hereditary multiple exostoses ( hme ) , also known as multiple osteochondroma ( mo ) , is an autosomal dominant bone disorder with an incidence of 1 in 50,000 live births in western populations ( 1 ) . the morbidity rate is greater in males than females , with a ratio of 1.5:1 , due to the mutations exerting a weaker phenotypic effect in females ( 2 ) .
hme is characterized by the presence of multiple benign cartilage - capped tumors , localized primarily in the long tubular bones , particularly in the humerus ( 1050% ) , forearm ( 3960% ) , knee ( 33% ) and ankle ( 25% ) .
the majority of patients with hme ( ~70% ) have a family history of the condition ( 1,3,4 ) .
hme is a genetically heterogeneous disorder with two disease - causing genes identified , exostosin glycosyl transferase-1 ( ext1 ) and exostosin glycosyl transferase-2 ( ext2 ) located at chromosomes 8q24 and 11p11-p12 , respectively ( 5,6 ) .
the proteins encoded by human ext1 and ext2 are type ii transmembrane glycoproteins , localized in the endoplasmic reticulum .
the ext1/ext2 complex is involved in the biosynthesis of heparin sulphate ( hs ) proteoglycan ( hspg ) ( 7 ) .
prior to deacetylation , the ext1/ext2 complex catalyzes the elongation of the hs chain . ext1 and ext2 are ubiquitously expressed in developing limb buds , and in osteochondromas their expression was decreased in correlation with mutation status ( 8,9 ) . to date , ~422 separate pathogenic mutations in ext1 and ~221 mutations in ext2 have been identified .
mutations in ext1 account for 5678% of cases in mo families , whereas ext2 mutations have been identified in 2144% of cases ( 4,1015 ) .
however , in china , ext2 mutations are identified more frequently than ext1 mutations ( 16 ) .
the majority of patients have been identified in a single family or as sporadic cases .
nonsense , frameshift and splice - site mutations , which represent the majority of mo - causing mutations ( 80% ) , have been predicted to lead to the premature translational termination of the associated amino acids , and the subsequent production of a truncated protein ( 17 ) .
mutations in ext1 are dispersed along the gene , and may occur in various exons ( 14,18 ) ; however , ext2 mutations do not appear to occur in the final third of the gene - coding region ( 17 ) .
other than ext1 and ext2 , the ext3 gene has been mapped to chromosome 19p ( 19 ) .
it appears to be a minor locus in hme families and no causative mutations in ext3 have been identified ( 20 ) .
three additional ext - like genes , designated extl1 , extl2 and extl3 have been identified and mapped to chromosomes 1 ( 1p36 , 1p11-p12 ) and 8 ( 8p12 ) ( 2123 ) .
although the extl genes are considered strong candidate genes for mo , to date no hme family has been associated with these loci .
the present study investigated a rare large family with mo , and identified a novel splice - site mutation in ext2 .
written consent was obtained from all study participants , and the present study was approved by the ethics committee of the second xiangya hospital ( changsha , china ) .
the proband was admitted to the second xiangya hospital in 2012 , and presented with a large osteochondroma .
the five - generation chinese family of the proband was subsequently investigated and a pedigree constructed based on clinical and radiographical evaluations of all family members ( fig .
there were 13 affected individuals ( 11 males and 2 females ) aged 880 years ( average age , 52 years ) .
the incidence rate was therefore 40% within the family , and mo occurred in each generation .
the affected individuals had 616 exostoses , typically located at the juxtaepiphyseal regions of long bones ; however , these were not as large as those present in the proband .
there were no other lesions that were atypical of mo and no evidence of short stature ( average height of adult male and female was 165 and 156 cm , respectively ) .
all affected individuals had lesions detected prior to age 10 , but had never received surgery , with the exception of the proband .
the proband , family member iv-2 , a 42-year - old male , presented with 41-year history of multiple osteochondroma . physical examination revealed a large mass on the left side of the back ( fig .
