gaucher disease ( gd ) is an autosomal recessive lysosomal storage disorder caused by a profound deficiency in the enzyme glucocerebrosidase [ gba ( glucosylceramidase , acid beta - glucosidase ) ec.3.2.1.45 ] ( brady et al . , 1965 ) .
the deficiency in gba results in the accumulation of its lipid substrate glucocerebroside in reticuloendothelial cells with cardinal clinical findings of hepatosplenomegaly , pancytopenia from bone marrow infiltration and osteonecrosis .
three clinical forms or subtypes of gd have been reported : type i ( non - neuronopathic , omim 230800 ) , type ii ( acute neuronopathic , omim 230900 ) , and type iii ( sub - acute neuronopathic , omim 231000 ) ( beutler , e and grabowski , ga , 1995 , mikosch , p , 2011 ) .
gaucher disease is a panethnic disease with an overall prevalence of 1/100,000 , but higher prevalence in the ashkenazi jewish population of 1/855 with predominantly the n370s allele ( guggenbuhl et al . , 2008 ) .
mutations in the gba gene of patients with gd can result in deficiency or absence of gba enzyme activity .
over 350 mutations causal to gd have been reported , including missense and nonsense mutations , insertions , deletions , complex recombinant alleles , and splice site mutations ( hruska et al . , 2008 ) .
the four most common mutations found in the gba gene are c.1226 g  > 
a ( n370s ) , c.1448 t  >  a ( l444p ) , c.84dupg ( 84gg ) and ivs2  +  1 g  >  a ( ivs2  +  1 ) , and account for >  90% of ashkenazi jewish population gd alleles , and 70% of general population gd alleles ( mao et al . , 2001 ) .
although studies of genotype  phenotype correlations have revealed significant heterogeneity , some consistent patterns have emerged for prognostic and therapeutic decisions .
for example , a milder phenotype is associated with the n370s allele ( beutler and gelbart , 1996 ) .
alternatively , null ( i.e. , completely non - functional ) alleles such as 84gg in combination with a severe mutation such as l444p in the second gba allele , are commonly associated with severe clinical manifestations and symptoms of the central nervous system . to date , there has been no reported case of a gaucher patient homozygous for two null alleles , e.g. 84gg/84gg , suggesting that this genotype is presumably lethal prenatally ( beutler and grabowski , 1995 ) .
this report describes the identification of a novel splice site mutation ivs9  +  1 g  >  a , and a novel complex allele g355r / r359x , in two unrelated patients with type i gd , who are both heterozygous for the common missense mutation , n370s .
the entire gba coding sequence of both patients was sequenced and the results were analyzed .
we have also established a restriction fragment length polymorphism ( rflp ) analysis using hph1 restriction endonuclease digest to confirm the presence of the ivs9  +  1 mutation and to screen for its presence in other patients . to demonstrate that mutations g355r and r359x are in cis arrangement , i.e. within the same allele on the same chromosome , gba exon 8 in which the mutations are located was cloned and sequence analyzed .
patient 1 is of english ancestry and was diagnosed at 3  years of age with gd from a bone biopsy showing  gaucher  cells but treatment was never sought . at 70  years of age , he pursued medical treatment of his gd .
he had severe hepatosplenomegaly , thrombocytopenia ( platelets 15    10/l ) with a history of easy bleeding and easy bruising from minor injuries , leukopenia ( leukocytes 2    10/l ) and anemia [ hemoglobin ( hb)128 
g / l ] , marrow replacement on spinal mri ( magnetic resonance imaging ) and an elevated chitotriosidase [ 19,322  nmol / h / ml ( normal 4120 , integrated genetics , labcorp specialty testing group , santa fe , nm , u.s.a . ) ] .
his peripheral t - lymphocyte beta - glucosidase activity was 0.3 ( reference 8.921.5  nmol / h / mg protein , floyd snyder , alberta children 's hospital , calgary , ab , canada ) .
he also had a monoclonal gammopathy of uncertain significance ( mgus ) with only a mild elevation of immunoglobulin g kappa and free light chains .
patient 2 was diagnosed in 1983 at age 20 , when hepatosplenomegaly and mild thrombocytopenia led to a bone marrow biopsy that showed  gaucher  cells .
acid beta - glucosidase level and mutation analysis were not done at that time . over the next 30  years , her hb and platelet count gradually decreased , the former from 125  g / l to 91  g / l by 2013 , and the latter from 84    10/l to 34    10/l .
past history also included easy bruising , frequent nosebleeds , menorrhagia , and mild hypertension .
