers of mitochondria were normal in the subsarcolemmal space, and only few mitochondria showed abnormal christae.

Patient 2. This boy was normal until the age of 4 y when amblyopia was suspected. Ophthalmologic examinations revealed impaired vision(0.6 and 0.7), visual field defects, and accumulated pigment granules of the retina. Bilateral sensorineural hearing impairment was detected at age 6. Aged 8 10/12 y he experienced an acute hearing loss on the right side during a febrile illness. While treated parenterally with corticosteroids and pentoxiphylline he developed hypocalcemic tetany with carpopedal spasms. Clinical examination showed small stature (124 cm, P2) and deterioration of his vision and hearing, but no myopathic or neurologic symptoms. ECG and computed tomography of the brain were normal. The boy was treated with 1,25-(OH)2-cholecalciferol (22.5 ng/kg/d). Doses could be reduced continuously to 8 ng/kg/d, and calcium supplementation (0.5 g/d) was stopped 3 y after onset. At the time of the last assessment, at age of 12 1/12 y, the patient had developed mild bilateral ptosis, restricted eye movements, dysmetric signs, and a right bundle branch block on ECGs.

Patient 3. This boy developed normally in infancy and childhood; his height was at P50 before disease onset. At age 5 8/12 he suffered from hypocalcemic tetany with painful paresthesia in his hands and carpopedal spasms. Clinical examination revealed impaired hearing and decelerated growth rate of his height (4 cm/y). Oral administration of 1,25-(OH)2-cholecalciferol (27.5 ng/kg/d) and calcium (0.5 g/d) was initiated. Since his 9th y the doses were decreased to 4 ng/kg/d and 0.25 g/d, respectively. At age 9½ y ptosis and external ophthalmoplegia were noticed during “acute keratoconjunctivitis.” Audiometric studies revealed moderate deafness due to sensorineural hearing loss. No retinal changes were seen but electroretinography demonstrated significantly delayed A waves, indicating tapetoretinal degeneration. Despite administration of coenzyme Q (250 mg/d) and riboflavin (300 mg/d) his neurologic course progressively deteriorated with muscular weakness, ataxia, and severe scoliosis. At present, his vision is reduced to 0.1 and 0.2. There is a striking light sensitivity due to generalized microcystic degeneration of the cornea. Electrocardiographic records show a prolonged Q-T interval. Magnetic resonance imaging of the brain demonstrates multiple bilateral lesions within the thalamus and brain stem similar to those seen in Leigh syndrome.

Patient 4. Early development of this girl was normal but it was noted that she was never able to run fast. At the age of 3 y she developed bilateral ptosis and sensorineural deafness requiring hearing aids. Aged 4 11/12 y hypocalcemic tetanic convulsions occurred with headache, generalized stiffness, carpopedal spasms, and brief loss of consciousness. Clinical examination demonstrated short stature (98 cm, <P3), proximal muscular weakness, bilateral ptosis, and external ophthalmoplegia. Funduscopy showed retinal “pepper-and-salt” changes. She was placed on cholecalciferol (1000 IU/d) and calcium (3.0 g/d). At the age of 7 6/12 y the girl presented with complete external opthalmoplegia, tremor in her legs, gait ataxia, and a right bundle branch block on ECGs. Magnetic resonance imaging and computed tomography of the brain were normal. Cerebrospinal fluid studies showed increased protein (1.33 g/L) and lactate (4.0 mmol/L) concentrations. Histologic analysis of muscle tissue taken by needle biopsy revealed mild myopathic changes with increased variation in fiber size and numerous ragged red fibers, one third of them being cytochrome C oxidase-negative. Ultrastructural studies showed subsarcolemmal accumulations of mitochondria with enlarged and malformed christae. At that time cholecalciferol was exchanged by 1,25-(OH)2-cholecalciferol (25 ng/kg/d) and calcium supplementation was stopped. Due to hypercalcemia and hypercalciuria the dose of 1,25-(OH)2-cholecalciferol was continuosly reduced, and 7 mo later the girl remained normocalcemic despite stopping treatment. At age 9 she showed latent hypothyroidism (TSH 5.16 mU/L, normal <4 mU/L) and prediabetes with normal glucose levels preprandial but elevated levels after oral glucose load (14.3 mmol/L at 60 min). No autoantibodies were detectable in blood against parathyroid, thyroid, and pancreatic tissue antigens. Despite treatment with riboflavin and coenzyme Q10 (300 mg each/d), her neurologic condition continued to deteriorate.

