Neurofibromatosis type 1 is an autosomal dominantly inherited disease predisposing to a multitude of tumors, most characteristically benign plexiform neurofibromas and diffuse cutaneous neurofibromas. We investigated the presence and distribution of somatic mitochondrial DNA (mtDNA) mutations in neurofibromas and in nontumor tissue of neurofibromatosis type 1 patients. MtDNA alterations in the entire mitochondrial genome were analyzed by temporal temperature gradient gel electrophoresis followed by DNA sequencing. Somatic mtDNA mutations in tumors were found in 7 of 19 individuals with cutaneous neurofibromas and in 9 of 18 patients with plexiform neurofibromas. A total of 34 somatic mtDNA mutations were found. All mutations were located in the displacement loop region of the mitochondrial genome. Several plexiform neurofibromas from individual patients had multiple homoplasmic mtDNA mutations. In cutaneous neurofibromas, the same mtDNA mutations were always present in tumors from different locations of the same individual. An increase in the proportion of the mutant mtDNA was always found in the neurofibromas when compared with nontumor tissues. The somatic mtDNA mutations were present in the Schwann cells of the analyzed multiple cutaneous neurofibromas of the same individual. The observed dominance of a single mtDNA mutation in multiple cutaneous neurofibromas of individual patients indicates a common tumor cell ancestry and suggests a replicative advantage rather than random segregation for cells carrying these mutated mitochondria.

Neurofibromatosis type 1 (NF1) is an autosomal dominantly inherited disorder with an estimated prevalence range from 1:2,190 to 1:7,800 (1). There are no known ethnic groups in which NF1 does not occur or is unusually common. NF1 is caused by mutations in the NF1 tumor suppressor gene, resulting in reduced Ras-GTPase-activating protein activity of neurofibromin (2-5). Clinical features of NF1 include café au lait spots, axillary freckling, iris hamartomas (Lisch nodules), skeletal abnormalities, and learning deficiencies (4). The most prevalent clinical manifestations are the development of benign plexiform and cutaneous neurofibromas. Plexiform neurofibromas can progress at low frequency to highly malignant peripheral nerve sheath tumors. Cutaneous neurofibromas typically start to develop around puberty, and the number of these tumors increases with age. Other tumor types less frequently associated with NF1 include optical gliomas, myeloid leukemias, phaeochromocytomas, and astrocytomas.

Loss of heterozygosity is thought to be the underlying cause for development of benign neurofibromas (6-10). Indeed, loss of heterozygosity at the NF1 locus has been shown in Schwann cells of cutaneous and plexiform neurofibromas and in malignant peripheral nerve sheath tumors (11-14), whereas other studies were unable to confirm loss of heterozygosity for NF1 in astrocytomas and neurofibromas of NF1 patients (15, 16). The variable expressivity, the plethora of mostly benign tumors, and the variability in loss of heterozygosity for NF1 suggest that the threshold for tumorigenesis is perhaps reduced by systemic modulators (17-19).

The diffuse cutaneous neurofibromas typical for NF1 may arise at multiple sites due to the independent occurrence of tumorigenic mutations. Alternatively, an early mutation in a disseminating precursor cell during development may be responsible for the observed phenotype. Here, we use mutational analysis to identify somatic changes in mitochondria in benign neurofibromas and unaffected tissue of NF1 patients. Characterization of somatic mitochondrial DNA (mtDNA) mutations in multiple tumors of the same patient may illuminate the cellular origin of multiple tumors in NF1 from a common precursor.

The role of mitochondria in tumor development has gained much attention with recent reports of somatic mtDNA mutations in brain, ovarian, esophageal, breast, and colorectal human cancers (20-27). Mitochondria contain multiple copies of circular double-stranded DNA molecules that have a high degree of sequence variations among different individuals (28). In addition to energy production by oxidative phosphorylation, mitochondria play a crucial role in programmed cell death (29-32) and generate DNA-damaging reactive oxygen species as side products of normal function. MtDNA is an easy target for oxidative DNA damage due to the close proximity to reactive oxygen species production, the lack of protective histone proteins, and the limited repair capabilities. The accumulation of reactive oxygen species might also contribute to increased nuclear gene mutagenesis (33).

The characteristics of multisystemic manifestation, variable expressivity, and somatic mosaicism of NF1 as well as the reported associations of neurofibromin with highly energy-dependent microtubules (34) and mitochondria (35) may indicate the presence of specific somatic mtDNA mutations in tumors of NF1 patients. We analyzed the mtDNA mutations in neurofibromas and compared mutations between several distinct tumors of the same individual. The presence of somatic mtDNA mutations in nontumor cells and tumor cells was compared.

Somatic mtDNA mutations in neurofibromas were detected by parallel analysis of mtDNA from pairs of matched tumor and normal blood samples by temporal temperature gradient gel electrophoresis (TTGE). Multiple PCR products comprising the entire mitochondrial genome were used. Somatic nucleotide alterations are identified as differences in the banding patterns between the DNA from matched normal and tumor tissues. These analyses were carried out on a total of 19 patients with cutaneous neurofibromas and 18 patients with plexiform neurofibromas. Figure 1 illustrates the results from TTGE and sequencing analyses.

FIGURE 1.

Detection of somatic mtDNA mutations in plexiform and cutaneous neurofibromas by TTGE and sequence analysis. A. Comparison of PCR-amplified mtDNA D-loop region from plexiform neurofibroma T173 and paired blood sample B174. Sequencing revealed multiple homoplasmic nucleotide substitutions in plexiform neurofibroma mtDNA. Direct sequencing of the blood and tumor mtDNA PCR products revealed five homoplasmic tumor-specific nucleotide substitutions in this region, including three novel ones (A16163G, C16186T, and C16221T). The detected T16189C and T16519C alterations have been reported previously (22, 25, 27). B. Comparison of mtDNA D-loop region from plexiform neurofibroma T191 and paired blood sample B192. Sequencing revealed two changes, T199C and G207A, from homoplasmic in normal to heteroplasmic in tumor and one heteroplasmic to heteroplasmic T204C change in the same region. Sequencing revealed two changes from homoplasmic T199 and G207 to heteroplasmic T199C and G207A. Interestingly, a heteroplasmic change in the proportion of T204C mutation between B192 and T191 was also detected, revealing a shift in the degree of heteroplasmy at T204C in the tumor sample. C. Comparison of mtDNA D-loop region from two cutaneous neurofibromas T104 and T105 from the same individual and paired blood sample B106. Sequencing revealed a gradual change from a heteroplasmic 303 to 309 C8/C9 to a homoplasmic C8 in both cutaneous neurofibromas.

