eight members with schwannomatosis from four consecutive generations of a family included in this study were selected according to the following criteria : age : > 30 years ; two or more nonintradermal schwannomas , at least one with histological confirmation ; no evidence of vestibular tumor on high - quality magnetic resonance imaging scan ; and no known constitutional nf2 mutation . the ages at initial symptoms of body pains and numbness were ~3749 years for the disease - affected members . among five individuals with spinal imaging , the spinal schwannoma manifested as intradural , extramedullary nodules in areas of the lumbar , thoracic , and cervical spine . genomic dna ( 3 g ) from peripheral blood mononuclear cells was used for ( i ) the exon capture using the sureselect human all exon kit ( agilent technologies , santa clara , ca ) and ( ii ) sequencing and cluster generation using the illumina genome analyzer iix ( illumina , san diego , ca ) , as we previously reported . single - base substitutions were detected based on minimum total coverage of 10 and minimum variant coverage of 3 . small insertions / deletions and the log2 copy number variation were calculated for tumor versus normal tissue and were median centered , as we previously reported . the saccharomyces cerevisiae coq6-null mutant w303g63 ( mat  ade2 - 1 , his 3 - 1,15 leu2 - 3,112 trp1 - 1 ura3 - 1 coq6:his3 ) , yeast coq6 gene expression plasmid , and a low - copy - number ( pqm ) or a high - copy - number ( prcm ) yeast expression plasmid containing the human coq6 open reading frame with an in - frame amino terminal yeast mitochondrial leader sequence ( as positive control for yeast rescuing studies ) were generous gifts of dr catherine f. clarke ( university of california , los angeles ) . the d208h mutation of human coq6 was introduced by site - directed mutagenesis ( stratagene , la jolla , ca ) . the s. cerevisiae coq6-null mutants were unable to survive in media containing a nonfermentable carbon source unless a functional coq6 gene was introduced into them , and the yeast complementation experiment was performed as previously reported . the coq10 level in cells was measured using a human coq10 elisa kit , and this level was converted to nanograms per microgram of total cellular protein . mitochondrial membrane potential ( m ) was measured using the standard mitochondrial - specific dual - fluorescence probe jc-1 ( 5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolylcarbocyanine iodide ) , as previously reported . rat coq6 gene  specific small interfering rna and scrambled small interfering rna were transfected into rat schwann cells to decrease coq6 , and then production of intracellular reactive oxygen species ( ros ) in rat schwann cells was also monitored by the fluorescence emission of 2,7-dichlorofluorescein at 72 hours posttransfection . hemagglutinin - tagged human wild - type coq6 and d208h coq6 open reading frames were cloned into the pcdna3.1 ( + ) mammalian expression plasmid and introduced into human fibroblast imr90 cells to assess rescue of coq6 knockdown by small interfering rna . eight members with schwannomatosis from four consecutive generations of a family included in this study were selected according to the following criteria : age : > 30 years ; two or more nonintradermal schwannomas , at least one with histological confirmation ; no evidence of vestibular tumor on high - quality magnetic resonance imaging scan ; and no known constitutional nf2 mutation . the ages at initial symptoms of body pains and numbness were ~3749 years for the disease - affected members . among five individuals with spinal imaging , the spinal schwannoma manifested as intradural , extramedullary nodules in areas of the lumbar , thoracic , and cervical spine . genomic dna ( 3 g ) from peripheral blood mononuclear cells was used for ( i ) the exon capture using the sureselect human all exon kit ( agilent technologies , santa clara , ca ) and ( ii ) sequencing and cluster generation using the illumina genome analyzer iix ( illumina , san diego , ca ) , as we previously reported . single - base substitutions were detected based on minimum total coverage of 10 and minimum variant coverage of 3 . small insertions / deletions and the log2 copy number variation were calculated for tumor versus normal tissue and were median centered , as we previously reported . the saccharomyces cerevisiae coq6-null mutant w303g63 ( mat  ade2 - 1 , his 3 - 1,15 leu2 - 3,112 trp1 - 1 ura3 - 1 coq6:his3 ) , yeast coq6 gene expression plasmid , and a low - copy - number ( pqm ) or a high - copy - number ( prcm ) yeast expression plasmid containing the human coq6 open reading frame with an in - frame amino terminal yeast mitochondrial leader sequence ( as positive control for yeast rescuing studies ) were generous gifts of dr catherine f. clarke ( university of california , los angeles ) . the d208h mutation of human coq6 was introduced by site - directed mutagenesis ( stratagene , la jolla , ca ) . the s. cerevisiae coq6-null mutants were unable to survive in media containing a nonfermentable carbon source unless a functional coq6 gene was introduced into them , and the yeast complementation experiment was performed as previously reported . the coq10 level in cells was measured using a human coq10 elisa kit , and this level was converted to nanograms