glycogen storage diseases ( gsd ) are a group of disorders caused by alteration of the enzymes involved in glycogen metabolism , producing an accumulation of glycogen in several tissues .
the most frequent type of glycogen storage disease is gsd ix , which incidence is 1:100,000 births and is responsible for 25% of all gsd cases .
gsd ix is caused by defects in the activity of the phosphorylase kinase complex ( phk , e.c .
2.7.1.38 ) , which plays a critical role in regulating glucose release from glycogen , causing an excess of glycogen storage .
patients with gsd ix generally present hepatomegaly , short stature , raised transaminases and triglycerides and sometimes ketotic hypoglycemia in early childhood .
phk is a decahexameric enzyme composed by four subunits :  ,  ,  and . subunits  and  have regulatory functions , the -subunit has catalytic function and -subunit is identical to calmodulin .
subunit  has two isoforms encoded by two different genes : phka1 and phka2 , which encode the muscular and liver isoforms , respectively .
subunits  ,  and  are encoded by phkb , phkg2 and calm1 , respectively .
phka1 and phka2 deficiencies are x - linked , while phkb and phkg2 deficiencies are inherited according to an autosomal recessive pattern .
the most common gsd ix subtype is the subtype ixa , caused by mutations in phka2 , accounting for approximately 75% of cases .
gsd ixa has been split biochemically in two further types : ixa1 ( gsd9a1 ) with loss of phk enzyme activity in peripheral blood cells and liver tissue , and ixa2 ( gsd9a2 ) with normal phk activity in blood cells .
, patients with gsd ixa , can develop growth retardation , hepatomegaly , and ketosis with or without hypoglycemia , increases in triglycerides , cholesterol and transaminases .
therefore , gsd ixa has been described as a benign form since often does not need treatment .
however , genetic variants in phka2 are associated with a broad phenotypic spectrum   and even cases of cirrhosis have been described .
the first reported case was a white 18  months old boy who had micronodular liver cirrhosis and presented the mutation p.arg298pro .
later reviews have described more patients with hepatic fibrosis at diagnosis who presented mutations in phka2 
 .
other uncommon phenotypes have been reported in the literature , such as kidney dysfunctions due to renal tubular acidosis and cns affectation with delayed cognitive and speech abilities as a result of phka2 pathogenic variants . to date the human gene mutation database ( hgmd )
has reported over 80 mutations in phka2 associated with gsd ixa , including missense , splicing , small deletions , small insertions and gross deletions variants . in this paper
the patient of spanish origin was born after 40  weeks and 5  days of gestation by normal vaginal delivery .
the paternal great - grandmother , two brothers of the great - grandmother and one sister of the grandmother had transaminase elevation .
his birth weight was 3.450  kg and birth length 50  cm . from 8  months onwards failure to thrive
was noted and at age one year a routine laboratory analysis revealed an increase in liver enzymes .
subsequent analysis showed : alt 113(1049  u / l ) and ast 134(037  u / l ) .
he showed hepatomegaly and , slight hyperechogenicity . a complete biochemical profile done at 2  years 5  months , revealed the following results : glucose 52  mg / dl ( 60100 ) , total cholesterol ( tc ) 228  mg / dl ( <  200 ) , hdl - c 26  mg / dl ( >  40 ) , ldl - c 166 ( <  160 ) , triglycerides ( tg ) 181  mg / dl ( <  150 ) , lactate dehydrogenase 351  ui / l ( 110295 ) , ast 108  ui / l ( 1560 ) , alt 77  ui / l ( <  45 ) , gamma - gt 39  ui / l ( 3    22 ) , creatine kinase ( ck ) 415  ui / l ( <  171 ) , serum iron 73  g / dl ( 100250 ) , vsg 40  mm / h ( 015 ) , mean corpuscular volume 85,8  fl ( 7085 ) , mean corpuscular hemoglobin concentration 30,9  g / dl ( 31,236,0 ) , high levels of the ketonic bodies : acetoacetate 1.46  mmol / l ( 0.000.10 ) , beta - hydroxy - butyrate 2.92  mmol / l ( 0.000.20 ) , and plasma lactate 1.92  mmol / l ( 0.501.80 ) .
the lysosomal enzymes activity in lymphocytes was within the normal range . during follow - up
he got tired easily , was less active than other children and complained of muscle weakness . in view of the biochemical results and the lack of neurological or cardiological manifestations
, gsd iii was felt to be less likely , considering in the differential diagnosis a gsd vi or ix . finally , and after informed consent from the family , genetic testing of the genes associated with gsd vi and gsd ix : pygl , phka1 , phka2 , phkb and phkg2 was arranged .
