A Novel Unstable Mutation in Mitochondrial DNA Responsible for Maternally Inherited Diabetes and Deafness

The patient is a normal-weight (BMI, 22.5 kg/m²), 77-year-old male suffering from diabetes since the age of 45. Insulin had to be introduced at the age of 69 because of poor metabolic control under maximal oral treatment. At admission, his HbA_1c was 9% despite two injections of NPH insulin. He was switched to an intensified insulin treatment by injections of premixed insulin analogs three times a day (0.87 units/kg/day, Lispro/NPH, 50/50 and 25/75; Lilly, Indianapolis, IN). Thereafter, HbA_1c ranged between 6.8 and 7.6%. He reported a history of myocardial infarction 20 years ago and an acute congestive heart failure episode 12 months ago. Since this episode, he was treated with an ACE inhibitor, anticalcic drug, and furosemide. At admission, the heart echography and probrain natriuretic peptide value (39 pg/mL) were in the normal range. Plasma creatinine (86 μmol/L) and microalbuminuria (<2 mg/L) were normal. Fundus examination detected only signs of mild background diabetic retinopathy. Macular pattern dystrophy was absent. The patient suffered from chronic bilateral hearing impairment and required permanent use of a conventional hearing aid. Audiogram showed that the defect preferentially affected high frequency perception (Supplementary Fig. 1).

Respiratory chain complexes I–V and citrate synthase activities were measured in fibroblasts obtained from the patient’s forearm skin explant. Fibroblasts were spun down (2,500g × 5 min) through a digitonin (0.01%) and Percoll (5%) solution. Rotenone-sensitive NADH quinone reductase (complex I), malonate-sensitive succinate quinone 2,6-dichloroindophenol sodium salt hydrate (DCPIP) reductase (complex II), antimycin-sensitive quinol cytochrome c reductase (complex III), cyanide-sensitive cytochrome c oxidase (complex IV), oligomycin-sensitive ATP hydrolase (complex V), and citrate synthase were measured by spectrophotometry using a dual-beam spectrophotometer (Cary 50 UV-visible spectrophotometer; Agilent Technologies, Massy, France) with standard procedures (5). Decylubiquinol quinone derivative was used to measure complex III (5). All measurements were performed at 37°C. Protein levels were determined by the Bradford method using BSA as a standard.

Blood and tissue samples were obtained after the patient had given informed consent. PCR restriction fragment–length polymorphism analysis was used to look for the m.3243A>G mutation (6). Surveyor analysis was performed as previously described (7). For quantitation of heteroplasmy, the forward unlabeled primer spanned mtDNA nucleotides 14443–14462 and the reverse labeled primer spanned nucleotides 14578–14559. Total DNA was amplified by PCR for 20 cycles (95°C/30 s, 60°C/30 s, and 72°C/30 s) (8). Fluorescent PCR products were separated by electrophoresis using an ABI 3130 genetic analyzer (Applied Biosystems, Foster City, CA) and were studied using the GeneMapper v4.0 software.

Total protein extracts (50 μg) were separated on an acrylamide gel by SDS-PAGE and transferred to a polyvinylidene fluoride membrane (Millipore, Saint-Quentin, France). A cocktail of anti-human total oxidative phosphorylation complex antibodies (Mitosciences, Eugene, OR) was used at 1/1,000, and detection was performed as previously described (9).

Our patient was suspected to suffer of mitochondrial diabetes based on the combination of a high-frequency sensorineural hearing impairment and other cases of diabetes and/or deafness in the family (Fig. 1A). We did not find the m.3243A>G mutation in leukocytes, fibroblasts, hair follicles, buccal mucosa, or urinary cells. Enzymatic spectrophotometric measurements of the individual respiratory chain complexes revealed a decrease in complex I activity in fibroblasts (Fig. 1B). Western blot analysis did not reveal any significant reduction in the amount of respiratory chain proteins as compared with the control (Supplementary Fig. 2). By sequencing the mitochondrial genome in fibroblasts, we found a heteroplasmic insertion of a cytosine residue (m.14535_14536insC) in the MT-ND6 coding region (Fig. 1C). The presence of this mutation, which was also detected in leukocytes and buccal mucosa, was confirmed by digestion with the Surveyor nuclease (7) (Supplementary Fig. 3). In hair follicles and urinary cells, we found a supplementary insertion of two cytosines (m.14535_14536insCC) (Fig. 1C). In all cases, insertions cause a frameshift at the 46th amino acid, leading to a premature stop codon in MT-ND6 (F46fsX61 and F46fsX53, respectively). This results in truncated polypeptides that lack the three last transmembrane domains of ND6. These mutations likely lead to unstable truncated products and abolish assembly of complex I (10). In fibroblasts, complex I deficiency was secondary to a high level of mutations (68%) (Fig. 1D). We also found the homoplasmic polymorphism (m.14530T>C) in all tissues.

Determination of the mitochondrial origin of diabetes is important for genetic counseling and clinical care (6). Nevertheless, it is frequently impossible to confirm the diagnosis when the m.3243A>G mutation is not found (4). Muscle biopsy is an invasive method that cannot be used as a routine examination, especially in diabetic patients. Our observation reveals that analysis of fibroblasts can identify a respiratory chain deficiency and direct molecular screening. Identification of an isolated complex deficiency led us to sequence mtDNA genes coding for the complex I subunits. We found that complex I deficiency is associated with an insertion of cytosine residues in the coding region of the MT-ND6 gene (m.14535_14536insC). This insertion is heteroplasmic and unstable because of the number of supplementary cytosine residues, which differs from one somatic tissue to another. This novel mutation is associated with a homoplasmic polymorphism at nucleotide 14530 that replaces a thymine by a cytosine residue, leading to the formation of an 8–base pair (bp) polyC tract. Several polyC repeats are found in the D-loop or in intergenic regions of mtDNA (11,12) and are unstable by a genetic standpoint. In the MIDD family, Janssen et al. (13) have shown that two repeats, a 6-bp polycytidine tract at nucleotide 568 in the D-loop and a 9-bp CCCCCTCTA sequence in the intergenic COII-tRNA^Lys region, are variable in length. Both repeats are stably inherited in a maternal way, but in proband’s fibroblasts, the average length of the polycytidine tracts is increased at both locations, indicating a fibroblast-specific instability. Nevertheless, the contribution of mtDNA length variants to the phenotype was difficult to determine in this family because they were located in noncoding regions. In contrast, the insertion of cytosine residue(s) found in our patient is responsible for a frameshift in MT-ND6, leading to a deficiency in complex I. The patient also carries the m.14530T>C polymorphism corresponding to a neutral mutation (G48G) that is found in 1 of 2,704 individuals in the Human Mitochondrial Genome Database (14,15). It is likely that the formation of the polyC repeat secondary to this polymorphism in the MT-ND6 coding region plays a role in the genetic instability leading to the observed complex I deficiency.

In conclusion, we describe for the first time an unstable mutation in mtDNA leading to the MIDD phenotype. This observation also illustrates the interest to study fibroblasts when the m.3243A>G mutation is not found.

This work was made possible by grants to V.P.-F. from the French Ministry of Health and Association Française contre les Myopathies.

No potential conflicts of interest relevant to this article were reported.

S.B., M.A., R.V., K.F., and N.D. performed experiments and contributed to discussion. B.V. contributed to the writing of the manuscript. V.P.-F. wrote the manuscript.

The authors thank Gaelle Augé and Alexia Figueroa (Archet 2 Hospital, Centre Hospitalier Universitaire de Nice) for technical help.