Single large-scale mitochondrial DNA deletion in a patient with mitochondrial myopathy associated with multiple symmetric lipomatosis

Multiple symmetric lipomatosis (MSL) (Launois-Bensaude syndrome, Madelung's disease) is a rare disorder of middle life characterized by large nonencapsulated lipomas distributed around the neck, shoulders, and other axial regions. ^[1] Neurologic involvement, particularly peripheral neuropathy, is considered an integral part of the MSL syndrome. ^[1] Alcoholism is frequently associated with MSL. ^[1] Recently, Berkovic et al. ^[2] reported an association between MSL and mitochondrial dysfunction. Moreover, further recent reports documented that some patients with MSL harbor mutations in the mitochondrial genome, in particular multiple mtDNA deletions, ^[3,4] or the mutation at nucleotide position (np) 8344 in the tRNA^Lys gene of mtDNA. ^[5-8] Here, we present the first report of a patient with MSL harboring a single large-scale mtDNA deletion.

Case report.

A 60-year-old nonalcoholic woman had a 28-year history of slowly progressive proximal muscle weakness and bilateral ptosis. She had neurosensory hearing loss and tension headache, but no other neurologic symptoms. At age 41 years, large confluent symmetric lipoma developed around the neck in a "horse-collar" distribution. Examination at age 59 years revealed bilateral ptosis without diplopia, and symmetric proximal weakness without wasting, affecting muscles in her upper limbs and especially in her lower limbs. She had exercise intolerance and complained of muscle pain on repeated muscle testing. Deep tendon reflexes were normal.

Cranial nerve examination was normal aside from hearing loss. Extraocular muscles were apparently uninvolved. Ophthalmoscopic examination was normal. Tibial and sural nerve conduction studies were normal, and needle electromyography showed a myopathic pattern. Electrocar-diographic studies showed right bundle branch block. Blood chemistry, including uric acid, and hematologic studies were normal.

A recent clinical reevaluation revealed mild extraocular muscle weakness.

Methods.

Results.

Sections stained with modified Gomori trichrome revealed ragged-red fibers (RRF). These fibers showed strong SDH activity, representing about 10% of fibers. COX activity was absent or deficient in about 20% of fibers. All RRF stained negative for COX, but some COX-negative fibers were not RRF.

Muscle biochemistry was normal; in particular, COX activity was within normal range. Southern blot analysis showed the presence of two populations of mtDNA, one corresponding to full-length genome (16.5 Kb) and the other corresponding to a shorter molecule of approximately 13.9 Kb Figure 1. The proportion of deleted mtDNA was 47%. The deletion spanned from np 10256-10300, at the ND3 gene, to np 12550-12680, at the ND5 gene, encompassing ND3, ND4L, ND4, and ND5 subunits, and several tRNA genes (see Figure 1).

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Figure 1. (a) Southern blot analysis. DNA was digested with BamHI, electrophoresed through a 0.8% agarose gel, transferred to nitrocellulose, and probed with labeled mtDNA. Sizes, in Kb, are shown on left (calculated on the basis of lambda digested with HindIII as markers). In muscle, we found two hybridizing bands: a normal-sized mtDNA of 16.5 Kb and a more rapidly migrating band (13.9 Kb) due to a deletion of 2.6 Kb. (b) Linearized physical map of the human mitochondrial genome and the deleted regions of mutant mtDNA in the patient. The dotted region is the extent of deletion and the hatched regions define upper and lower limits. 12S and 16S = ribosomal RNA genes; ND = NADH dehydrogenase (complex I) subunits; CO = cytochrome c oxidase (complex IV) subunits; ATPase 8/6 = complex V subunits; cyt b = apocytochrome b.

Discussion.

We studied a nonalcoholic woman with MSL, ptosis, proximal weakness, neurosensory hearing loss, mitochondrial dysfunction in skeletal muscle, and no peripheral neuropathy. Molecular genetic analysis revealed a single mtDNA deletion. MSL is sometimes associated with mtDNA mutations, in particular with multiple deletions ^[3,4] and with the point mutation at np 8344 in the tRNA^Lys gene ^[5-8] Table 1. However, there is no report on patients with MSL harboring a single mtDNA deletion. Berkovic et al. ^[2] described four MSL patients with mitochondrial abnormalities. Of them, one had proximal weakness, bilateral ptosis, and hearing loss, taking part of a more complex syndrome involving peripheral nerve and CNS. Klopstock et al. ^[3] documented muscle mitochondrial dysfunction in 7 of 11 patients with MSL. Of them, one harbored multiple deletions of mtDNA in muscle and showed peripheral neuropathy, ataxia, and dysarthria as the most prominent clinical features. Moreover, some patients with MERRF syndrome harboring the 8344 np mutation presented with cervical lipomas resembling those of MSL. ^[5,6,8]

Respiratory chain was normal, but there was focal COX deficiency, particularly in RRF, indicating that 20% of fibers had impairment of mitochondrial protein synthesis. Although the patient had 47% of mutant genomes in muscle, the presence of COX-deficient fibers suggests the accumulation in RRF of deleted mtDNA to levels over 60%.

MSL is a heterogeneous disorder frequently associated with alcoholism. ^[1] A recent report documented normal respiratory chain in muscle from chronic alcoholic patients, thus alcoholism seems unrelated to mitochondrial metabolism in alcoholic MSL patients. ^[12] Among nonalcoholic patients, there is a subgroup of patients without mitochondrial abnormalities, ^[3,13] while another shows mitochondrial dysfunction in muscle. ^[2,3] Even among patients with mitochondrial involvement, the clinical spectrum has a wide range, varying from severe encephalomyopathy, e.g., MERRF, to ocular myopathy. On the other hand, clinical variability occurs in patients with mtDNA deletions. We suggest that MSL is an additional, uncommon manifestation of the wide clinical spectrum of mitochondrial disorders, in particular of those associated with mtDNA deletions.

The reasons for this broad variability may relate to the different distribution of deleted and wild-type mtDNA in different tissues. Our patient conceivably had higher percentages of mutant genomes in clinically affected organs like lipomatous tissue or muscle. In fact, Holme et al. ^[7] found a high proportion of mutant mtDNA in lipomas from a MERRF patient. However, the relation between MSL and mitochondrial dysfunction is unclear. Although the microscopic appearance of lipomas in MSL is similar to that of white fat, brown fat is normally located in the cervical region where lipomas develop in MSL. ^[14] Moreover, brown adipose tissue has abundant mitochondria and a great fat turnover coupled with a particularly high activity of the mitochondrial respiratory chain. ^[14] A mitochondrial dysfunction could therefore lead to progressive lipid storage, making brown fat similar to white fat, thus explaining why the microscopic appearance of MSL adipose tissue resembles that of white fat.

Acknowledgment

We are indebted to Dr. Juan Trueba, consultant neurologist of the Hospital 12 de Octubre.