FIGURE 1.

Detection of somatic mtDNA mutations in plexiform and cutaneous neurofibromas by TTGE and sequence analysis. A. Comparison of PCR-amplified mtDNA D-loop region from plexiform neurofibroma T173 and paired blood sample B174. Sequencing revealed multiple homoplasmic nucleotide substitutions in plexiform neurofibroma mtDNA. Direct sequencing of the blood and tumor mtDNA PCR products revealed five homoplasmic tumor-specific nucleotide substitutions in this region, including three novel ones (A16163G, C16186T, and C16221T). The detected T16189C and T16519C alterations have been reported previously (22, 25, 27). B. Comparison of mtDNA D-loop region from plexiform neurofibroma T191 and paired blood sample B192. Sequencing revealed two changes, T199C and G207A, from homoplasmic in normal to heteroplasmic in tumor and one heteroplasmic to heteroplasmic T204C change in the same region. Sequencing revealed two changes from homoplasmic T199 and G207 to heteroplasmic T199C and G207A. Interestingly, a heteroplasmic change in the proportion of T204C mutation between B192 and T191 was also detected, revealing a shift in the degree of heteroplasmy at T204C in the tumor sample. C. Comparison of mtDNA D-loop region from two cutaneous neurofibromas T104 and T105 from the same individual and paired blood sample B106. Sequencing revealed a gradual change from a heteroplasmic 303 to 309 C8/C9 to a homoplasmic C8 in both cutaneous neurofibromas.

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When the analyzed mtDNA sequences from blood were compared with those of the published sequence (36), 71 germ line sequence variations were revealed (Table 1). These do not represent all the variations because only the mtDNA PCR products of regions that showed somatic mutations in the paired tumor sample were sequenced. Nine of the germ line variations detected are novel. Many of the remaining reported polymorphisms occurred in multiple individuals. Among them, A73G and T16519C are common polymorphisms in various ethnic groups. The apparent high frequencies of A263G and 303-309insC are because these are polymorphisms in the Cambridge reference sequence (37, 38). Although germ line variations are generally considered silent, missense mutations such as the novel A265V (C5263T) alteration in the mitochondrial electron transport chain complex NADH dehydrogenase subunit 2 may have a functional effect.

Table 1.

Germ Line Sequence Variations

Gene/RegionGerm Line MutationFrequency*Significance
A. Novel    
D-loop T10C 7S DNA 
D-loop T55C 7S DNA 
D-loop T57C Hypervariable segment 2 
D-loop T408A L-strand promoter 
16S A2706G 16S RNA 
ND2 C5263T GCC-GTC, A265V 
COI G6917A GTG-GGG, V338V 
ND4 A11947G ACA-ACG, T396T 
D-loop C16465T  
B. Reported    
D-loop T72C Hypervariable segment 2 
D-loop A73G 12 Hypervariable segment 2 
D-loop T146C H-strand origin 
D-loop C150T H-strand origin 
D-loop T152C H-strand origin 
D-loop A189G H-strand origin 
D-loop C194T H-strand origin 
D-loop T195C H-strand origin 
D-loop T199C H-strand origin 
D-loop T204C H-strand origin 
D-loop G207A H-strand origin 
D-loop C242T mtTF1 binding site 
D-loop A263G 16 H-strand origin 
D-loop C271T H-strand origin 
D-loop C295T mtTF1 binding site 
D-loop 303-309insC 15 CSB II 
D-loop C462T  
D-loop T489C  
D-loop A508G  
D-loop 514insCA  
D-loop 514insCACA  
D-loop 568insCCC  
12s A663G 12S RNA 
12s G709A 12S RNA 
ND2 G4580A ATG-ATA, M37M 
ND2 A4769G ATA-ATG, M100M 
COI T6776C CAT-CAC, H291H 
ND4 G11914A ACG-ACA, T385T 
ND4 G12007A TGG-TGA, W416W 
ND5 A12612G GTA-GTG, V92V 
ND5 A12693G AAA-AAG, K119K 
ND5 C12705T ATC-ATT, I123I 
ND6 A14233G ATC-GTC, I29V 
CytB T14798C TTC-CTC, F18L 
D-loop G16145A  
D-loop C16186T Hypervariable segment 1 
D-loop C16188T Hypervariable segment 1 
D-loop T16189C Hypervariable segment 1 
D-loop T16192T Hypervariable segment 1 
D-loop C16193T Hypervariable segment 1 
D-loop C16195T Hypervariable segment 1 
D-loop C16222T Hypervariable segment 1 
D-loop C16223T Hypervariable segment 1 
D-loop C16278T Hypervariable segment 1 
D-loop C16290T Hypervariable segment 1 
D-loop C16292T Hypervariable segment 1 
D-loop C16294T Hypervariable segment 1 
D-loop C16296T Hypervariable segment 1 
D-loop T16298C Hypervariable segment 1 
D-loop T16304C Hypervariable segment 1 
D-loop A16309G Hypervariable segment 1 
D-loop T16311C Hypervariable segment 1 
D-loop T16362C Hypervariable segment 1 
D-loop G16390A Hypervariable segment 1 
D-loop T16519C  
Gene/RegionGerm Line MutationFrequency*Significance
A. Novel    
D-loop T10C 7S DNA 
D-loop T55C 7S DNA 
D-loop T57C Hypervariable segment 2 
D-loop T408A L-strand promoter 
16S A2706G 16S RNA 
ND2 C5263T GCC-GTC, A265V 
COI G6917A GTG-GGG, V338V 
ND4 A11947G ACA-ACG, T396T 
D-loop C16465T  
B. Reported    
D-loop T72C Hypervariable segment 2 
D-loop A73G 12 Hypervariable segment 2 
D-loop T146C H-strand origin 
D-loop C150T H-strand origin 
D-loop T152C H-strand origin 
D-loop A189G H-strand origin 
D-loop C194T H-strand origin 
D-loop T195C H-strand origin 
D-loop T199C H-strand origin 
D-loop T204C H-strand origin 
D-loop G207A H-strand origin 
D-loop C242T mtTF1 binding site 
D-loop A263G 16 H-strand origin 
D-loop C271T H-strand origin 
D-loop C295T mtTF1 binding site 
D-loop 303-309insC 15 CSB II 
D-loop C462T  
D-loop T489C  
D-loop A508G  
D-loop 514insCA  
D-loop 514insCACA  
D-loop 568insCCC  
12s A663G 12S RNA 
12s G709A 12S RNA 
ND2 G4580A ATG-ATA, M37M 
ND2 A4769G ATA-ATG, M100M 
COI T6776C CAT-CAC, H291H 
ND4 G11914A ACG-ACA, T385T 
ND4 G12007A TGG-TGA, W416W 
ND5 A12612G GTA-GTG, V92V 
ND5 A12693G AAA-AAG, K119K 
ND5 C12705T ATC-ATT, I123I 
ND6 A14233G ATC-GTC, I29V 
CytB T14798C TTC-CTC, F18L 
D-loop G16145A  
D-loop C16186T Hypervariable segment 1 
D-loop C16188T Hypervariable segment 1 
D-loop T16189C Hypervariable segment 1 
D-loop T16192T Hypervariable segment 1 
D-loop C16193T Hypervariable segment 1 
D-loop C16195T Hypervariable segment 1 
D-loop C16222T Hypervariable segment 1 
D-loop C16223T Hypervariable segment 1 
D-loop C16278T Hypervariable segment 1 
D-loop C16290T Hypervariable segment 1 
D-loop C16292T Hypervariable segment 1 
D-loop C16294T Hypervariable segment 1 
D-loop C16296T Hypervariable segment 1 
D-loop T16298C Hypervariable segment 1 
D-loop T16304C Hypervariable segment 1 
D-loop A16309G Hypervariable segment 1 
D-loop T16311C Hypervariable segment 1 
D-loop T16362C Hypervariable segment 1 
D-loop G16390A Hypervariable segment 1 
D-loop T16519C  