after dna extraction the sample was analyzed by ngs using the illumina truesight one ( tso ) assay .
library preparation and exome enrichment steps were performed according to manufacturer 's workflow and sequenced in a miseq system .
the tso assay covers 12  mb of the human genome , containing 4813 clinically relevant genes .
we choose a specific subset of genes for sequence analysis : pygl , phka1 , phka2 , phkb and phkg2 .
briefly , sequences were mapped to the crch37/hg19 human reference sequence and data bases used for analysis were human gene mutation database ( hgmd professional ) ( www.biobaseinternational.com/hgmd ) from biobase corporation ; the human gene mutation database , online mendelian inheritance in man ( www.omim.org ) ; gene tests ( www.genetests.org ) .
variant annotation was carried out with ensembl 's variant effect predictor tool and was based on the transcripts enst00000216392 ( pygl ) , enst00000339490 ( phka1 ) , enst00000379942 ( phka2 ) , enst00000323584 ( phkb ) , enst00000563588 ( phkg2 ) .
the in silico predictors of pathogenicity used were cadd ( combined annotation dependent depletion ) , polyphen ( polymorphism phenotyping ) , mutassesor , fasthmm and vest .
scores of conservation used : gerp2 , phascons , phylop , maxentscan , nnsplice , genesplicer and human splicing finder were used as splicing predictors .
the files were uploaded in bam format for analysis using alamut visual v.2.8.0 ( interactive biosoftware ; france ) .
after dna extraction the sample was analyzed by ngs using the illumina truesight one ( tso ) assay .
library preparation and exome enrichment steps were performed according to manufacturer 's workflow and sequenced in a miseq system .
the tso assay covers 12  mb of the human genome , containing 4813 clinically relevant genes .
we choose a specific subset of genes for sequence analysis : pygl , phka1 , phka2 , phkb and phkg2 .
briefly , sequences were mapped to the crch37/hg19 human reference sequence and data bases used for analysis were human gene mutation database ( hgmd professional ) ( www.biobaseinternational.com/hgmd ) from biobase corporation ; the human gene mutation database , online mendelian inheritance in man ( www.omim.org ) ; gene tests ( www.genetests.org ) .
variant annotation was carried out with ensembl 's variant effect predictor tool and was based on the transcripts enst00000216392 ( pygl ) , enst00000339490 ( phka1 ) , enst00000379942 ( phka2 ) , enst00000323584 ( phkb ) , enst00000563588 ( phkg2 ) .
the in silico predictors of pathogenicity used were cadd ( combined annotation dependent depletion ) , polyphen ( polymorphism phenotyping ) , mutassesor , fasthmm and vest .
scores of conservation used : gerp2 , phascons , phylop , maxentscan , nnsplice , genesplicer and human splicing finder were used as splicing predictors .
the files were uploaded in bam format for analysis using alamut visual v.2.8.0 ( interactive biosoftware ; france ) .
we identified the previously undescribed hemizygous missense variant nm_000292.2(phka2):c.1963 g  >  a , p.(glu655lys ) in phka2 exon 18 .
1 ) . in silico analysis showed two possible consequences of this change : first , a missense change in the amino acid glutamate to lysine in position 655 of the protein p.(glu655lys ) .
two out of five predictors of pathogenicity classified this variant as damaging or possible damaging ( fasthmm   2.68 ( s  <    1 damaging ) and vest 0.597 ) and three further predictors classified it as benign ( cadd 6.9 ( s  <  11 benign ) , polyphen 0.01 ( s  <  0.03 benign ) , mutassesor 0.415 ( s  <  1.12 benign ) . all proved conservation : gerp.2 5.78 ( >  2.45 conserved ) , phastcons ( placenta ) 0.96 ( range 01 ) , phastcons ( vertebrate ) 1 ( range 01 ) , phylop ( placenta ) 2.5620 ( score    2 ) and phylop ( vertebrate ) 3.3290 ( score    2).second , since the mutation affects the last coding nucleotide of phka2 exon 18 ; the splicing predictors maxentscan , nnsplice , genesplicer , human splicing finder - like revealed that this variant may disrupt canonical splice donor site of exon 18 , probably resulting in an aberrant protein .
splicing predictions at nearest natural junction predicted a decreased splicing efficiency at the canonical donor site by   68,9% .
the variant was absent in the mother , thus , it is likely to have arisen as  de novo  event .
first , a missense change in the amino acid glutamate to lysine in position 655 of the protein p.(glu655lys ) .
