gaucher 's disease ( gd ) includes several clinical subtypes with a continuum of clinical findings from a perinatal lethal disorder to an asymptomatic type .
the clinical diagnosis of gd currently relies on demonstration of a deficient lysosomal enzyme acid -glucosidase ( glucocerebrosidase [ gba ] ) which is responsible for the hydrolysis of glucocerebroside ( glucosylceramide ) into glucose and ceramide .
gba gene is the only gene in which mutations are known to cause the gd .
mutations of the gene encoding -gba ( gcase ) cause varying degrees of decreased gcase activity .
the accumulated substrates lead to organ damage of diseased individuals , particularly in the liver , spleen , bone marrow , and brain .
three major clinical forms of gd are mostly distinguished by clinical manifestations , age onset , and rate of disease progression .
gd type 1 is characterized by the presence of clinical or radiographic evidence of bone symptoms , hepatosplenomegaly , anemia , and thrombocytopenia , and the absence of primary central nervous systemic symptoms .
mature acid  - gba is a 59 kd , 497 amino acid membrane glycoprotein encoded by the gba gene ( genbank accession # j03059 ) .
gba gene is localized on chromosome 1q21 , spans 7.6 kb and is composed of 11 exons and ten introns . to date , more than 420 mutations have been reported in gba gene , including point mutations ( missense and nonsense ) , deletions and insertions , splice junction mutations , frame shifts alterations , and recombinant alleles .
it is not enough of these known mutations for the study of gd , and much more novel mutations remain to be discovered .
in jewish descent , n370s ( c.1226a > g ) was found to be the most common mutation .
this study aimed to discover more mutations and provide data for the genetic pattern of gba gene , which will help the development of quick and accurate genetic diagnostic tools for this disease .
all procedures were approved by the institutional review board of shanghai children 's hospital , china .
experiments were carried out only after written informed consent had been obtained from the parents of subjects younger than 18 years .
a 5-year - old boy was full - term delivered by his mother at the age of 36-year - old with a procedure of cesarean .
physical examination identified that liver was located about 3 cm under the rib cage , and spleen was located about 15 cm under the left side of the rib cage . no touch pain was observed .
x - ray showed no abnormal periosteal elevation or other signs except that slight endosteal scalloping enlargement was noted .
bone marrow examination revealed the presence of lipid - engorged macrophages , typical gaucher cells appeared with 
fibrillary crumpled silk  in the cytoplasm and eccentrically placed nucleus . to confirm the diagnosis of gd
the enzyme activity in peripheral blood leukocytes was 1.94 nmolhmg , about 3.529.5% of normal activity ( 6.5655.10 nmolhmg ) .
genomic dna was obtained from peripheral blood leukocytes using the qiaamp dna blood mini kit ( qiagen , hilden , germany ) according to the manufacturer 's instructions .
gba gene is localized on chromosome 1q21 , its pseudogene gbap1 is 16 kb downstream of gba .
the similarity of them was 55% ( 9750 bp/17,575 bp ) with a gap of 7025 bp .
we designed long polymerase chain reaction ( pcr ) primers shown in table 1 to distinguish gba gene from gbap1 .
pcr was performed as follows : dna was predenatured for 5 min at 94c , followed by 25 cycles of denaturation at 94c for 30 s , annealing at 61c for 30 s and elongation at 72c for 2 min , and another 25 cycles of denaturation at 94c for 30 s , annealing at 58c for 30 s and elongation at 72c for 3 min , finally 72c for 2 min .
then , designed primers are shown in table 2 for each exon to sequence long pcr segments .
primers for long fragment pcr in this study pcr : polymerase chain reaction ; gba : glucocerebrosidase . primers for sanger sequencing of gba gene gba : glucocerebrosidase .
sequencing reactions were performed with sanger sequencing chemistry using the big dye terminator v3.1 cycle sequencing kit ( applied biosystems inc . , foster city , ca , usa ) on an abi 3130 automated sequencer ( applied biosystems inc .
sequencing reaction conditions were performed as follows : predenatured for 1 min at 96c , followed by 25 cycles of denaturation at 96c for 10 s , annealing at 50c for 5 s , and elongation at 60c for 4 min .
data from sequencing were analyzed by sequencher demo 3.0 ( gene codes corp , usa ) and mutation surveyor demo version 4.0 ( softgenetics llc , usa ) with the reference sequences from ncbi ( nm_001005741.2 for gba ) .
phylogenetic analysis was performed by clustal  1.83 programs to compare the human wild - type gcase protein sequence ( np_001005742.1 ) with orthologs from rat , mouse , pig , sheep , and cow .