Hypocalcemia and hyperphosphatemia were detected in all children at the time of first tetanic episodes caused by hypoparathyroidism with serum magnesium levels being normal or slightly diminished (Table 1). Determination of the following parameters yielded normal results in all patients studied: red and white blood cell counts, platelets, sodium, chloride, serum osmolarity, glucose, creatinine, creatinine clearance, uric acid, lactate, creatine kinase, aspartate aminotransferase, alanine aminotransferase, γ-glutamyl transferase, alkaline phosphatase, coagulation screening, and 25-OH- and 1,25 (OH)2-cholecalciferol(before therapy), triiodothyronine, and thyroxine. Oral glucose load and TSH were normal in patients 1-3.

Table 1 Laboratory findings in serum at the first major tetanic episode (prior to treatment)
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Biochemical analysis. Frozen muscle specimens obtained by needle biopsy were used to prepare homogenates. Activities of rotenone-sensitive NADH:Q1 oxidoreductase (complex I) and succinate:cytochromec oxidoreductase (complex II/III) were measured as previously described(17). COX (complex IV) and citrate synthase activities were determined according to Cooperstein and Lazarow(18) and Srere(19), respectively. The method of Lowry et al.(20) was used to measure the protein content of muscle homogenates.

Molecular DNA analysis. Total genomic DNA was isolated from venous blood cells using standard protocols.

Southern blot analysis. DNA, 0.5-2.0 μg, was digested withPvu II or SnaBI/BglII (10 U each), respectively, for 4-6 h in reaction buffer supplied by the manufacturer. Separation of the DNA fragments by 0.8% agarose gel electrophoresis, transfer to positively charged nylon membrane (Boehringer Mannheim, Germany) and hybridization were performed as described previously(21). Two different mtDNA probes (S1, S3) generated by PCR were used for hybridization experiments(see below). Probe labeling and detection of hybridized DNA were carried out by using the Digoxigenin labeling and detection kit (Boehringer Mannheim, Germany) according to the manufacture's recommendations. The visualized mtDNA bands were densitometrically analyzed using the One Dscan software(Scanalytics; Billerica, MA).

PCR amplification. PCR-based deletion screening of the mtDNA and regional mapping procedures were performed as described elsewhere(21). To generate the mtDNA probes (S1, S3) and the template DNA suitable for sequencing of the patients' individual breakpoints, 0.5 μg of total DNA was submitted to long distance PCR amplification with the following pairs of oligonucleotides: probe S1, 5′-AAT CTG AGG AGG CTA CTC AGT A-3′ (nt 15 235-15 256) and 5′-TCC ATA GGG TCT TCT CGT CTT-3′ (nt 2734-2714); probe S3, 5′-TCT GTG GAG CAA ACC ACA GTT-3′ (nt 8181-8201) and 5′-GAA GCT TAG GGA GAG CTG GG-3′(nt 12 576-12 557); patients' template DNA 5′-CAG AAG CTG CCA TCA AGT AT-3′ (nt 4627-4646) and 5′-ACA GTT CAC TTT AGC TAC CC-3′(nt 16 491-16 472). After addition of 20-50 pmol of each primer, DNA was amplified on a programmable heating block (Hybaid) by a Taq DNA polymerase mix (3.5 U) containing 0.1:0.1 Pwo polymerase (Boehringer Mannheim, Germany) in 50 μL of its buffer. Cycling conditions, after an initial step at 94°C for 2 min, were: 30 cycles each consisting of 20 s at 94°C, 30 s at 60°C, and 8 min at 68°C with an increment of 20 s in the last 20 cycles, followed by a single step of 7 min at 68°C. The amplified DNA was electrophoresed through 1% agarose and visualized by ethidium bromide staining. Appropriate PCR fragments were isolated from excised gel slices according to standard protocols (Qiagen, Inc., Chatsworth, CA).