two out of five predictors of pathogenicity classified this variant as damaging or possible damaging ( fasthmm   2.68 ( s  <    1 damaging ) and vest 0.597 ) and three further predictors classified it as benign ( cadd 6.9 ( s  <  11 benign ) , polyphen 0.01 ( s  <  0.03 benign ) , mutassesor 0.415 ( s  <  1.12 benign ) . all proved conservation : gerp.2 5.78 ( >  2.45 conserved ) , phastcons ( placenta ) 0.96 ( range 01 ) , phastcons ( vertebrate ) 1 ( range 01 ) , phylop ( placenta ) 2.5620 ( score    2 ) and phylop ( vertebrate ) 3.3290 ( score    2 ) .
second , since the mutation affects the last coding nucleotide of phka2 exon 18 ; the splicing predictors maxentscan , nnsplice , genesplicer , human splicing finder - like revealed that this variant may disrupt canonical splice donor site of exon 18 , probably resulting in an aberrant protein .
splicing predictions at nearest natural junction predicted a decreased splicing efficiency at the canonical donor site by   68,9% .
the variant was absent in the mother , thus , it is likely to have arisen as  de novo  event .
gsd ix is considered as a benign condition , due to the fact that the patients may become asymptomatic when they grow up and frequently the treatment is not necessary .
however , recent studies have shown that untreated children may develop undesirable effects such as morning sickness , which could affect their school performance , and growth delay , which may result in psychological distress . in adult patients
the risk of bone fracture may be higher due to the reduced bone density secondary to delayed puberty . in this study we identified a novel phka2 variant in in a patient with clinical suspicious of gsd .
the patient showed a wide range of associated clinical features : hepatomegaly , growth delay , muscle weakness , fasting ketosis and high levels of liver enzymes and total cholesterol .
. genetic analysis of phka2 identified a hemizygous missense variant nm_000292.2(phka2):c.1963 g  >  a , p.(glu655lys ) in exon 18 , which could explain this phenotype . in silico analysis revealed the possible pathogenicity of this variant .
bionformatics tools for predicting splicing effects showed that this variant disrupts exon 18 canonical splice donor site , probably resulting in an aberrant protein . according to the acmg criteria for variant classification we classify this variant as pathogenic .
gsd ix is a significant subgroup of gsds , however its biochemical and genetic diagnosis has been complicated due to its genetic heterogeneity and phenotypic overlapping with others gsds .
normoglycemic ketonemia is a usual biochemical feature in patients with gsd types vi and ix , and ketonemia can occur with hypoglycemia in all gsd types .
therefore , gsd vi and gsd ix must be incorporated into the differential diagnosis of ketotic normoglycemia , and kb ( kb is the sum of blood acetoacetate and -hydroxybutyrate concentrations ) must be regularly measured in ketotic gsd patients .
also the clinical manifestations of gsd ix practically overlap with those of gsd vi except for the inheritance pattern . as a result of this ,
the novel phka2 variant identified in this study confirms the diagnosis of gsd ix allowing the differential diagnosis with regard to other types of gsd .
phka2 missense mutations can affect the n - terminal glucoamylase domain or the c - terminal calcineurin b - like domain
. variants associated with n - terminal domain principally lead to gsdixa2 and are clustered into the predicted active site of the phk . they might have a direct impact on the predicted hydrolytic activity of the  subunit .
the variants at the c - terminal domain lead to gsdixa1 and are clustered in the region predicted to interact with the regulatory domain of the catalytic subunit in a region covering this interaction site leading to unstable phk complex or less regulated holoenzyme . in our patient
, it was not possible to distinguish between gsdixa1 and gsd1xa2 since the enzymatic assays was not available .
moreover , we can not predict whether the variant c.1963 g  >  a , p.glu655lys is affecting n - terminus or c - terminus domain .
we can conclude that all in silico tools predict a deleterious impact on protein function and genetic test helps avoiding the use of much more invasive techniques such as quantification of glycogen content and pkh activity in liver biopsy samples .
 glucogenosis type ix is caused by pathogenic variants of the phka2 gene . herein , we report a patient with clinical symptoms compatible with glycogen storage disease type ixa . 
 pygl , phka1 , phka2 , phkb and phkg2 genes were analyzed by next generation sequencing ( ngs ) . 
 we identified the previously undescribed hemizygous missense variant nm_000292.2(phka2):c.1963 g  >  a , p.(glu655lys ) in phka2 exon 18 . in silico analyses showed two possible pathogenic consequences : it affects a highly conserved amino acid and disrupts the exon 18 canonical splice donor site . 
 the variant was found as a  de novo  event . 
Introduction
Case report
Methods
Genetic analysis by Next Generation Sequencing (NGS)
Bioinformatic analysis
Results
Discussion