we used swiss - model ( http://swissmodel.expasy.org ) to predict possible structural changes caused by t219a mutation and analyzed with pymol ( schrdinger , japan ) .
the further analysis of the stability of gcase was based on the prediction of the hydrophobic property of the protein by protscale ( http://web.expasy.org/protscale/ ) .
the pcdh plasmid vector was digested with ecori and noti for 2.5 h at 37c and used as the target fragment .
gba and pcdh plasmid were ligated with neb t4 dna ligase ( neb#m0202 ) at 16c overnight .
g and c.1448t > c were successfully obtained using the kod - plus - mutagenesis kit ( toyobo , osaka , japan ) .
the two plasmids carrying the mutated loci and a recombinant plasmid without mutations were transfected into cos7 cells ( african green monkey kidney fibroblast - like cell line , cell bank of chinese academy of sciences ) cultured in a 6 cm dish using polyethylenimine . collected cells after 48 h and added cell lysis buffer to collected the protein .
the 20 l lysate was used to carry on western analyses using flag tag antibody ( dykddddk tag ) to verify recombinant plasmid could express in cos7 cells [ figure 1 ] .
then , other two groups wild and mutated recombinant plasmids were expressed to test enzyme activity .
after transfection , cells were collected in a new 2 ml centrifuge tube with 2 ml 1 phosphate buffered saline ( pbs ) buffer , and then washed with 1 ml 1 pbs buffer .
the mixture was centrifuged ( 4000 g for 2 min ) to separate cells from the supernatant .
the final concentration of the homogenate was 12 g/l determined by the protein quantitative analysis kit from shanghai biocolor bioscience & technology company ( shanghai , china ) .
the activity of acid  - gba was measured using the artificial fluorogenic substrate 4-methylumbelliferyl - glucopyranoside ( sigma , usa ) .
mixtures of 0.2 mmol / l citrate - phosphate buffer ( ph 5.5 ) and 0.6% ( w / v ) sodium taurocholate were added to the substrate for a final concentration of 5 mmol / l .
twenty microliters of the homogenates were mixed with 10 l of substrate and incubated for 1 h at 37c .
the reaction was stopped by adding 200 l glycine / nahco3 buffer ( ph 10.5 ) .
the fluorescence was read at an excitation wavelength of 365 nm and an emission wavelength of 450 nm using 4-mu to calibrate the fluorescence spectrometer .
( b ) other two groups prove activity of the variant is about 77% lower than the wild .
all procedures were approved by the institutional review board of shanghai children 's hospital , china .
experiments were carried out only after written informed consent had been obtained from the parents of subjects younger than 18 years .
a 5-year - old boy was full - term delivered by his mother at the age of 36-year - old with a procedure of cesarean .
physical examination identified that liver was located about 3 cm under the rib cage , and spleen was located about 15 cm under the left side of the rib cage . no touch pain was observed .
x - ray showed no abnormal periosteal elevation or other signs except that slight endosteal scalloping enlargement was noted .
bone marrow examination revealed the presence of lipid - engorged macrophages , typical gaucher cells appeared with 
fibrillary crumpled silk  in the cytoplasm and eccentrically placed nucleus . to confirm the diagnosis of gd
the enzyme activity in peripheral blood leukocytes was 1.94 nmolhmg , about 3.529.5% of normal activity ( 6.5655.10 nmolhmg ) .
genomic dna was obtained from peripheral blood leukocytes using the qiaamp dna blood mini kit ( qiagen , hilden , germany ) according to the manufacturer 's instructions .
gba gene is localized on chromosome 1q21 , its pseudogene gbap1 is 16 kb downstream of gba .
the similarity of them was 55% ( 9750 bp/17,575 bp ) with a gap of 7025 bp .
we designed long polymerase chain reaction ( pcr ) primers shown in table 1 to distinguish gba gene from gbap1 .
pcr was performed as follows : dna was predenatured for 5 min at 94c , followed by 25 cycles of denaturation at 94c for 30 s , annealing at 61c for 30 s and elongation at 72c for 2 min , and another 25 cycles of denaturation at 94c for 30 s , annealing at 58c for 30 s and elongation at 72c for 3 min , finally 72c for 2 min .
then , designed primers are shown in table 2 for each exon to sequence long pcr segments .
primers for long fragment pcr in this study pcr : polymerase chain reaction ; gba : glucocerebrosidase . primers for sanger sequencing of gba gene gba : glucocerebrosidase .