Sequencing of the PCR products. Approximately 100 ng of purified DNA were sequenced on an ABI 377 DNA sequencer (Applied Biosystems, Foster City, CA) using the ABI dye terminator kit (Applied Biosystems) in accordance with the manufacturer's recommendations. DNA sequence analysis including breakpoint identification was carried out using the DNASIS software (Hitachi, San Bruno, CA).

RESULTS

Muscle biochemistry. The results of the biochemical study on the respiratory chain in muscle homogenates are summarized in Table 2. Enzyme activities expressed in relation to citrate synthase were normal for complex I, II/III, and IV in patient 1. In patient 4, complex I and IV activities were reduced to 75 and 58% of the lower limit, respectively, whereas complex II/III activity was within the normal range.

Table 2 Biochemical analysis of the respiratory chain in muscle homogenate
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mtDNA studies. Southern blot analysis ofPvu II-digested blood cell DNA (single cleavage site at nt 2652) by hybridization with probe S1 revealed a two-banding pattern in all four patients (Fig. 1a, lanes 2, 4, 6, and 8). The upper bands corresponded to the normal 16.5-kb mtDNA population of a control (lane 1). The lower bands of 7.1-8.8 kb in size were not detected by hybridization with the probe S3 (Fig. 1b, lanes 2, 4, 6, and 8). These results are consistent with partial deletions (deleted monomers) present in blood cells of all patients.

Figure 1
figure 1

Southern blot analysis of leukocyte mtDNA restricted with PvuII (lanes 1, 2, 4, 6, and 8) or SnaBI (lanes 3, 5, 7, and 9). Lane 1, control DNA; lanes 2 and 3, patient 1; lanes 4 and 5, patient 2; lanes 6 and 7, patient 3; lanes 8 and 9, patient 4. S1 (nt 15 235-2734) was used in a as hybridization probe which detects partially duplicated (A), wild-type mtDNA (B) and deleted monomers (C). S3 (nt 8181-12 576) was used in b as hybridization probe which detects partially duplicated (A) and wild-type mtDNA (B).

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Southern blot analysis of DNA double-digested with SnaBI (single restriction site at nt 10 736) and BglII (nuclear DNA restriction only), and hybridization with the probe S1 revealed a quite different banding pattern consisting of three major bands (Fig. 1a, lanes 3, 5, 7, and 9). The middle band of each triplet was of identical size as the control (lane 1), thus corresponding to the normal 16.5-kb mtDNA. The upper bands ranging from 23.6-25.3-kb in size were detected by the probe S3(Fig. 1b, lane 3, 5, 7, and 9). Thus, these rearranged mtDNA populations contain sequences, which are absent in the deleted molecules generated by PvuII cleavage. The results are consistent with an insertion of mtDNA sequences which encompass areas outside the deleted regions(compare Fig. 2). Because these partially duplicated molecules were cleaved by PvuII into two fragments, a 16.5-kb full-length fragment and a smaller (deleted) fragment, as demonstrated above, they must harbor two PvuII sites, separated by the smaller fragment and thus must share the same region with deleted monomers.

Figure 2
figure 2

Schematic diagram showing the partially duplicated mtDNA molecule (A), the normal mtDNA (B), and a partially deleted mtDNA monomer (C). Regions which correspond to deleted sequences are shown as open arc of circle in A and B, whereas retained regions are shown as filled arc of circle in A, B, and C. Duplicated regions in A are shown as stippled arc of circle. O(H) and O(L) denote the localization of the origins of heavy and light strand replication. The triangles mark the restriction sites of PvuII (▸) and of SnaBI (▸▸), respectively, beams (|) mark the breakpoints of rearrangements inA and C.

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The major bands below the 16.5-kb band were not detected by the S3 probe (Fig. 1,a and b, lanes 3, 5, 7, and 9), suggesting that the same sequences were lacking as in the partial deleted monomers generated by PvuII. Further, this population migrated slower than the corresponding deleted monomers generated by PvuII (at 9.8 kb in patient 1, at 7.5 kb in patient 2, at 8.8 kb in patient 3, and at 10.9 kb in patient 4). This was due to the loss of the SnaBI restriction site, which is located within the deleted region in all patients. Consequently, the deleted monomers are not linearized and show an altered mobility during gel electrophoresis caused by their circular form. The minor bands below correspond most likely to deleted monomers that have been linearized by mechanical shearing during DNA processing.