2a ) , ( 393319 cm ) , and > 14 osseous nodules of varying sizes located on the prothorax wall , left scapula , bilateral forearms , knees and left ankle .
the patient had had an operation at age 22 due to a rapidly enlarging lumbar spinal osteochondroma .
x - ray analysis revealed a diffuse flocculent shadow with high density in the left lung field and multiple bony protrusions on limbs , as presented in fig .
2b . computed tomography ( ct ) angiography did not reveal any imaging of large arteries , indicating a potential chondroma ( fig .
ct revealed the left back mass was of mixed density and multiple flecked calcifications , which spread into the left side of the chest ( fig .
the patient was subsequently followed up once every three months in the first year , and once every year thereafter .
dna of all affected family members was extracted from peripheral blood as previously described ( 24 ) .
the coding regions of the ext1 and ext2 genes were amplified by polymerase chain reaction ( pcr ) using primer sets , as previously described ( 25 ) .
the pcr products were purified by bigdye terminator version 1.1 ( applied biosystems ; thermo fisher scientific , inc . ,
waltham , ma , usa ) followed by direct dna sequencing with an abi 3100 automatic sequencer ( applied biosystems ; thermo fisher scientific , inc . ) , using forward and reverse primers .
total rna was extracted from the tumor tissue of the proband and healthy lung tissue of an unrelated individual , using the qiagen rneasy mini kit ( qiagen , inc . ,
cdna synthesis was performed using revertaid first strand cdna synthesis kit ( fermentas ; thermo fisher scientific , inc . ) , using the following primers : forward , 5-aaccagaacacactgcgcatcaag and reverse , 5-agctccacgaagaaccacacagaa for exons 25 .
amplification of cdna was performed and products were purified by bigdye terminator version 1.1 followed by direct dna sequencing with an abi 3100 automatic sequencer .
written consent was obtained from all study participants , and the present study was approved by the ethics committee of the second xiangya hospital ( changsha , china ) .
the proband was admitted to the second xiangya hospital in 2012 , and presented with a large osteochondroma .
the five - generation chinese family of the proband was subsequently investigated and a pedigree constructed based on clinical and radiographical evaluations of all family members ( fig .
there were 13 affected individuals ( 11 males and 2 females ) aged 880 years ( average age , 52 years ) .
the incidence rate was therefore 40% within the family , and mo occurred in each generation .
the affected individuals had 616 exostoses , typically located at the juxtaepiphyseal regions of long bones ; however , these were not as large as those present in the proband .
there were no other lesions that were atypical of mo and no evidence of short stature ( average height of adult male and female was 165 and 156 cm , respectively ) .
all affected individuals had lesions detected prior to age 10 , but had never received surgery , with the exception of the proband .
the proband , family member iv-2 , a 42-year - old male , presented with 41-year history of multiple osteochondroma . physical examination revealed a large mass on the left side of the back ( fig .
2a ) , ( 393319 cm ) , and > 14 osseous nodules of varying sizes located on the prothorax wall , left scapula , bilateral forearms , knees and left ankle .
the patient had had an operation at age 22 due to a rapidly enlarging lumbar spinal osteochondroma .
x - ray analysis revealed a diffuse flocculent shadow with high density in the left lung field and multiple bony protrusions on limbs , as presented in fig .
2b . computed tomography ( ct ) angiography did not reveal any imaging of large arteries , indicating a potential chondroma ( fig .
ct revealed the left back mass was of mixed density and multiple flecked calcifications , which spread into the left side of the chest ( fig .
the patient was subsequently followed up once every three months in the first year , and once every year thereafter .
dna of all affected family members was extracted from peripheral blood as previously described ( 24 ) .
the coding regions of the ext1 and ext2 genes were amplified by polymerase chain reaction ( pcr ) using primer sets , as previously described ( 25 ) .
the pcr products were purified by bigdye terminator version 1.1 ( applied biosystems ; thermo fisher scientific , inc . ,
waltham , ma , usa ) followed by direct dna sequencing with an abi 3100 automatic sequencer ( applied biosystems ; thermo fisher scientific , inc . ) , using forward and reverse primers .