sequencing reactions were performed with sanger sequencing chemistry using the big dye terminator v3.1 cycle sequencing kit ( applied biosystems inc . , foster city , ca , usa ) on an abi 3130 automated sequencer ( applied biosystems inc .
sequencing reaction conditions were performed as follows : predenatured for 1 min at 96c , followed by 25 cycles of denaturation at 96c for 10 s , annealing at 50c for 5 s , and elongation at 60c for 4 min .
data from sequencing were analyzed by sequencher demo 3.0 ( gene codes corp , usa ) and mutation surveyor demo version 4.0 ( softgenetics llc , usa ) with the reference sequences from ncbi ( nm_001005741.2 for gba ) .
phylogenetic analysis was performed by clustal  1.83 programs to compare the human wild - type gcase protein sequence ( np_001005742.1 ) with orthologs from rat , mouse , pig , sheep , and cow .
we used swiss - model ( http://swissmodel.expasy.org ) to predict possible structural changes caused by t219a mutation and analyzed with pymol ( schrdinger , japan ) .
the further analysis of the stability of gcase was based on the prediction of the hydrophobic property of the protein by protscale ( http://web.expasy.org/protscale/ ) .
the pcdh plasmid vector was digested with ecori and noti for 2.5 h at 37c and used as the target fragment .
gba and pcdh plasmid were ligated with neb t4 dna ligase ( neb#m0202 ) at 16c overnight .
g and c.1448t > c were successfully obtained using the kod - plus - mutagenesis kit ( toyobo , osaka , japan ) .
the two plasmids carrying the mutated loci and a recombinant plasmid without mutations were transfected into cos7 cells ( african green monkey kidney fibroblast - like cell line , cell bank of chinese academy of sciences ) cultured in a 6 cm dish using polyethylenimine . collected cells after 48 h and added cell lysis buffer to collected the protein .
the 20 l lysate was used to carry on western analyses using flag tag antibody ( dykddddk tag ) to verify recombinant plasmid could express in cos7 cells [ figure 1 ] .
then , other two groups wild and mutated recombinant plasmids were expressed to test enzyme activity .
after transfection , cells were collected in a new 2 ml centrifuge tube with 2 ml 1 phosphate buffered saline ( pbs ) buffer , and then washed with 1 ml 1 pbs buffer .
the mixture was centrifuged ( 4000 g for 2 min ) to separate cells from the supernatant .
the final concentration of the homogenate was 12 g/l determined by the protein quantitative analysis kit from shanghai biocolor bioscience & technology company ( shanghai , china ) .
the activity of acid  - gba was measured using the artificial fluorogenic substrate 4-methylumbelliferyl - glucopyranoside ( sigma , usa ) .
mixtures of 0.2 mmol / l citrate - phosphate buffer ( ph 5.5 ) and 0.6% ( w / v ) sodium taurocholate were added to the substrate for a final concentration of 5 mmol / l .
twenty microliters of the homogenates were mixed with 10 l of substrate and incubated for 1 h at 37c .
the reaction was stopped by adding 200 l glycine / nahco3 buffer ( ph 10.5 ) .
the fluorescence was read at an excitation wavelength of 365 nm and an emission wavelength of 450 nm using 4-mu to calibrate the fluorescence spectrometer .
( b ) other two groups prove activity of the variant is about 77% lower than the wild .
a novel heterozygous c.655a > g ( p.t219a ) mutation and a heterozygous c.1448t > c ( p.l483p ) were identified by sanger sequencing in this patient .
the c.655a > g ( p.t219a ) mutation was identified in his mother , while c.1448t > c ( p.l483p ) was identified in his father [ figure 2a ] .
we found that c.655a > g ( p.t219a ) mutation has never been reported neither in chinese patient nor other ethnic populations .
( b ) amino acids on 219 and 483 are well conserved in human , rat , mouse , pig , sheep , and cow .
a comparison of the human wild - type gcase protein sequence ( np_001005742.1 ) with orthologs from rat , mouse , pig , sheep , and cow indicated that gcase was conserved at p.t219a and p.l483p [ figure 2b ] .
protein structure models for the wild - type and c.655a > g mutant and c.1448t > c mutant proteins were generated by swiss - model and analyzed by pymol .
the c.1448t > c ( p.l483p ) mutation   is located in domain 2 of gcase which can influence the catalytic activity of gcase by changing the stability of the protein indirectly , and c.655a > g ( p.t219a ) is located in domain three which contains the catalytic center of the enzyme .
p.t219a does not have an effect on enzyme activity directly because there are other acid , amino residues among p.t219 , loop 1 , 2 , 3 , and catalytic center e235 , e340 .
t219 is near the surface of the protein and turns to be more hydrophobic when threonine changes to alanine .
hydroxyl of t219 could bind to k225 and c287 to form hydrogen bonding [ figure 4a and 4b ] .
once mutation happened , the hydrogen bonding would disappear which lead to decrease of the stability of gcase in the area that results in activity declining [ figure 4c4e ] .