her spleen and liver were palpated 11 and 7  cm below the respective costal margins .
beta - glucosidase level was 1  nmol / mg protein ( normal 816 ) .
mri of the abdomen showed liver and spleen volumes of 1802 and 1141  cm , respectively , as well as the presence of cholelithiasis .
mri of the femurs showed the classic erlenmeyer flask abnormality and evidence of marrow packing , but no necrosis or infarcts .
the patient was begun on imiglucerase 30  mg / kg every 2  weeks ; as of may 2015 her hb was 129 
g / l , platelets 90    10/l , and chitotriosidase 1125  nmol / h / ml .
the methods for collecting dot blood samples on filter paper cards , ( devost and choy , 2000 ) pcr amplification of gba genomic dna for sequence analysis using gba specific primers , and rflp analysis of gba mutations are described in supplementary information . in order to confirm that mutations g355r
/ r359x were in cis arrangement i.e. present in the same allele , exon 8 was cloned into the sequencing vector pjet1.2 .
one shot top10 chemically competent e. coli ( life technologies , carlsbad , ca ) was transformed with pjet1.2/exon 8 and transformants were selected for on low sodium lb agar containing 100  g / ml ampicillin .
colony pcr was performed on 24 clones , 15 of which contained the exon 8 insert and were sequenced analyzed by europhins mwg operon .
sequencing results were compared to functional gba genomic sequence from genbank ( gi183011).the results revealed that in patient 1 at nucleotide position c.1226 ( g.4824 ) , there was a heterozygous a to g transition that resulted in asparagine codon ( aac ) being substituted by that for serine ( agc ) in gba amino acid residue position 370 ( n370s ) ( fig . 1 , left column and fig .
c.1328  +  1 , the sequence shows a heterozygous g to a transition ( fig 1 , right column and fig .
although this substitution is intronic , it is located at the intron - exon boundary and within the spliceosome recognition sequence ( mount , 1982 ) , specifically the donor sequence .
the chromatogram of both regions revealed a double peak depicting the normal and mutant sequence at the respective locations at c.1226 and c.1328  +  1 , which is consistent with a heterozygous mutation ( fig .
these findings were confirmed by performing sequence analysis in both the forward and reverse directions ( data not shown ) .
sequence analysis of the rest of the gba coding region and intron - exon boundaries showed identity with the genbank wild type sequence .
mismatch pcr using primer set m370/pbii was performed , resulting in an amplified fragment of 105  bp . as first described by beutler et al .
( 1990 ) , the mutation n370s creates a cleavage site in this fragment and upon digestion by xhoi restriction endonuclease and electrophoresis in 6% polyacrylamide , dna fragment lengths from patient 1 at 105 , 86 and 19  bp were noted , while the normal control showed only a single band at 105  bp ( data not shown ) .
nebcutter software ( new england biolabs ) was used to determine various restriction sites in normal and ivs9  +  1 g  >  a mutant alleles .
the enzyme hphi was determined to cleave in 2 positions on the normal allele , but 3 on the mutant allele as mutation ivs9 
after digestion with hphi , samples were run in an 8% acrylamide gel and visualized with ethidium bromide .
the normal control has fragment lengths of only 145 , 74 and 33  bp ( fig 3 , lane 4 ) .
the fragment lengths for patient 1 were revealed to be 145 , 107 , 74 , 38 and 33  bp , which is consistent with ivs9  +  1 g  >  a in the heterozygous form ( fig 3 , lane 3 ) .
sequence analysis of patient 2 showed that mutation n370s is present in the heterozygous form with two other heterozygous point mutations : g to c at c.1180 , g.4396 , and t to c at c. 1192 , g.4408 ) substituting glycine by arginine ( g355r ) and arginine by a premature termination ( r359x ) , respectively . to find out whether mutations r355r and r359x are present in cis- or trans - arrangement ( i.e. within the same gba allele or in different gba alleles ) , pcr - amplified gba exon 8 genomic dna of patient 2 was cloned into the pjet1.2 vector in e. coli top10 strain and subjected to sequence analysis .
of the 15 clones sequenced , 5 contained the exon 8 mutations g355r and r359x showing both mutations in cis arrangement ( i.e. on the same chromosome ) as a g355r / r359x complex allele , while 10 contained the wild type exon 8 sequence indicating that the cloned fragments were from the other ( exon 9 mutation n370s ) gaucher allele ( fig .
genotyping of the gaucher mutations n370s and g353r / r359x was also performed from dot blood genomic dna of the asymptomatic mother and sister of patient 2 .