NOTE: The total number of distinct germ line sequence variations is 71 (9 novel and 62 reported). Missense substitutions are in bold.

*

Number of tumors that carry germ line variation.

ND2, NADH dehydrogenase subunit 2.

The complete results of our analysis of NF1-associated cutaneous and plexiform neurofibromas are listed in Table 2. The overall percentage of neurofibromas with somatic mtDNA mutations was similar to that found for glioblastoma and medulloblastoma but lower than those in lung, breast, and oral cancers (20-27).

Table 2.

Somatic mtDNA Mutations in NF1-Associated Neurofibromas

Case No.Gene/RegionSomatic MutationCambridge Sequencenl to tu Pattern*FunctionPreviously Reported in Tumors
NF1-associated plexiform neurofibromas       
T159 D-loop A73G Homo-homo Hypervariable segment 2 eso 
T159 D-loop C16193T Homo-homo Hypervariable segment 1 Novel 
T159 D-loop C16278T Homo-homo Hypervariable segment 1 ov 
T159 D-loop C16519T Homo-homo  Lung, glioblastoma 
T165 D-loop T64C Hetero-hetero Hypervariable segment 2 Novel 
T171 D-loop 303-309delC, C9/C8-C8/C9 C7 Hetero-hetero CSB crc, gastric, eso, ov, brca 
T173 D-loop C64T Homo-homo Hypervariable segment 2 Novel 
T173 D-loop A73G Homo-homo Hypervariable segment 2 eso 
T173 D-loop T152C Homo-homo H-strand origin ov 
T173 D-loop T195C Homo-homo H-strand origin Lung, glioblastoma 
T173 D-loop A16163G Homo-homo Termination-associated sequence Novel 
T173 D-loop C16186T Homo-homo 7S DNA Novel 
T173 D-loop T16189C Homo-homo 7S DNA brca 
T173 D-loop C16221T Homo-homo Hypervariable segment 1 Novel 
T173 D-loop T16519C Homo-homo  Lung, glioblastoma 
T179 D-loop 303-309insC, C7/C8-C8 C7 Hetero-homo CSB crc, gastric, eso, ov, brca 
T185 D-loop C204T Homo-homo H-strand origin gastric, glioblastoma 
T185 D-loop A207G Hetero-homo H-strand origin brca 
T187 D-loop 303-309delC, C7/C8-C7 C7 Hetero-homo CSB crc, gastric, eso, ov, brca 
T189 D-loop T204C Hetero-homo H-strand origin gastric, glioblastoma 
T189 D-loop G207A Homo-hetero H-strand origin brca 
T189 D-loop 303-309insC, C7-C7/C8 C7 Homo-hetero CSB crc, gastric, eso, ov, brca 
T191 D-loop A73G Homo-hetero Hypervariable segment 2 eso 
T191 D-loop T199C Homo-hetero Hypervariable segment 2 ov 
T191 D-loop T204C Hetero-hetero H-strand origin gastric, glioblastoma 
T191 D-loop G207A Homo-hetero H-strand origin brca 
T191 D-loop 303-309insC, C7-C7/C8 C7 Homo-hetero CSB crc, gastric, eso, ov, brca 
NF1-associated cutaneous neurofibromas       
T104 D-loop 303-309delC, C9/C8-C8 C7 Hetero-homo CSB crc, gastric, eso, ov, brca 
T105 D-loop 303-309delC, C9/C8-C8 C7 Hetero-homo CSB crc, gastric, eso, ov, brca 
T107 D-loop 303-309insC, C7-C7/C8 C7 Homo-hetero CSB crc, gastric, eso, ov, brca 
T108 D-loop 303-309insC, C7-C7/C8 C7 Homo-hetero CSB crc, gastric, eso, ov, brca 
T119 D-loop T16304C Homo-homo Hypervariable segment 1 ov 
T120 D-loop T16304C Homo-homo Hypervariable segment 1 ov 
T590 D-loop 303-309insC, C7/C8-C8/C7 C7 Hetero-hetero CSB crc, gastric, eso, ov, brca 
T591 D-loop 303-309insC, C7/C8-C8/C7 C7 Hetero-hetero CSB crc, gastric, eso, ov, brca 
T583 D-loop 303-309insC, C7/C8-C8/C7 C7 Hetero-homo CSB crc, gastric, eso, ov, brca 
T584 D-loop 303-309insC, C7/C8-C8/C7 C7 Hetero-homo CSB crc, gastric, eso, ov, brca 
T468 D-loop T196C Hetero-hetero CSB Novel 
T469 D-loop T196C Hetero-homo CSB Novel 
T554 D-loop 303-309insC, C7/C8-C8/C7 C7 Hetero-hetero CSB crc, gastric, eso, ov, brca 
T555 D-loop 303-309insC, C7/C8-C8/C7 C7 Hetero-hetero CSB crc, gastric, eso, ov, brca 
Case No.Gene/RegionSomatic MutationCambridge Sequencenl to tu Pattern*FunctionPreviously Reported in Tumors
NF1-associated plexiform neurofibromas       
T159 D-loop A73G Homo-homo Hypervariable segment 2 eso 
T159 D-loop C16193T Homo-homo Hypervariable segment 1 Novel 
T159 D-loop C16278T Homo-homo Hypervariable segment 1 ov 
T159 D-loop C16519T Homo-homo  Lung, glioblastoma 
T165 D-loop T64C Hetero-hetero Hypervariable segment 2 Novel 
T171 D-loop 303-309delC, C9/C8-C8/C9 C7 Hetero-hetero CSB crc, gastric, eso, ov, brca 
T173 D-loop C64T Homo-homo Hypervariable segment 2 Novel 