( a and b ) integral and part view of the three - dimensional structure of gcase before mutation .
( c and d ) integral and part view of the three - dimensional structure of gcase after mutation .
t219 provides hydroxyl to form hydrogen bonds with k225 and c287 but a219 can not .
( a and b ) oh ( hydroxyl ) of t219 can promote the formation of hydrogen bonds between t219 and c287 and k225 .
( c - e ) oh disappears after mutation ( carbon atoms are green , nitrogen atoms are blue , oxygen atom are red , and hydrogen atoms are gray ) . after verifying that the recombinant plasmid could express gcase in cos7 cells [ figure 1a ]
the activity of recombinant gcase analyzed by fluorescence spectrophotometry showed that the average activity of the wild - type was 114.69 nmolhmg , while the mutation was approximately 77% lower , at 26.37 nmolhmg [ figure 1b ] .
these data indicated that the heterozygous point mutation we identified in gba gene led to a gcase mutation that reduced its activity .
this led to the accumulation of glucocerebroside in the phagocyte system , which led to the formation of gaucher cells , induced other manifestations of gd , and eventually led to gd .
a novel heterozygous c.655a > g ( p.t219a ) mutation and a heterozygous c.1448t > c ( p.l483p ) were identified by sanger sequencing in this patient .
the c.655a > g ( p.t219a ) mutation was identified in his mother , while c.1448t > c ( p.l483p ) was identified in his father [ figure 2a ] .
we found that c.655a > g ( p.t219a ) mutation has never been reported neither in chinese patient nor other ethnic populations .
( b ) amino acids on 219 and 483 are well conserved in human , rat , mouse , pig , sheep , and cow .
a comparison of the human wild - type gcase protein sequence ( np_001005742.1 ) with orthologs from rat , mouse , pig , sheep , and cow indicated that gcase was conserved at p.t219a and p.l483p [ figure 2b ] .
protein structure models for the wild - type and c.655a > g mutant and c.1448t > c mutant proteins were generated by swiss - model and analyzed by pymol .
the c.1448t > c ( p.l483p ) mutation   is located in domain 2 of gcase which can influence the catalytic activity of gcase by changing the stability of the protein indirectly , and c.655a > g ( p.t219a ) is located in domain three which contains the catalytic center of the enzyme .
p.t219a does not have an effect on enzyme activity directly because there are other acid , amino residues among p.t219 , loop 1 , 2 , 3 , and catalytic center e235 , e340 .
t219 is near the surface of the protein and turns to be more hydrophobic when threonine changes to alanine .
hydroxyl of t219 could bind to k225 and c287 to form hydrogen bonding [ figure 4a and 4b ] .
once mutation happened , the hydrogen bonding would disappear which lead to decrease of the stability of gcase in the area that results in activity declining [ figure 4c4e ] .
( a and b ) integral and part view of the three - dimensional structure of gcase before mutation .
( c and d ) integral and part view of the three - dimensional structure of gcase after mutation .
t219 provides hydroxyl to form hydrogen bonds with k225 and c287 but a219 can not .
( a and b ) oh ( hydroxyl ) of t219 can promote the formation of hydrogen bonds between t219 and c287 and k225 .
( c - e ) oh disappears after mutation ( carbon atoms are green , nitrogen atoms are blue , oxygen atom are red , and hydrogen atoms are gray ) .
after verifying that the recombinant plasmid could express gcase in cos7 cells [ figure 1a ] , we further investigated the effect of the mutation through other group tests .
the activity of recombinant gcase analyzed by fluorescence spectrophotometry showed that the average activity of the wild - type was 114.69 nmolhmg , while the mutation was approximately 77% lower , at 26.37 nmolhmg [ figure 1b ] .
these data indicated that the heterozygous point mutation we identified in gba gene led to a gcase mutation that reduced its activity .
this led to the accumulation of glucocerebroside in the phagocyte system , which led to the formation of gaucher cells , induced other manifestations of gd , and eventually led to gd .
it is estimated that the global morbidity of gd is between 1:40,000 or 50,000 and up to approximately 1:450 in the ashkenazi jew population .
however , this treatment is expensive and not effective for patients with types 2 and 3 gd
a diagnosis of gd is suspected when there is an unexplained manifestation , such as hepatosplenomegaly , anemia , thrombocytopenia , or a bone marrow smear showing gaucher cells .