rflp and dna sequence analysis showed that the mother is heterozygous for mutation n370s and negative for g353r / r359x , while the sister was negative for both n370s and g353s / r359x , i.e. normal homozygous .
these findings indicated that the parents of patient 2 had transmitted mutation n370s and the complex g353s / r359x allele to her , and the normal gba allele to her unaffected sister .
there have been more than 350 mutations recorded to date , including 16 splice site mutations that cause gaucher disease ( http://www.hgmd.org ) . many of these mutations , including ivs9  +  1(g  >  a ) and g355r / r359x , are present in only one or a small number of individuals .
a splice site mutation can have a range of consequences including exon skipping , activation of cryptic splice sites , creation of a pseudo - exon within a gene or intron retention ( nakai and sakamoto , 1994 ) . in the case of the highly prevalent splice site mutation
ivs2  +  1 ( g  >  a ) , exon 2 is entirely removed from the spliced mrna ( he and grabowski , 1992 ) . in order to assess the possible impact of the ivs9  +  1 ( g  >  a ) mutation found in patient 1 on gba mrna splicing
, we used the regrna 2.0 program ( http://regrna2.mbc.nctu.edu.tw/ ) , the neural network program ( http://www.fruitfly.org/seq_tools/splice.html ) , and the maximum entropy program ( http://genes.mit.edu/burgelab/maxent/xmaxentscan_scoreseq.html ) to predict how this mutation would affect rna processing .
all three programs indicate that the ivs9  +  1 mutated sequence would no longer be recognized as donor splice site ( please refer to supplementary information for details of the in silico analyses ) .
additionally , the neural network and maximum entropy programs predicted that the mutation would result in a much lower splice site signal below recognition threshold .
thus , the ivs9  +  1 mutation will be very disruptive for gba mrna processing and enzyme function .
an alternative possibility is the activation of cryptic donor splice sites . in a study describing another exon 9 splice site mutation ( c.1389 - 1 g  >  a ) , the neural network and maximum entropy programs predicted the mutated sequence would no longer be recognized as an acceptor splice site .
however , when rt - pcr analysis was performed on mrnas from cultured fibroblasts , it was found that the mutation led to exon slippage of 4 nucleotides and activation of a cryptic acceptor splice site ( malini et al . , 2014 ) .
we are unable to perform similar rt - pcr analysis of fibroblast gba mrnas from patient 1 because dried dot blood on filter paper cards is the only available cell source for our study .
the structure of gba has been elucidated by x - ray crystallography and provides some insight to the role of the 497 residues making up the three domains of gba polypeptide .
domain i ( gba amino acid residues 127 and 383414 ) is a -sheet containing two disulfide bridges and a glycosylation at n19 , which is required for activity in vivo .
domain ii ( gba amino acid residues 3075 and 431497 ) consists of two -sheets resembling an immunoglobulin fold that may interact with saposin c , an essential activator of gba ( aerts , jmfg , et al .
, 1990 , dvir , h , et al . , 2003 , tamargo , rj , et al . ,
domain iii ( gba amino acid residues 76381 and 416430 ) consists of a ( /a)8 tim barrel containing the catalytic site : e340 , the nucleophile , and e235 , the acid / base catalyst ( dvir et al . , 2003 ) .
exon 9 codes for gba amino acid residues 370424 , and therefore plays a major role in the formation of the both domain i and the catalytic domain .
an absence of exon 9 because of aberrant splicing would be detrimental to gba function by abolishing its activity completely . in this case , however , the second gaucher allele is the relatively mild mutation n370s ( beutler , e and grabowski , ga , 1995 , grace , me , et al . , 1990 ) which modulates the clinical expression to a mild to moderate type 1 form .
other splicing mutations have been reported in compound heterozygosity with n370s in patients with type 1 gd ( dominissini , s , et al . , 2006 ,
the novel complex allele g355r / r359x in patient 2 will result first in a substitution of glycine by arginine , followed by a protein truncation at gba amino acid residue 359 . complex alleles are often the result of reciprocal or non - reciprocal recombination events with the pseudogene ( hruska et al . , 2008 ) .
the human gba pseudogene is 96% homologous to the gba functional gene but contains a 55  bp deletion in exon 9 and a number of exonic point mutations as well as large deletions in several introns ( horowitz et al . ,
g355r / r359x , however , neither mutation is pseudogene - derived , and therefore , there is no evidence to suggest this is the result of a recombination event .