T173 D-loop A73G Homo-homo Hypervariable segment 2 eso 
T173 D-loop T152C Homo-homo H-strand origin ov 
T173 D-loop T195C Homo-homo H-strand origin Lung, glioblastoma 
T173 D-loop A16163G Homo-homo Termination-associated sequence Novel 
T173 D-loop C16186T Homo-homo 7S DNA Novel 
T173 D-loop T16189C Homo-homo 7S DNA brca 
T173 D-loop C16221T Homo-homo Hypervariable segment 1 Novel 
T173 D-loop T16519C Homo-homo  Lung, glioblastoma 
T179 D-loop 303-309insC, C7/C8-C8 C7 Hetero-homo CSB crc, gastric, eso, ov, brca 
T185 D-loop C204T Homo-homo H-strand origin gastric, glioblastoma 
T185 D-loop A207G Hetero-homo H-strand origin brca 
T187 D-loop 303-309delC, C7/C8-C7 C7 Hetero-homo CSB crc, gastric, eso, ov, brca 
T189 D-loop T204C Hetero-homo H-strand origin gastric, glioblastoma 
T189 D-loop G207A Homo-hetero H-strand origin brca 
T189 D-loop 303-309insC, C7-C7/C8 C7 Homo-hetero CSB crc, gastric, eso, ov, brca 
T191 D-loop A73G Homo-hetero Hypervariable segment 2 eso 
T191 D-loop T199C Homo-hetero Hypervariable segment 2 ov 
T191 D-loop T204C Hetero-hetero H-strand origin gastric, glioblastoma 
T191 D-loop G207A Homo-hetero H-strand origin brca 
T191 D-loop 303-309insC, C7-C7/C8 C7 Homo-hetero CSB crc, gastric, eso, ov, brca 
NF1-associated cutaneous neurofibromas       
T104 D-loop 303-309delC, C9/C8-C8 C7 Hetero-homo CSB crc, gastric, eso, ov, brca 
T105 D-loop 303-309delC, C9/C8-C8 C7 Hetero-homo CSB crc, gastric, eso, ov, brca 
T107 D-loop 303-309insC, C7-C7/C8 C7 Homo-hetero CSB crc, gastric, eso, ov, brca 
T108 D-loop 303-309insC, C7-C7/C8 C7 Homo-hetero CSB crc, gastric, eso, ov, brca 
T119 D-loop T16304C Homo-homo Hypervariable segment 1 ov 
T120 D-loop T16304C Homo-homo Hypervariable segment 1 ov 
T590 D-loop 303-309insC, C7/C8-C8/C7 C7 Hetero-hetero CSB crc, gastric, eso, ov, brca 
T591 D-loop 303-309insC, C7/C8-C8/C7 C7 Hetero-hetero CSB crc, gastric, eso, ov, brca 
T583 D-loop 303-309insC, C7/C8-C8/C7 C7 Hetero-homo CSB crc, gastric, eso, ov, brca 
T584 D-loop 303-309insC, C7/C8-C8/C7 C7 Hetero-homo CSB crc, gastric, eso, ov, brca 
T468 D-loop T196C Hetero-hetero CSB Novel 
T469 D-loop T196C Hetero-homo CSB Novel 
T554 D-loop 303-309insC, C7/C8-C8/C7 C7 Hetero-hetero CSB crc, gastric, eso, ov, brca 
T555 D-loop 303-309insC, C7/C8-C8/C7 C7 Hetero-hetero CSB crc, gastric, eso, ov, brca 
*

nl, normal (blood); tu, tumor; homo, homoplasmic; hetero, heteroplasmic.

The letter before the np number indicates the nucleotide found in the normal tissue, and the letter after the np number indicates the nucleotide found in tumor tissue. The homoplasmic or heteroplasmic status of the mutation in normal and tumor tissue is indicated accordingly in the nl to tu Pattern column.

crc, colorectal cancer; eso, esophageal cancer; ov, ovarian cancer; brca, breast cancer.

Somatic mtDNA mutations were detected in 9 of 18 plexiform neurofibromas (Table 2). Four of the plexiform tumors with mtDNA mutations harbored a single alteration; five cases had >1 mutation. A total of 27 different somatic mtDNA mutations were identified. All mutations were found in the displacement loop (D-loop) region. Insertions or deletions in the nucleotide positions (np) 303 to 309 poly(C) region were detected in multiple tumors. This region has been reported to be the somatically unstable mutation hotspot of breast cancer (25, 39). All nucleotide substitutions were transitions between T/C and A/G, consistent with oxidative DNA damage. Fourteen of 27 of the somatic mtDNA mutations in the plexiform tumors were alterations from homoplasmic state in blood to homoplasmic state in tumor. A shift from homoplasmy in blood to heteroplasmy in the corresponding tumor was found in six cases, and a shift from heteroplasmy in blood to homoplasmy in tumor was detected in four cases. Three pairs of matched samples had heteroplasmic nucleotide substitutions in both blood and tumor, but there were detectable differences in the degree of heteroplasmy as assessed by quantitative comparison of the nucleotide peak amplitudes in the corresponding sequence profiles (Table 2).