total rna was extracted from the tumor tissue of the proband and healthy lung tissue of an unrelated individual , using the qiagen rneasy mini kit ( qiagen , inc . ,
cdna synthesis was performed using revertaid first strand cdna synthesis kit ( fermentas ; thermo fisher scientific , inc . ) , using the following primers : forward , 5-aaccagaacacactgcgcatcaag and reverse , 5-agctccacgaagaaccacacagaa for exons 25 .
amplification of cdna was performed and products were purified by bigdye terminator version 1.1 followed by direct dna sequencing with an abi 3100 automatic sequencer .
written consent was obtained from all study participants , and the present study was approved by the ethics committee of the second xiangya hospital ( changsha , china ) .
the proband was admitted to the second xiangya hospital in 2012 , and presented with a large osteochondroma .
the five - generation chinese family of the proband was subsequently investigated and a pedigree constructed based on clinical and radiographical evaluations of all family members ( fig .
there were 13 affected individuals ( 11 males and 2 females ) aged 880 years ( average age , 52 years ) .
the incidence rate was therefore 40% within the family , and mo occurred in each generation .
the affected individuals had 616 exostoses , typically located at the juxtaepiphyseal regions of long bones ; however , these were not as large as those present in the proband .
there were no other lesions that were atypical of mo and no evidence of short stature ( average height of adult male and female was 165 and 156 cm , respectively ) .
all affected individuals had lesions detected prior to age 10 , but had never received surgery , with the exception of the proband .
the proband , family member iv-2 , a 42-year - old male , presented with 41-year history of multiple osteochondroma . physical examination revealed a large mass on the left side of the back ( fig .
2a ) , ( 393319 cm ) , and > 14 osseous nodules of varying sizes located on the prothorax wall , left scapula , bilateral forearms , knees and left ankle .
the patient had had an operation at age 22 due to a rapidly enlarging lumbar spinal osteochondroma .
x - ray analysis revealed a diffuse flocculent shadow with high density in the left lung field and multiple bony protrusions on limbs , as presented in fig .
2b . computed tomography ( ct ) angiography did not reveal any imaging of large arteries , indicating a potential chondroma ( fig .
ct revealed the left back mass was of mixed density and multiple flecked calcifications , which spread into the left side of the chest ( fig .
the patient was subsequently followed up once every three months in the first year , and once every year thereafter .
dna of all affected family members was extracted from peripheral blood as previously described ( 24 ) .
the coding regions of the ext1 and ext2 genes were amplified by polymerase chain reaction ( pcr ) using primer sets , as previously described ( 25 ) .
the pcr products were purified by bigdye terminator version 1.1 ( applied biosystems ; thermo fisher scientific , inc . , waltham , ma , usa ) followed by direct dna sequencing with an abi 3100 automatic sequencer ( applied biosystems ; thermo fisher scientific , inc . ) , using forward and reverse primers .
total rna was extracted from the tumor tissue of the proband and healthy lung tissue of an unrelated individual , using the qiagen rneasy mini kit ( qiagen , inc . ,
cdna synthesis was performed using revertaid first strand cdna synthesis kit ( fermentas ; thermo fisher scientific , inc . ) , using the following primers : forward , 5-aaccagaacacactgcgcatcaag and reverse , 5-agctccacgaagaaccacacagaa for exons
amplification of cdna was performed and products were purified by bigdye terminator version 1.1 followed by direct dna sequencing with an abi 3100 automatic sequencer .
dna was extracted from family members with hme ( fig . 1a , solid squares ) , and the possible mutations residing in the ext genes were scanned in the exon and intron junctions .
dna sequence analysis revealed a heterozygous mutation , c.939 + 1 g > t in ext2 ( fig .
3 ) in all family members with hme ; none of the unaffected family members carried this mutation .