per microgram of total cellular protein . mitochondrial membrane potential ( m ) was measured using the standard mitochondrial - specific dual - fluorescence probe jc-1 ( 5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolylcarbocyanine iodide ) , as previously reported . rat coq6 gene  specific small interfering rna and scrambled small interfering rna were transfected into rat schwann cells to decrease coq6 , and then production of intracellular reactive oxygen species ( ros ) in rat schwann cells was also monitored by the fluorescence emission of 2,7-dichlorofluorescein at 72 hours posttransfection . hemagglutinin - tagged human wild - type coq6 and d208h coq6 open reading frames were cloned into the pcdna3.1 ( + ) mammalian expression plasmid and introduced into human fibroblast imr90 cells to assess rescue of coq6 knockdown by small interfering rna . the disease - affected members of the family were a father and his daughter in the first and second generations , respectively , and five of eight siblings in the third generation ( named as s1s8 ) ; in addition , one member of the fourth generation has been diagnosed with schwannomatosis thus far ( figure 1a ) . the schwannomatosis tissues of s2 and s7 displayed similar morphological features under hematoxylin - and - eosin staining ( upper panel in figure 1b ) and positive staining for s-100 protein , clearly indicating their schwann cell origin ( lower panel in figure 1b ) . constitutional mutations in smarcb1 , lztr1 , nf1 , and nf2 genes  including point mutations , intragenic insertions / deletions , and gene duplication  were not found in the family by whole - genome sequencing analysis . consistent with this observation , genetic abnormalities of smarcb1 , nf1 , and nf2 in the schwannomatosis tissues of s2 and s7 were not discovered by fluorescence in situ hybridization analysis ( supplementary figure s1a  c online ) and sanger sequencing . immunohistochemical analysis further showed that smarcb1 ( supplementary figure s2a online ) , nf1 ( supplementary figure s2b online ) , and nf2 ( supplementary figure s2c online ) proteins were normally expressed in these tissues . the distribution of schwannomatosis - affected members in the family indicated an autosomal dominant trait . to explore the genetic differences between the affected and unaffected members , we performed whole - genome sequencing on the genomic dna samples of s4 ( schwannomatosis - affected ) and s5 ( normal ) . comparative analysis of whole - genome sequencing showed that there were no deleted / amplified dna regions in s4 compared with the dna of s5 ( supplementary figure s3 online ) . no genetic abnormalities in the smarcb1 , lztr1 , nf1 , and nf2 genes were further identified by whole - genome sequencing analysis ( supplementary table s2 online ) . we further performed whole - exome sequencing on the genomic dna samples of s2 , s3 , and s7 ( supplementary figure s4 online ) . twelve shared heterozygous variants , including nine missense variants ( supplementary table s3 online ) , were identified by these methods ( supplementary figure s4 online ) . variants of hrnr and gstt2 proteins were assessed as benign by polyphen-2 ( polymorphism phenotyping software , version 2 ) . the de novo heterozygous variants of mypn , coq6 , ckmt1a , cyp11a1 , duox1 , and triobp genes were commonly found in all affected members . on the basis of intensive literature review and mutation prediction of these genetic variants , we focused on a heterozygous mutation ( p.asp208his/d208h ; c.622g > c ) , reference sequence nm_182476.2 ) of the coq6 gene , whose function is essential but not redundant . sanger sequencing analysis showed that the d208h allele was found in the disease - affected but not in normal family members ( figure 1c ) . the messenger rna transcript of the d208h coq6 allele was found in the schwannomatosis tissues of s2 and s7 by reverse transcription  polymerase chain reaction ( figure 1d ) . in addition to the heterozygous d208h mutation , molecular analysis did not discover other coq6 genetic variants in these schwannomatosis tissues . coq6 monooxygenase is evolutionarily conserved , and the d208 residue is located at the highly conserved flavin adenine dinucleotide ( fad)-binding domain . the missense mutation altered an amino acid residue that is uniformly conserved from escherichia coli to homo sapiens ( humans ) ( figure 1e , upper panel ) . structural modeling of the fad - binding domain of human coq6 using composer in sybyl ( tripos , st louis , mo ) indicated that the h208 residue would clash with the residues s212 and v214 in the fad - binding domain ( figure 1e , lower panel ) , indicating that h208 may negatively affect coq6 function by interfering with its binding to fad . as shown in figure 2a ( left panel ) , all the transformed coq6-null yeast mutants grew normally in sd - ura selective media ( synthetic defined media with uracil dropout , in which glucose acts as fermentable carbon source ) . the yeast coq6 gene ( prs1-yq6 , positive control ) and wild - type human coq6 ( hwt ) gene , but not the d208h coq6 mutant ( d208h - q6 ) , in both high - copy - number ( prcm ) and low - copy - number ( pqm ) plasmids ( the empty vectors were negative controls ) rescued the growth of coq6-null yeasts plated on ypg media ( yeast extract / peptone / glycerol media , in