One patient with plexiform tumor, T173, harbored nine somatic mtDNA mutations. To rule out an error in DNA sampling, identity analysis on tumor T173 and corresponding blood sample B174 samples was done. Identical alleles were detected at five polymorphic sites: the short tandem repeat in intron 3 of the PAH gene (chromosome 12), the CTG repeats of the myotonin protein kinase gene (disease gene for myotonic dystrophy, chromosome 19), the CAG repeats of the androgen receptor gene (X chromosome), and the SCA1 (chromosome 6) and SCA3 (chromosome 4) genes (data not shown). These results support that tumor T173 indeed had nine somatic mtDNA mutations, four of which are novel. The cause for this unusually large number of tumor-specific somatic mtDNA mutations is not clear. Apparently, the point mutations in the origin of H-strand replication and the termination-associated sequence regulate the mtDNA synthesis and transcription in the tumor. A large number (>6) of somatic mtDNA mutations also occurred in ∼5% to 10% of medulloblastomas and breast and lung cancers (25, 40).

Cutaneous neurofibromas were analyzed from a total of 19 patients. Seven of these tumors had somatic mtDNA mutations (Table 2), and all of them occurred in the hypervariable D-loop region. NF1 patients usually have multiple cutaneous neurofibromas ranging from <10 to >1000. Surprisingly, multiple cutaneous neurofibromas resected from distinct anatomic sites on an affected individual always shared identical somatic mtDNA mutations (Tables 2 and 3): Analysis of tumor samples T104 and T105 (Fig. 2C) revealed a change in a short poly(C) sequence at np 303 to 309 in the conserved sequence block (CSB), which shifted from C8/C9 heteroplasmy in the blood sample (B106) of the same patient to near homoplasmy of C8 in both tumor samples (Table 3). Analysis of mtDNA samples from tumors T107 and T108 showed a shift from np 303 to 309 C7 homoplasmy in the matched blood sample B109 to C7/C8 heteroplasmy in each of the tumors. Tumors T119 and T120 both harbor the same homoplasmic T16304C mutation when compared with the corresponding blood DNA B121, which is homoplasmic for the wild-type T16304. Comparable changes were detected in tumors and blood of patients 4, 5, and 6 (Table 3). In patient 7, we detected a high proportion of np 303 to 309 C8 in blood and in tumor samples, whereas no D-loop mutations were found in patient 8. Tumor tissues from different parts of the same tumor also showed the identical mtDNA mutations with similar degrees of plasmy (Table 3, patients 4 and 5).

Table 3.

Somatic mtDNA Mutations in Two Separate Cutaneous Neurofibromas

PatientSampleSpecimen TypeLocationSomatic Mutation% Heteroplasmy
B106 Blood  303-309insC C9/C8 C9 50%, C8 50% 
 T105 Tumor 1  303-309delC C9/C8 C9 0%, C8 100% 
 T104 Tumor 2  303-309delC C9/C8 C9 5%, C8 95% 
B109 Blood  303-309insC C7/C8 C7 100%, C8 0% 
 T108 Tumor 1  303-309insC C7/C8 C7 60%, C8 40% 
 T107 Tumor 2  303-309insC C7/C8 C7 40%, C8 60% 
B121 Blood  T16304 T 100%, C 0% 
 T120 Tumor 1  T16304C T 0%, C 100% 
 T119 Tumor 2  T16304C T 0%, C 100% 
B587 Blood  303-309insC C7/C8 C7 50%, C8 50% 
 S586 Skin Distal from tumor 303-309insC C7/C8 C7 40%, C8 60% 
 S585 Skin Overlaying the tumor 303-309insC C7/C8 C7 30%, C8 70% 
 T583 Tumor 1 Thorax 303-309insC C7/C8 C7 10%, C8 90% 
 T584 Tumor 2, part 1 Abdomen 303-309insC C7/C8 C7 10%, C8 90% 
 T584 Tumor 2, part 2 Abdomen 303-309insC C7/C8 C7 5%, C8 95% 
B594 Blood  303-309insC C7/C8 C7 50%, C8 50% 
 S588 Skin Distal from tumor 303-309insC C7/C8 C7 40%, C8 60% 
 S589 Skin Overlaying the tumor 303-309insC C7/C8 C7 40%, C8 60% 
 T590 Tumor 1 Thorax right side 303-309insC C7/C8 C7 20%, C8 80% 
 T591 Tumor 2, part 1 Abdomen 303-309insC C7/C8 C7 0%, C8 100% 
 T592 Tumor 2, part 2 Abdomen 303-309insC C7/C8 C7 20%, C8 80% 
 T593 Tumor 2, part 3 Abdomen 303-309insC C7/C8 C7 0%, C8 100% 
B276 Blood  T196C T 50%, C 50% 
 T468 Tumor 1  T196C T 30%, C 70% 
 T469 Tumor 2  T196C T 5%, C 95% 
B9 Blood  303-309insC C8/C7 C8 70%, C7 30% 
 T554 Tumor  303-309insC C8/C7 C8 95%, C7 5% 
 T555 Tumor  303-309insC C8/C7 C8 70%, C7 30% 
B598 Blood    
 S597 Skin 2 cm away from 595 No mutation found  
 T596 Tumor 1 Thorax right side No mutation found  
 T595 Tumor 2 Thorax right side No mutation found  
PatientSampleSpecimen TypeLocationSomatic Mutation% Heteroplasmy
B106 Blood  303-309insC C9/C8 C9 50%, C8 50% 
 T105 Tumor 1  303-309delC C9/C8 C9 0%, C8 100% 
 T104 Tumor 2  303-309delC C9/C8 C9 5%, C8 95% 
B109 Blood  303-309insC C7/C8 C7 100%, C8 0% 
 T108 Tumor 1  303-309insC C7/C8 C7 60%, C8 40% 
 T107 Tumor 2  303-309insC C7/C8 C7 40%, C8 60% 
B121 Blood  T16304 T 100%, C 0% 
 T120 Tumor 1  T16304C T 0%, C 100% 
 T119 Tumor 2  T16304C T 0%, C 100% 
B587 Blood  303-309insC C7/C8 C7 50%, C8 50% 
 S586 Skin Distal from tumor 303-309insC C7/C8 C7 40%, C8 60% 
 S585 Skin Overlaying the tumor 303-309insC C7/C8 C7 30%, C8 70% 
 T583 Tumor 1 Thorax 303-309insC C7/C8 C7 10%, C8 90% 
 T584 Tumor 2, part 1 Abdomen 303-309insC C7/C8 C7 10%, C8 90% 
 T584 Tumor 2, part 2 Abdomen 303-309insC C7/C8 C7 5%, C8 95% 
B594 Blood  303-309insC C7/C8 C7 50%, C8 50% 
 S588 Skin Distal from tumor 303-309insC C7/C8 C7 40%, C8 60% 
 S589 Skin Overlaying the tumor 303-309insC C7/C8 C7 40%, C8 60% 
 T590 Tumor 1 Thorax right side 303-309insC C7/C8 C7 20%, C8 80% 
 T591 Tumor 2, part 1 Abdomen 303-309insC C7/C8 C7 0%, C8 100% 
 T592 Tumor 2, part 2 Abdomen 303-309insC C7/C8 C7 20%, C8 80% 
 T593 Tumor 2, part 3 Abdomen 303-309insC C7/C8 C7 0%, C8 100% 
B276 Blood  T196C T 50%, C 50% 
 T468 Tumor 1  T196C T 30%, C 70% 
 T469 Tumor 2  T196C T 5%, C 95% 
B9 Blood  303-309insC C8/C7 C8 70%, C7 30% 
 T554 Tumor  303-309insC C8/C7 C8 95%, C7 5% 
 T555 Tumor  303-309insC C8/C7 C8 70%, C7 30% 
B598 Blood    
 S597 Skin 2 cm away from 595 No mutation found  
 T596 Tumor 1 Thorax right side No mutation found  
 T595 Tumor 2 Thorax right side No mutation found  