the diagnosis can be confirmed by reduced gba activity ( usually < 30% of normal ) in peripheral blood leukocytes or skin fibroblasts .
the diagnosis of gd should be distinguished from leukemia , lymphadenoma , multiple myeloma , and other diseases .
we diagnosed a pediatric patient from the children 's hospital who was suspected of suffering from gd by identifying gaucher cells in a bone marrow smear .
because the gba activity was 1.9 nmolhmg , at least 70% lower than normal , the patient was diagnosed with gd . at present , there have been more than 420 mutations identified in gba gene according to the human gene mutation database .
genetic examinations of both the patient and his parents were necessary to ascertain the type of mutation , and sanger sequencing identified heterozygous point mutations at c.655a > g ( maternal disease haplotype ) and c.1448t > c ( paternal disease haplotype ) of the patient . in combination with the clinical manifestations , we confirmed the patient 's diagnosis of type 1 gd .
homozygous c.1448t > c ( p.l483p ) had been reported early in 1987 and was related to type 2 gd . compared to patients with this compound heterozygous mutation , patients with homozygous c.1448t > c mutation developed earlier in 13 years old .
the pathogenic predictions for the point mutations c.655a > g and c.1448t > c were made by mutation taster ( http://www.mutationtaster.org ) , which indicated that it was a clear mutation type and that both were disease causing .
we found changes in two amino acids that altered the primary structure of gcase , which will be discussed later .
the prediction of evolutionary conservation was based on human , rat , mouse , pig , sheep , and cow . as expected ,
the two amino acids are well conserved in most mammals . as is known to all ,
mature acid -gba is consists of two chains involving 497 amino acids according to rcsb database ( http://www.rcsb.org/pdb/home/home.do ) .
gcase has three domains including domain 1 ( residues 129 and 384414 ) , domain 2 ( residues 3075 and 431497 ) , and domain 3 ( residues 76382 and 425430 ) [ figure 3a ] .
catalytic center e235 and e340 and could be seen in figure 3c surrounded by loop 1 ( residues 345350 ) , loop 2 ( residues 393399 ) , and loop 3 ( residues 312319 ) .
mutant c.1448t > c is located in -strand ( d482m489 ) and changed hydrophobic -sheet of d443d445 .
the spatial relationships of residue 219 , catalytic center , and three loops before and after mutation were analyzed by pymol [ figure 3 ] .
it was obvious there were residues between t219 and catalytic center as well as a219 . as a result , mutation t219a did not have an effect on e235 and e340 , and the cap around them .
the prediction of hydrophobic property of gcase by protscale was based on kyte 's method .
this would directly lead to reaction changing of acid amino between mutation sites and others .
our laboratory was focused on the mechanisms of neuronopathic gd and the development of therapeutic strategies .
epigenetic studies on mouse models of neuronopathic gd   are in progress , and the results will contribute to our understandings of gd .
this study was supported by grants from natural science foundation of china ( no . 81371269 ) and shanghai research program ( no .
this study was supported by grants from natural science foundation of china ( no . 81371269 ) and shanghai research program ( no . 14140902600 , no .
 background : gaucher 's disease ( gd ) is an autosomal recessive disorder caused by a deficiency of acid -glucosidase ( glucocerebrosidase [ gba ] ) that results in the accumulation of glucocerebroside within macrophages . 
 many mutations have been reported to be associated with this disorder . 
 this study aimed to discover more mutations and provide data for the genetic pattern of the gene , which will help the development of quick and accurate genetic diagnostic tools for this disease.methods:genomic dna was obtained from peripheral blood leukocytes of the patient and sanger sequencing is used to sequence gba gene . 
 sequence alignments of mammalian -gba ( gcase ) and three - dimensional protein structure prediction of the mutation were made . 
 a construct of this mutant and its compound heterozygous counterpart were used to measure gcase in vitro.results:gcase is relatively conserved at p.t219a . 
 this novel mutation differs from its wild - type in structure . 
 moreover , it also causes a reduction in gcase enzyme activity.conclusion:this novel mutation ( c.655a > g , p.t219a ) is a pathogenic missense mutation , which contributes to gd . 
I
M
Patient and clinical data
Molecular analysis
Phylogenetic analysis and structural predictions
Mutagenesis of 
R
DNA sequencing identifies a novel missense mutationin 
GCase is conserved at two mutated loci
Structural prediction of mutant alleles
GCase activity 
D
Financial support and sponsorship
Conflicts of interest