the missense mutation g355r has not previously been reported ; however , two studies have characterized another mutation , g355d , in which glycine at gba amino acid residue 355 is substituted by aspartic acid instead of arginine . in one study ,
g355d in combination with l444p resulted in the neuronopathic type ii gd ( tsai et al . ,
these findings suggest that g355r may also have varying consequences , depending on the identity of the mutation in the second allele . in silico analysis of this mutation on the stability and function of gba using the foldx program ( http://foldxsuite.crg.eu ) and polyphen 2 program ( http://genetics.bwh.harvard.edu/pph2/ ) predict that g355r would severely destabilize and damage the structure of gba ( please refer to supplementary information for details of the analysis ) .
the nonsense mutation r359x has been described as a perinatal lethal mutation when in combination with other lethal mutations such as v398f ( stone et al . , 1999 ) , and as a parkinsonism - associated mutation ( sunwoo et al . , 2011 ) . because r359x results in premature termination of gba translation , amino acid residues 359497 that constitute gba domain i ( residue 383414 ) , domain ii ( residue 431497 ) , and domain iii ( residue 359381 ) that constitute the catalytic and saposin activator binding domains ( dvir , h , et al .
in addition , the transcript resulting from nonsense mutation may also be prematurely degraded via nonsense - mediated mrna decay , a post - transcriptional quality control process in eukaryotes that degrades transcripts carrying disease - causing premature termination codons ( turner , ka and choy , fym , 2015 , popp , mw and maquat , le , 2013 ) .
these will be detrimental to gba function and it is reasonable to postulate that the complex allele g355r / r359x might also be lethal when in combination with another severe or lethal gaucher allele .
however , the second gaucher allele is the relatively mild mutation n370s which ameliorates the clinical severity and as such ( as in the case of patient 1 ) , patient 2 presents with type 1 gd , which again demonstrates the rescuing properties of n370s as we previously reported ( jack et al . , 2014 ) .
the nonsense mutation r359x has been reported in patients heterozygous for n370s , who also presented with type 1 gd ( alfonso et al . , 2007 ) .
we have identified a novel splice site mutation ( ivs9  +  1 g  >  a ) and a novel complex allele g355r / r359x in two unrelated patients with type
we have also developed a rflp procedure that utilizes hph1 restriction endonuclease digest for confirmation and efficient identification of the ivs9  +  1 mutation in future patients . in silico analysis of these novel gaucher alleles showed that they are detrimental to either gba splicing and/or structure and function .
however , compound heterozygosity with mutation n370s in both patients has modulated the clinical expression to a mild to moderate type 1 gaucher phenotype .
 gaucher disease is an autosomal recessive lysosomal storage disorder resulting from deficient glucocerebrosidase activity . 
 more than 350 mutations that cause gaucher disease have been described to date . 
 novel mutations can potentially provide insight into the glucocerebrosidase structure  function relationship and biochemical basis of the disease . here , we report the identification of two novel mutations in two unrelated patients with type i ( non - neuronopathic ) gaucher disease : 1 ) a splice site mutation ivs9  +  1 g  >  a ; and ( 2 ) a complex allele ( cis ) g355r / r359x . both patients have a common n370s mutation in the other allele . 
 the splice site mutation results from an intronic base substitution ( g to a , c.1328  +  1 , g.5005 ) at the donor splice site of exon and intron 9 . 
 the complex allele results from two point mutations in exon 8 of glucocerebrosidase ( g to c at c.1180 , g.4396 , and t to c at c. 1192 , g.4408 ) substituting glycine by arginine ( g355r ) and arginine by a premature termination ( r359x ) , respectively . in order to demonstrate that g355r / r359x are in cis arrangement , pcr - amplified glucocerebrosidase exon 8 genomic dna from the patient was cloned into the vector pjet1.2 in escherichia coli top10 strain . 
 out of the 15 clones that were sequence analyzed , 10 contained the normal allele sequence and 5 contained the complex allele g355r / r359x sequence showing both mutations in cis arrangement . 
 restriction fragment length polymorphism analysis using hph1 restriction endonuclease digest was established for the ivs9  +  1 g  >  a mutation for confirmation and efficient identification of this mutation in future patients . 
 past literature suggests that mutations affecting splicing patterns of the glucocerebrosidase transcript as well as mutations in gaucher complex alleles are detrimental to enzyme activity . 
 however , compound heterozygosity with n370s , a mild mutation , will lead to a mild phenotype . 
 the cases reported here support these past findings . 
Introduction
Case reports
Materials and methods
Results
Discussion
Conclusion