NOTE: Somatic mtDNA mutations in two separate cutaneous neurofibromas from each of eight patients and in paired skin samples of patients 4, 5, and 8. In all samples from patients 4 to 8, only D-loop region was analyzed. % Heteroplasmy was estimated from the sequencing chromatogram. They do not represent the actual proportion. However, the trend of progressive alteration was obvious (patients 4 and 5). For samples 104 and 105, TTGE gel chromatogram was used to estimate the percentage of mutant heteroplasmy, which was too low to be revealed by sequencing (Fig. 1C).

FIGURE 2.

Cell specificity of mtDNA mutations in cutaneous neurofibromas and skin of the same patient. A to C. Cutaneous neurofibroma 583 with selected Schwann cells (A), microdissected cells (B), and sequence results of np 303 to 309 C7/C8 D-loop region (C). Arrow, C8. D to F. The same neurofibroma as in A with selected endothelial cells (D), microdissected cells (E), and sequencing results (F). Arrow, C7. Magnification is 200× in all images. G. Sequencing results of epithelial cells from a skin sample of the same patients as in A. Arrow, C7. H. Sequencing results of dermal fibroblasts from a skin sample of the same patient as in A. Arrow, C8 (70%)/C7(30%).

FIGURE 2.

Cell specificity of mtDNA mutations in cutaneous neurofibromas and skin of the same patient. A to C. Cutaneous neurofibroma 583 with selected Schwann cells (A), microdissected cells (B), and sequence results of np 303 to 309 C7/C8 D-loop region (C). Arrow, C8. D to F. The same neurofibroma as in A with selected endothelial cells (D), microdissected cells (E), and sequencing results (F). Arrow, C7. Magnification is 200× in all images. G. Sequencing results of epithelial cells from a skin sample of the same patients as in A. Arrow, C7. H. Sequencing results of dermal fibroblasts from a skin sample of the same patient as in A. Arrow, C8 (70%)/C7(30%).

Close modal

These results suggest that the tumor-specific mtDNA mutations may have already been present in nontumor cells and accumulated in the neurofibromas. To examine this, we analyzed mtDNA isolated from unaffected skin and matched cutaneous neurofibromas from three NF1 patients. Mutation analysis of these skin and tumor samples was focused on the D-loop and its surrounding region, because the data obtained from 37 neurofibromas revealed that all somatic mtDNA mutations occurred in the D-loop region. In one patient (patient 8), no somatic mtDNA D-loop mutations were found in the tumor and skin samples (Table 3). The other two sets of tumors displayed somatic mtDNA mutations (patients 4 and 5). A progressive increase in mutant mtDNA content was showed among blood, skin, and the neurofibromas in both of these patients (Table 3). In addition, the identical mutation was present in the skin, in different parts of the tumor, and in separate tumors from different locations of the same individual.

Neurofibromas are mixed-cell tumors composed predominantly of Schwann cells and fibroblasts, with a minority of perineureal cells, neurons, mast cells, and endothelial cells. To determine which cell type in an apparently homoplasmic cutaneous neurofibroma contains the mutated mitochondria, mutational analysis was done on laser capture microdissected cells. S100-positive Schwann cells, S100-negative cells, and endothelial cells were dissected from neurofibromas 583 and 584 (patient 4) and 592 and 593 (patient 5; Table 3). Fibroblasts and epithelial cells were also dissected from unaffected skin of the same patients. MtDNA from the separate cell types was analyzed for the presence of the previously identified D-loop mutation 303-309insC C7/C8.

In tumors 583 and 584, S100-positive Schwann cells contained almost exclusively the C8 mtDNA mutation (Fig. 2A-C). S100-negative tumor-derived cells, presumably fibroblasts, were also highly enriched for the C8 mtDNA mutation (data not shown). Although carefully selected, we cannot completely exclude the possibility that the S100 cell fraction contains neoplastic Schwann cells, as it is notoriously difficult to distinguish between the different cell types in neurofibromas. Tumor-derived nonneoplastic endothelial cells contained only C7 mtDNA (Fig. 2D-F), as did epithelial cells obtained from the skin sample 586 of the same patient (Fig. 2G). Dermal fibroblasts from skin sample 586 contained C7 and C8 mtDNA at a ratio of ∼30:70 (Fig. 2H). Similar results were obtained for the different cell types of tumor samples 592 and 593 and matched skin sample 588. The results of the cell type–specific mtDNA analysis are summarized in Table 4.