which glycerol acts as a nonfermentable carbon source ) , which indicated that the d208h coq6 mutant lacked complementation in coq6-deficient yeast mutants . furthermore , we also verified that knockdown of coq6 dramatically decreased coq10 production in human fetal lung fibroblast imr90 cells over a period of 72 hours ( figure 2b ) . knockdown of coq6 in imr90 cells dramatically decreased mitochondrial membrane potential , which may cause cell death by energy depletion , and concurrently increased ros levels over 48 hours ( supplementary figure s5a , b online ) . furthermore , we showed that only reconstitution of wild - type human coq6 , not the d208h coq6 mutant , rescued the imr90 cells after knockdown of coq6 ( figure 2c ) . moreover , we also modeled the haploinsufficiency of coq6 by knockdown of coq6 gene in rat schwann cells and found that ros production increased in a dosage - dependent manner ( figure 2d ) , which might suggest that haploinsufficiency of coq6 may cause chronic ros overproduction in schwann cells . the disease - affected members of the family were a father and his daughter in the first and second generations , respectively , and five of eight siblings in the third generation ( named as s1s8 ) ; in addition , one member of the fourth generation has been diagnosed with schwannomatosis thus far ( figure 1a ) . the schwannomatosis tissues of s2 and s7 displayed similar morphological features under hematoxylin - and - eosin staining ( upper panel in figure 1b ) and positive staining for s-100 protein , clearly indicating their schwann cell origin ( lower panel in figure 1b ) . constitutional mutations in smarcb1 , lztr1 , nf1 , and nf2 genes  including point mutations , intragenic insertions / deletions , and gene duplication  were not found in the family by whole - genome sequencing analysis . consistent with this observation , genetic abnormalities of smarcb1 , nf1 , and nf2 in the schwannomatosis tissues of s2 and s7 were not discovered by fluorescence in situ hybridization analysis ( supplementary figure s1a  c online ) and sanger sequencing . immunohistochemical analysis further showed that smarcb1 ( supplementary figure s2a online ) , nf1 ( supplementary figure s2b online ) , and nf2 ( supplementary figure s2c online ) proteins were normally expressed in these tissues . the distribution of schwannomatosis - affected members in the family indicated an autosomal dominant trait . to explore the genetic differences between the affected and unaffected members , we performed whole - genome sequencing on the genomic dna samples of s4 ( schwannomatosis - affected ) and s5 ( normal ) . comparative analysis of whole - genome sequencing showed that there were no deleted / amplified dna regions in s4 compared with the dna of s5 ( supplementary figure s3 online ) . no genetic abnormalities in the smarcb1 , lztr1 , nf1 , and nf2 genes were further identified by whole - genome sequencing analysis ( supplementary table s2 online ) . we further performed whole - exome sequencing on the genomic dna samples of s2 , s3 , and s7 ( supplementary figure s4 online ) . twelve shared heterozygous variants , including nine missense variants ( supplementary table s3 online ) , were identified by these methods ( supplementary figure s4 online ) . variants of hrnr and gstt2 proteins were assessed as benign by polyphen-2 ( polymorphism phenotyping software , version 2 ) . the de novo heterozygous variants of mypn , coq6 , ckmt1a , cyp11a1 , duox1 , and triobp genes were commonly found in all affected members . on the basis of intensive literature review and mutation prediction of these genetic variants , we focused on a heterozygous mutation ( p.asp208his/d208h ; c.622g > c ) , reference sequence nm_182476.2 ) of the coq6 gene , whose function is essential but not redundant . sanger sequencing analysis showed that the d208h allele was found in the disease - affected but not in normal family members ( figure 1c ) . the messenger rna transcript of the d208h coq6 allele was found in the schwannomatosis tissues of s2 and s7 by reverse transcription  polymerase chain reaction ( figure 1d ) . in addition to the heterozygous d208h mutation , molecular analysis did not discover other coq6 genetic variants in these schwannomatosis tissues . coq6 monooxygenase is evolutionarily conserved , and the d208 residue is located at the highly conserved flavin adenine dinucleotide ( fad)-binding domain . the missense mutation altered an amino acid residue that is uniformly conserved from escherichia coli to homo sapiens ( humans ) ( figure 1e , upper panel ) . structural modeling of the fad - binding domain of human coq6 using composer in sybyl ( tripos , st louis , mo ) indicated that the h208 residue would clash with the residues s212 and v214 in the fad - binding domain ( figure 1e , lower panel ) , indicating that h208 may negatively affect coq6 function by interfering with its binding to fad . as shown in figure 2a ( left panel ) , all the transformed coq6-null yeast mutants grew normally in sd - ura selective media ( synthetic defined media with uracil dropout , in which glucose acts as fermentable carbon source ) . the yeast coq6 gene ( prs1-yq6 , positive control ) and wild - type human coq6 ( hwt ) gene , but not the d208h coq6 mutant ( d208h - q6 ) , in both high - copy - number ( prcm ) and low - copy - number ( pqm ) plasmids ( the empty vectors were negative controls ) rescued the growth of coq6-null yeasts plated on ypg media ( yeast extract / peptone / glycerol media , in which glycerol acts as a nonfermentable carbon source ) , which indicated that the d208h coq6 mutant lacked complementation in coq6-deficient yeast mutants . furthermore , we also verified that knockdown of coq6 dramatically decreased coq10 production in human fetal lung fibroblast imr90 cells over a period of 72 hours ( figure 2b ) . knockdown of coq6 in imr90 cells dramatically decreased mitochondrial membrane potential , which may cause cell death by energy depletion , and concurrently increased ros levels over 48 hours ( supplementary figure s5a , b online ) . furthermore , we showed that only reconstitution of wild - type human coq6 , not the d208h coq6 mutant , rescued the imr90 cells after knockdown of coq6 ( figure 2c ) . moreover , we also modeled the haploinsufficiency of coq6 by knockdown of coq6 gene in rat schwann cells and found that ros production increased in a dosage - dependent manner ( figure 2d ) , which might suggest that haploinsufficiency of coq6 may cause chronic ros overproduction in schwann cells . coq10 is an electron carrier in the mitochondrial respiratory chain and is also a lipid - soluble antioxidant implicated in protecting cells from damage by ros . a previous study showed that severe coq10 deficiency ( < 30% ) caused a marked defect in bioenergetics , with decreased adenosine triphosphate production and sometimes decreased cell growth , but no increase in ros or oxidative stress  induced death . by contrast , intermediate decreases in coq10 ( 3050% ) caused mild defects in bioenergetics , with significant increases in ros and oxidative stress  induced cell death . our knockdown of coq6 in imr90 cells also resulted in a similar reduction of cellular coq10 and mitochondrial membrane potential . we also identified that knockdown of coq6 impaired mitochondrial function and increased ros overproduction in rat schwann cells , with a positive correlation between decreased coq6 protein and ros production . elevated levels of ros and decreased levels of ros scavengers and antioxidant enzymes are associated with various human diseases including cancers . for example , increased ros production caused by mitochondrial dna mutations has been linked to tumor progression in prostate cancer models . abnormally low plasma levels of coq10 have been found in a number of cancer types , including cervical cancer and melanoma . decreased levels of coq10 have been detected in human breast cancer tissues . however , whether deficiency of coq10 predisposes individuals to certain cancers is not known . similarly , the roles of coq10 biosynthesis gene mutations have not been appreciated in cancers . the association is only now becoming apparent with the identification of coq2 gene ( one of the coq10 biosynthesis enzymes ) mutations in human colon and rectal cancers , ovarian carcinoma , and glioblastoma multiforme by cancer genomic studies . in this study , we have identified a heterozygous loss - of - function coq6 missense mutation in familial schwannomatosis . a critical issue related to implication of the genetic alteration in the familial disease is whether the heterozygous loss - of - function coq6 displays haploinsufficiency . a large - scale screening study for genes in yeast displaying haploinsufficiency found that haploinsufficiency of coq6 resulted in a mild reduction of fitness in a medium containing glucose . a recent study demonstrated that both homozygous and compound heterozygous loss - of - function mutations in the human coq6 gene caused early - onset steroid - resistant nephritic syndrome with sensorineural deafness , which progressed to end - stage renal failure by a median age of < 2 years , and two single heterozygous nonsense mutations were also identified in two families with cyclosporin a therefore , it may be assumed that the coq6 haploinsufficiency may be conditional and tissue / cell specific . in the family under study , none of the members harboring the mutated allele had nephritic syndrome , but they did have schwannomatosis , indicating diversity and heterogeneity of clinical phenotypes caused by defects of the same gene . germline abnormalities associated with cancer may be detected in every cell in the body or only in the tumor cells . interestingly , despite the presence of a constitutional genetic abnormality that might affect growth regulatory pathways in all cells , people are generally predisposed to only certain tumor types . in this particular family , we consider that the d208h coq6 allele may lead to a chronic or conditional haploinsufficiency of coq6 in a cell / tissue - specific manner , causing chronic ros overproduction in schwann cells through an unknown mechanism , thus predisposing the family members to familial schwannomatosis . in summary , although the exact oncogenetic mechanism of the loss - of - function coq6 gene in the disease remains a challenging question to be elucidated , we have , for the first time , suggested an association of the defect in one of the coq10 biosynthesis genes , coq6 , with familial schwannomatosis . the mutated coq6 allele may lead to coq10 deficiency and chronic overproduction of ros in schwann cells , which may predispose individuals to the disease .