Thus, beside normal mtDNA, partial duplications and partial deletions were present in blood cells of every patient. Determination of the proportion of each mtDNA population (Table 3) demonstrated that duplicated mtDNA was predominant (38-53%) in all patients except for patient 1 in whom wildtype mtDNA prevailed. The deleted monomers varied from 5 to 30%, and the normal mtDNA from 25% in patient 4 to 70% in patient 1.

Table 3 Sizes and proportions of the different mtDNA populations in leukocytes (L)
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The breakpoints of the patients' rearrangements were localized within the region of nt 5800-15 800 by the use of appropriate PCR primers (Fig. 3). As indicated by sequence analysis, the deletion in patient 1 spanned 8348 bp, from nt 5941 to 14288; in patient 2, 9485 bp, from nt 6124 to 15 608; in patient 3, 8587 bp, from nt 6333 to 14 919; and in patient 4 spanned 7813 bp, from nt 7883 to 15 695 (Fig. 4). Thus all deletion types encompassed four genes of complex I (ND3, 4L, 4, 5, and part of 6), both genes of complex V (ATPase 8 and 6), two genes of complex IV (part of COXII and III), and six tRNA genes (tRNALys, tRNAGly, tRNAArg, tRNAHis, tRNAS-er(AGY), and tRNALeu(UCN)). Beyond that, the deletions of patients 1-3 included the complete COXII gene, parts of the COXI gene, and the tRNASer(UCN) and tRNAAsp genes. The deletions of patients 2-4 included the complete ND6 gene, different parts of the cytochrome b gene, and the tRNAGlu gene.

Figure 3
figure 3

Identification of mtDNA rearrangements by PCR breakpoint detection. Leukocyte mtDNA were subjected to long distance PCR analysis with the primers nt 4627-4646 and nt 16 491-16 472 followed by 1% agarose gel electrophoresis containing ethidium bromide. Lane 1, patient 4; lane 2, patient 1; lane 3, patient 3; lane 4, patient 2; lane M, molecular weight marker (λ HindIII). The DNA fragment of 11 865 bp corresponds to the normal mtDNA, and the fragments of smaller sizes correspond to the rearranged mtDNA populations.

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Figure 4
figure 4

Regional assignment of the deletions. Cytb, cytochrome b; O(H), O(L), P(H), and P(L), origins of replication (O) and promotors (P) of the heavy(H) and light (L) strand; single capital letters denote tRNA genes encoding corresponding amino acids; S1, S3, regional assignment of hybridization probes.

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The sequence analysis of the end points (Table 4) showed imperfect direct repeats present in all rearrangments which, however, do not precisely flank the breakpoints (imperfect direct repeats, class II repeats according to Mita et al.(22). Single repeated elements of 11 and 10 bp were identified in patients 1 and 2, respectively, whereas in patients 3 and 4, pairs of repeated elements (6 bp;17 bp and 12 bp;5 bp, respectively) were found.

Table 4 Nucleotide sequences of the L-strand at the boundaries of mtDNA rearrangements
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Gene fusion results in the disruption of the open reading frame in patients 1, 3, and 4 due to the introduction of a stop codon 3, 14, and 4 triplets after the breakpoint fusion, respectively. In patient 2, the first 73 amino acids of the predicted fusion protein are part of the COXI subunit and are extended by 99 amino acids until a stop codon terminates the open reading frame. Finally, PCR analysis with specific primers failed to demonstrate rearranged mtDNA molecules in the blood cells from the mothers of the patients.

DISCUSSION

In four unrelated children (three boys and one girl) with KSS and hypoparathyroidism and deafness as early symptoms, pleioplasmic large scale mtDNA rearrangements were identified in blood cells. All patients were sporadic cases without any family history of neurologic or endocrine disorders. Hypocalcemic tetany occurred between the ages of 6 and 13 y with paresthesia and carpopedal spasms or, more severely, with generalized convulsions and stiffness. Sensorineural hearing deficits were consistently present at onset, in two of them (patient 2 and 4) starting before manifestation of PTH deficiency. Myopathic symptoms, however, such as progressive external ophthalmoplegia and bilateral ptosis were present only in two children from the beginning (patients 1 and 4), whereas both the others(patient 2 and 3) showed a latency of 3-4 y between first symptoms due to hypocalcemia and ocular myopathy.