Table 4.

Somatic mtDNA Mutations in Cells Dissected from Cutaneous Neurofibromas and Paired Skin of Patients 4 and 5 of Table 2 

PatientSampleSpecimen TypeCell TypeSomatic Mutation% Heteroplasmy
586 Skin Epithelial 303-309insC C7/C8 C7 100%, C8 0% 
 586 Skin Dermal fibroblast 303-309insC C7/C8 C7 30%, C8 70% 
 583 Tumor 1 S100-positive Schwann cell 303-309insC C7/C8 C7 0%, C8 100% 
 583 Tumor 1 S100-negative cells 303-309insC C7/C8 C7 5%, C8 95% 
 583 Tumor 1 Endothelial cell 303-309insC C7/C8 C7 100%, C8 0% 
588 Skin Epithelial 303-309insC C7/C8 C7 100%, C8 0% 
 588 Skin Dermal fibroblast 303-309insC C7/C8 C7 35%, C8 65% 
 592 Tumor 2, part 2 S100-positive Schwann cell 303-309insC C7/C8 C7 5%, C8 95% 
 592 Tumor 2, part 2 S100-negative cells 303-309insC C7/C8 C7 10%, C8 90% 
 592 Tumor 2, part 2 Endothelial cell 303-309insC C7/C8 C7 100%, C8 0% 
PatientSampleSpecimen TypeCell TypeSomatic Mutation% Heteroplasmy
586 Skin Epithelial 303-309insC C7/C8 C7 100%, C8 0% 
 586 Skin Dermal fibroblast 303-309insC C7/C8 C7 30%, C8 70% 
 583 Tumor 1 S100-positive Schwann cell 303-309insC C7/C8 C7 0%, C8 100% 
 583 Tumor 1 S100-negative cells 303-309insC C7/C8 C7 5%, C8 95% 
 583 Tumor 1 Endothelial cell 303-309insC C7/C8 C7 100%, C8 0% 
588 Skin Epithelial 303-309insC C7/C8 C7 100%, C8 0% 
 588 Skin Dermal fibroblast 303-309insC C7/C8 C7 35%, C8 65% 
 592 Tumor 2, part 2 S100-positive Schwann cell 303-309insC C7/C8 C7 5%, C8 95% 
 592 Tumor 2, part 2 S100-negative cells 303-309insC C7/C8 C7 10%, C8 90% 
 592 Tumor 2, part 2 Endothelial cell 303-309insC C7/C8 C7 100%, C8 0% 

NOTE: Percentage of heteroplasmy was estimated from the sequencing chromatogram.

In the analysis of NF1-associated tumors, one single somatic mtDNA mutation was detected in each cutaneous neurofibroma, whereas an average of three mutations was detected per plexiform neurofibroma. This compares with an average of one to three somatic mtDNA mutations for other tumors (20, 22, 25, 27). It is noteworthy that 22 of 27 mutations in plexiform neurofibromas are nucleotide substitutions compared with only 2 of 7 mutations in cutaneous neurofibromas. One interesting observation is that mutations at np 204 and 207 occurred three times in three unrelated patients. This result suggests that either the np 204 and 207 are mutation hotspots or the mutant mitochondria have selective growth advantage.

All of the mtDNA somatic mutations identified in our study occurred in the hypervariable D-loop region of the mitochondrial genome. This is unique because numerous studies on lung, breast, ovarian, bladder, head and neck, glioblastoma, and oral cancers showed that 20% to 70% of somatic mtDNA mutations were found in coding regions (20-32, 40). The pathologic significance of mutations in noncoding regions of the mitochondrial genome is currently unknown. Apparently, mutations in the D-loop influence the origin of replication and promoter region and may affect the mitochondrial biogenesis, transcription, and protein expression (41, 42).

The most common somatic mtDNA mutations identified in our study are insertions or deletions at np 303 to 309. Mutations at np 303 to 309 were not observed in mtDNA from 40 muscle tissues of individuals ranging in age from 0 to 65 years.5

5

L-J.C. Wong, unpublished observation.

This suggests that the variability in the np 303 to 309 region is not due to a microsatellite instability. It is of interest to note that the np 303 to 309 C8 mutation was always dominant in the tumors, whereas the C7 variant was dominant in nontumor tissue and was either diminished or not detected in tumors.

MtDNA mutations may occur randomly, and multiple mutations may or may not occur simultaneously. To reach a homoplasmic state may require some mechanism for selection. Homoplasmy for mtDNA mutations in tumors can also be caused by random segregation after a sufficient number of cell divisions (43). Thus, mutations in the origin of replication (D-loop region) may provide a replicative advantage of these mutant mtDNAs, or homoplasmy for the D-loop mutation in tumors may be the result of random segregation. The last mechanism is unlikely to explain the observed distribution of mtDNA mutations in individual neurofibromas. The independent appearance of the same mtDNA mutation in distinct tumors of the same patient in six analyzed individuals argues against a random process. This is supported by the observation that multiple mutations in tumors of the same patient were almost always found in a homoplasmic state. In addition to random segregation and replicative advantage, mitochondrial segregation dynamics and maintenance may also be influenced by the genetic background of cells (44). Because neurofibromin is closely associated with microtubules and mitochondria (34, 35), haploinsufficiency of NF1 may perhaps affect the stability and promote segregation of specifically mutated mitochondria in neurofibromas.

The present data indicate the presence of somatic mtDNA mutations in a heteroplasmic state in cells of nontumor tissues of NF1 patients. The mutated mitochondria, particularly the np 303 to 309 C8 genotype, accumulate and ultimately dominate in the multiple cutaneous neurofibromas of a patient. This specific mitochondrial distribution pattern, in association with NF1 haploinsufficiency, may be indicative of a lowered tumorigenic threshold in NF1. Furthermore, the mitochondrial signatures of plexiform and cutaneous neurofibromas of NF1 patients vary, underlying their separate developmental origins. Although the present findings cannot exclude a mechanism of frequent mutational events in the NF1 gene, leading to multiple cutaneous neurofibromas, they support the notion that multiple cutaneous neurofibromas in NF1 patients may arise from a widely distributed cell carrying an early somatic mitochondrial mutation.