4 . following rt - pcr at exons 25 , two bands were identified in the proband ; one band of normal size ( ~500 bp ) and one smaller band ( ~300 bp ) .
the size of one band was revealed to be 429 bp , and the other , 296 bp .
compared with cdna of ext2 , sequence analysis confirmed exon 5 skipping in the aberrant allele , resulting in an in - frame deletion of the ext2 protein .
the mo family investigated in the present study exhibited a ratio of male and female patients at 5.5:1 , which differs from the ratio of 1.5:1 reported previously ( 26 ) .
therefore , the present study performed a detailed physical examination of all healthy females in the 3rd to 5th generation , to rule out misdiagnosis due to a weak phenotype .
linkage analysis has confirmed that hme is genetically heterogeneous , and the genes that have indicated the greatest levels of association are ext1 , ext2 and ext3 .
it has been revealed that mutations in ext1 or ext2 are responsible for the majority of hme cases .
the proteins ext1 and ext2 form a hetero - oligomeric complex that functions in hspg biosynthesis .
this complex has a substantially greater glycosyltransferase activity than homo - oligomers of ext1 or ext2 ( 27 ) ; therefore , a mutation present in ext1 or ext2 may result in a critical reduction in hspg ( 9,28,29 ) .
this may subsequently alter the balance of fibroblast growth factor and indian hedgehog homolog signals ( 3032 ) .
thus , the normal signaling pathway involved in bone development may be affected , leading to premature differentiation of cartilage , cartilage cell proliferation and abnormal bone growth in the adjacent areas ( 33 ) , resulting in hme . according to the mo mutation database ( medgen.ua.ac.be/lovdv.2.0/home.php ) ,
713 mutations of the ext1 gene and 386 mutations of the ext2 gene have been identified ; however , no mutations in ext3 have been reported . among the 386 ext2 mutations , the majority are nonsense mutations , followed by frameshift and substitution mutations .
the present study revealed a novel splicing mutation ( c.939 + 1 g > t ) leading to deletion of 196 bp in exon 5 of ext2 , which may result in a truncated and subsequently pathogenic protein .
this mutation resulted in deletion from codon 744 to 939 of exon 5 of the mrna , causing a shift in the codon - reading frame , followed by the synthesis of 266 novel amino acids that terminate with a stop codon at position 994 .
certain studies have suggested that no mutations exist downstream of exon 8 ; however , according to the human gene mutation database ( www.hgmd.cf.ac.uk/ac/index.php ) , 3 patients with mutations in exon 10 and 4 patients with mutations in exon 11 have been identified .
the truncated protein that arises due to the c.939 + 1 g > t mutation in the family investigated in the present study did not contain the amino acids encoded for by exon 7 to l4 .
this alteration may cause disease ; however , whether it is associated with the rare large osteochondroma that occurred on proband remains to be elucidated .
the present study suggested that knudson 's two hit hypothesis or potential mutations in ext3 or extl may explain the occurrence of the osteochondroma . in conclusion , the present study demonstrated that the c.939 + 1 g > t ( ext2 ) mutation , present in a five - generation 33-member mo family , resulted in the splicing out of exon 5 .
 multiple osteochondromas ( mo ) , also known as hereditary multiple exostoses , is an autosomal dominant bone disorder . 
 mutations in exostosin glycosyl transferase-1 ( ext1 ) and exostosin glycosyl transferase-2 ( ext2 ) , including missense , nonsense , frameshift and splice - site mutations , account for up to 80% of reported cases . 
 the proteins ext1 and ext2 form a hetero - oligomeric complex that functions in heparan sulfate proteoglycan biosynthesis . a heterozygous ext2 mutation , c.939 + 1g > t , was identified in a five - generation 33-member mo family , and was present in all 13 affected members . 
 the mutation results in deletion of exon 5 in the mrna , producing a frameshift that leads to a premature termination codon . 
 the present study extends the mutational spectrum of ext2 . 
Introduction
Materials and methods
None
Subjects
DNA analysis
Reverse transcription-PCR (RT-PCR)
Results
Discussion