Previous case reports confirm that hyoparathyroidism in KSS almost exclusively manifests during childhood and may precede myopathic and neurologic signs(4, 23–29). The mean age at onset is 9 ± 4 y with a wide range from 4 mo(30) to “late teenage”(31). As reported here, more than half of cases described so far first presented with tetanic symptoms due to hypocalcemia. In some patients other features of KSS (mainly ptosis) can predate the hypocalcemia(5), but they usually remain unrecognized.

Hypoparathyroidism was proven in our patients by reduced serum concentrations of intact PTH and hyperphosphatemia. None showed evidence of dietary, malabsorbent, or metabolic vitamin D disorders(31), or autoimmune mechanisms(32) which have been assumed to cause hypoparathyroidism in KSS. Severe hypomagnesemia, known to suppress PTH secretion, was observed in two patients(25, 32) but was absent at onset of tetany in our children. Hence it seems likely that factors inherent to parathyroid glands are involved in the pathogenesis of PTH deficiency at the level of PTH synthesis or secretion. For unknown reasons the requirement of 1,25-(OH)2-cholecalciferol and calcium decreased in all four patients with increasing age and one child (patient 4) even remained normocalcemic despite withdrawal of this therapy.

The mtDNA analysis of blood cells from all our patients revealed pleioplasmic rearrangements, consisting of partially duplicated and deleted genomes and normal mtDNA. The deletions are of different sizes ranging from 7813 to 9485 bp, and invariably retain the D-loop, both promotors and origins of replication, at least 11 of 22 tRNAs, and 2 of 16 encoding genes. The duplications involve one full-length and one partly deleted mitochondrial genome. Their sizes thus depend on the sizes of deleted sequences, as illustrated in Figure 2. Patient 2 for example harbors the smallest duplication (23 653 bp) and the largest deletion (9485 bp), whereas patient 4 carries the largest duplication (25 324 bp) but the smallest deletion (7814 bp). The sizes neither of duplicated nor of deleted mtDNA correlate with their amounts present in blood. The fact that the largest duplication (patient 4) was associated with the highest amount of duplicated mtDNA contrasts with data reported by Poulton et al.(16) who found a decrease of the amount of duplicated mtDNA with increased duplication size.

The breakpoints of all rearrangements are flanked by imperfect direct repeats of 10-17 bp in length (Table 4). In contrast to class I repeats, which are located at the edges, repeated elements in our patients are located imprecisely relative to the breakpoints (class II repeats). Additional direct repeats of smaller size (5 and 6 bp, respectively) are present in patients 3 and 4, which, however, are located further away from the breakpoints as the larger ones. Sequence analysis of patients 1 and 2 exemplifies that 5′ as well as 3′ imperfect repeats may be retained. These data support the assumption that slip replication mechanisms(33) or homologous recombination events(22, 34) may play a major role in the formation of partial duplications(11, 14). In mammalian cells a considerable fraction of normal mtDNA molecules is present as catenated circles formed by two or more circular mtDNAs(35) and as dimers sized 32 kb in mice and 33 kb in human(36, 37). Recombination between direct repeats of the same mtDNA genome with loss of the DNA among them would result in a partial duplication as observed in our patients. Alternatively, slip replication might result in the same rearrangement if the partially deleted circle is not separated from the normal circle. According to this model, deleted monomers might arise from the partially duplicated molecules due to subsequent steps that involve similar mechanisms(11). Once generated, the duplicated molecules may be prone to replicate even faster and accumulate in rapidly dividing cells due to the presence of two pairs of origin of replication(10, 12).

The mtDNA findings of our study are consistent with these hypothetic considerations. Determination of mtDNA populations in our patients shows that rearranged mtDNA is present in leukocytes at levels up to 75%. The amount of duplications exceeds the deleted mtDNA in all cases. Nevertheless, none of our patients has ever had signs or history of pancytopenia or blood cell dysfunction. This may be related to the small proportion of deleted mtDNA not exceeding 30%. In Pearson's bone marrow-pancreas syndrome, however, pancytopenia is usually associated with 60% and more deleted mtDNA present in blood cells(38, 39). Hence, partial duplications seem not to be as deleterious as the deleted counterparts. This assumption is supported by the observation that in patient 4 platelet morphology and function (including adhesion capabilities and thrombelastographs) are normal, although they harbor less than 5% wild-type mtDNA but huge amounts (>95%) of duplicated mtDNA (E. Wilichowski, unpublished data).