Tissue Samples

Patients with NF1 were recruited through the Departments of Neurosurgery and Neurogenetics, Massachusetts General Hospital, Harvard University and the Department of Neurology, Klinikum Nord Ochsenzoll (Hamburg, Germany) according to institutional review board–approved protocols. Patients were phenotypically characterized for features of NF1, including number, location, and size of cutaneous neurofibromas. Only patients with a clear diagnosis of NF1 according to NIH criteria (45) were included in this study. Cutaneous and plexiform neurofibromas were removed during routine surgery, dissected into multiple aliquots, and frozen immediately. Two or more cutaneous neurofibromas resected from different anatomic sites on each individual were obtained from 19 patients. For three of these patients, skin samples were biopsied from an area overlaying the resected cutaneous neurofibroma and from an area distal to the tumor (Table 2, patients 4, 5, and 6). A single plexiform neurofibroma sample was obtained from each of 18 patients.

The age of patients with cutaneous tumors ranges from 16 to 50 years with a mean age of 37.6 years. The age of patients with plexiform tumors ranges from 8 to 73 years with a mean age of 28.8 years.

DNA Isolation

DNA was isolated from frozen tissues using proteinase K and phenol/chloroform extraction method. DNA was extracted from peripheral blood lymphocytes using a modified nonenzymatic method (46). Total DNA was quantified using fluorescent Hoechst dye H33258 (Sigma, St. Louis, MO) with DyNA Quant 200 (Amersham Biosciences, Uppsala, Sweden)according to the manufacturer's protocol and diluted to 5 ng/μL to be used in PCR reactions (47).

Mutational Analysis of the Entire Mitochondrial Genome

DNA isolated from 19 pairs of matched blood and cutaneous neurofibroma samples and 18 pairs of matched blood and plexiform neurofibroma samples was used for mutational analysis of the mitochondrial genome by TTGE.

Thirty-two pairs of overlapping primers were used to amplify the entire mitochondrial genome by PCR (47). The PCR-amplified DNA fragments vary from 306 to 805 bp long. The positions and sequence of the PCR primers and the PCR and TTGE conditions were as described recently (47). Briefly, the DNA template, after the initial denaturation at 94°C for 5 minutes, was amplified with 35 cycles of 94°C for 45 seconds, 55°C for 45 seconds, and 72°C for 45 seconds and completed by 4 minutes of extension at 72°C. The PCR products were denatured at 95°C for 30 seconds and slowly cooled to 45°C for 45 minutes at a rate of 1.1°C/min. The reannealed homoduplexes and heteroduplexes were maintained at 4°C until TTGE analysis was done on a D-Code apparatus (Bio-Rad Laboratories, Hercules, CA). Five microliters of denatured and reannealed PCR products were loaded onto a polyacrylamide gel (acrylamide/bis 37.5:1) prepared in 1.25× Tris-acetate-EDTA buffer containing 6 mol/L urea. Electrophoresis was carried out at 145 V for 4 to 5 hours at a constant 1°C/h to 2°C/h temperature increment (47). The temperature range was determined by computer simulation from the melting curve of the analyzed DNA fragment (MacMelt software, Bio-Rad Laboratories). The gels were stained with 2 mg/L ethidium bromide for 5 minutes and imaged with a digital CCD gel documentation system (high-performance ultraviolet transilluminator, Ultraviolet Products, Upland, CA).

On TTGE analysis, a single-band shift represents a homoplasmic DNA alteration, and a multiple-band pattern represents a heteroplasmic mutation (48). Any DNA fragments showing different banding patterns between matched blood and tumor sample pairs were sequenced to identify the exact mutations.

To rule out the possibility that some mutations may not be detectable by TTGE, we randomly chose six samples that did not show TTGE-positive banding patterns and sequenced 10 coding regions containing stretches of six to eight homopolynucleotides. No mutations were found.

Sequence Analysis

Confirmation of the nucleotide alteration was carried out by direct DNA sequencing of the purified PCR product using the original PCR primers and a BigDye terminator cycle sequencing kit (Perkin-Elmer, Foster City, CA) and analyzed on ABI 377 (Applied Biosystems, Foster City, CA) automated sequencer. All mutations identified were confirmed by repeat PCR and sequencing. The results of DNA sequence analysis were compared with the published Cambridge sequence (37) using MacVector 7.0 (Oxford Molecular Ltd., Oxford, United Kingdom) software. Sequence variations found in both tumor and blood mtDNA were scored as germ line variations. Each was then checked against the MITOMAP database (36). Variations not recorded in the database were categorized as novel mtDNA variations, and those appearing in the database were reported as polymorphisms. Any mtDNA sequence differences found between a tumor sample and its corresponding blood sample were scored as somatic mtDNA mutations specific to the tumor.

Laser Capture Microdissection

Microdissection was done using a PixCell II (Arcturus, Mountain View, CA) laser capture microscope according to the manufacturer's instructions. Frozen 10 μm sections were prepared on uncoated slides from cutaneous neurofibromas and unaffected skin tissues from the same patients (Table 2, patients 4 and 5). The sections were stained in H&E immediately before microdissection. Adjacent control slides were stained with antibody to S100 protein to confirm histologic and morphologic differentiation of Schwann cells within the neurofibromas. S100-negative cells, S100-positive Schwann cells, and endothelial cells were dissected from neurofibromas. Epithelial skin cells and dermal fibroblasts were dissected from skin samples. About 200 to 300 S100-positive Schwann cells, S100 negative cells, and morphologically distinguishable endothelial cells from neurofibromas, epidermal epithelial cells, and dermal fibroblast from normal skin were dissected for DNA extraction (PicoPure extraction kit, Arcturus). The D-loop region of the mtDNA was amplified by PCR and directly sequenced.

We thank Dr. Mia MacCollin for valuable discussions and continuing support of this project.

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1

United States Medical Research Command Neurofibromatosis Research Program award DMAD17-00-1-0535 (A. Kurtz) and BCRP award DAMD 17-01-1-0258 (L-J.C. Wong).

Note: A. Kurtz and M. Lueth contributed equally to this study.