On the other hand duplications are almost invariably associated with multisystem manifestation(12, 13, 15, 16). In our patients the amount of rearranged genomes correlated with the age at onset of first tetanic symptoms such that the higher the amount of mutant mtDNA the earlier tetany appeared. Although these data obtained from blood cells have to be interpreted cautiously they indicate that rearrangements are implicated in the pathogenesis of hypoparathyroidism.

Due to the two sets of origins of replication duplicated molecules are thought to escape from selection in rapidly dividing cells, tend to spread out, and thus affect many tissues. Two recent autoptic studies confirmed wide tissue distribution in patients with multisystem disorders(40, 41). It is therefore reasonable to assume that the pleioplasmic mtDNA rearrangements detected in our patients are also present in the parathyroid glands and the hair cells of the inner ear causing PTH deficiency and sensorineural deafness, respectively. Up to now, there are only few reports in the literature on autoptic investigations and no molecular studies of parathyroid glands in KSS patients. No parathyroid glands were found in one case with hypoparathyroidism(42), and only one parathyroid gland of histologically normal appearance was found in another case(25). Immunohistochemical studies on parathyroid glands with adenomatous proliferation and hyperplasia showing randomly distributed cytochrome C oxidase deficiency of oxyphil cells indicate that mitochondrial abnormalities are expressed in cells involved in the regulation of serum PTH level and calcium homeostasis(43).

PTH is synthesized in the parathyroid clear and oxyphil cells, both of which are conspicuous for their high numbers of mitochondria(44). The regulation of PTH secretion involves exocytosis of secretory granules within seconds, PTH synthesis over hours, and changes in parathyroid cell numbers over days and months(45). The extracellular domain of the calcium-sensing receptor interacts with the blood ionized calcium and in case of reduced levels activates protein kinase C and/or coupled GTP binding polypeptides at the intracellular site. These G proteins in turn are coupled with effector proteins, such as adenylate cyclase that generate cAMP from ATP. This second messenger may act directly or indirectly at any stages of the PTH synthesis and release, including expression of the nuclear PTH gene(46). In this process ATP is required to mediate the cellular signaling at many sites (Fig. 5). As a consequence, the lack of ATP caused by mtDNA rearrangements may alter the signaling pathway and results in the inability of the parathyroid gland to release PTH at adequate amounts. Up to now, no data are available which support this hypothesis of disturbed signaling pathway, which might explain multiple symptoms in mitochondrial disorders in analogous fashions.

Figure 5
figure 5

Schematic representation of PTH secretion in the parathyroid cell according to Brown(45) and Watson and Hanley(46). The membrane-bound calcium receptor interacts with extracellular ionized calcium [Ca2+]e and transduces the signal by changes of intracellular calcium level[Ca2+]i or conformational changes of linked effector proteins. Low [Ca2+]e results in a decrease of[Ca2+]i. Protein kinase C is activated in this situation and mediates the release of newly synthesized PTH by an ATP-consuming liberation process. Furthermore, low[Ca2+]e causes via the calcium receptor conformational changes of a G protein which in turn, after binding of GTP, activates the coupled adenylcyclase. Increased cytosolic cAMP promotes PTH gene expression due to interaction with a cAMP response element (CRE) and mediates the release of stored PTH by activated kinases and an ATP-consuming process.

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In conclusion, this study provides evidence that KSS with hypoparathyroidism and deafness as the main initial presentation is associated with pleioplasmic mtDNA rearrangements. The presence of high amounts of duplicated and deleted populations in blood cells reflects their wide tissue distribution and indicates that ATP deficiency is involved in the pathogenesis of PTH deficiency and hearing loss. Furthermore, analysis of blood mtDNA allows a fast and reliable identification of this mitochondrial disorder even in the absence of neurologic symptoms and its separation from other types of hypoparathyroidism with deafness caused by nuclear gene defects(47).