the molybdenum cofactor ( moco ) is essential for the activity of these enzymes and is associated with amidoxime reductase in mitochondria .
moco deficiency ( mocd ) causes a severe progressive metabolic encephalopathy with neonatal convulsions , spasticity , opisthotonus , brain atrophy , altered facial morphology and severe developmental disability , spherophakia and dislocated lenses in survivors , associated with deficiency of both xanthine oxidase and sulfite oxidase .
histopathology of the brain shows severe loss of neocortical neurons , gliosis and areas of cystic necrosis in white matter , also seen in sulfite oxidase deficiency , suggesting that deficiency of sulfite oxidase causes much of the cerebral pathology .
the mocs1a and mocs1b genes encode an enzyme complex that forms cyclic pyranopterin monophosphate ( cpmp ) from gtp .
the mocs2a and mocs2b gene product , in association with a protein encoded by mocs3 , convert cpmp to molybdopterin ( mpt ) .
cpmp is deficient or absent when there are mutations in the mocs1 gene , while its oxidation product , compound z , is detectable in urine when there are mocs2 mutations ( fig .  1 ) .
the gephyrin gene product adenylates mpt and adds molybdenum to form moco . when urine amino acids are screened on suspicion of a metabolic disease , an elevated s - sulfo - l - cysteine level suggests either sulfite oxidase deficiency or mocd .
an elevation of urine xanthine and low blood uric acid indicate an additional deficiency of xanthine oxidase due to mocd .
patients with mocs1a or mocs1b deficiency have been treated with a stable injectable form of cpmp ( hitzert et al . , 2012 , veldman et al . , 2010 ) , but with mocs2 mutations supplementation with cpmp is ineffective .
pyridoxal-5-phosphate is sequestered by elevated levels of the cyclic form of alpha - amino adipic semialdehyde ( piperideine-6-carboxylate ) in mocd , so seizures in mocd might respond to pyridoxine supplementation ( struys et al . , 2012 ) .
a 3.2  kg full term newborn girl with apgar scores 9 at 1  min and 5  min appeared well until day 2 when refractory seizures , opisthotonus , startle reflexes and vomiting developed .
the pregnancy and family history were unremarkable ; the parents were of samoan and caucasian origin . despite treatment with intravenous glucose - saline , phenobarbitone and phenytoin ,
cranial ultrasound mri showed a thin anterior corpus callosum and mri showed severe hypoplasia of its body and splenium ; there was a slight parallel appearance of the lateral ventricles with mild prominence of the trigone and temporal horns .
there was subtle loss of gray / white matter differentiation and cerebral edema with attenuation of surface csf spaces .
the deep cerebral veins , the vein of galen and the internal cerebral veins were dilated .
a dilated serpiginious vein connected the superior sagittal sinus and vein of galen instead of the usual straight sinus and could indicate a persistent embryonic vein .
the weights and measurements were in keeping with the gestational age at autopsy , which confirmed normal external morphology and thinning of the corpus callosum , cerebral edema and congested tortuous meningeal vessels .
uric acid , s - sulfo - l - cysteine and xanthine in urine were measured by direct - injection electrospray tandem mass spectrography ( veldman et al . , 2010 ) .
urine sulfite was measured by a semi - quantitative dipstick test ( merckoquant test sulfite , merck chemicals , darmstadt , germany ) .
mutations in mocs2b were identified by pcr amplification of exons and neighboring intronic sequences of leukocyte genomic dna from the patient and her parents and sanger sequencing .
mocs2a and mocs2b wild - type ( wt ) proteins and the mocs2b - s140f variant were recombinantly expressed in escherichia coli and purified to homogeneity .
the small subunit of mpt synthase , mocs2a , was expressed and purified as intein - fusion protein with a chitin - domain for subsequent affinity purification and eluted with ammonium sulfide , resulting in the release of activated mocs2a protein with a thiocarboxylated c - terminal tail ( gutzke et al . , 2001 ) .
mocs2b wildtype and the mocs2b - s140f variant were cloned into pet15b , expressed in e. coli bl21 and purified by ammonium sulfate precipitation and subsequent gelfiltration using superdex 200 size exclusion column .
changes in three - dimensional structure of the mutant protein were analyzed by circular dichroism spectroscopy ( rudolph et al . , 2001 ) .
complex formation of mpt synthase was analyzed by isothermal titration calorimetry using mocs2a and either wt mocs2b or the mocs2b - s140f variant . in vitro mpt synthesis rates
were quantified as a function of the concentration of small mpt synthase subunit mocs2a ( llamas et al . , 2004 ) .
uric acid levels in urine were low during life and at autopsy , and urine xanthine was elevated , while the level of compound z which is normally undetectable in urine was found to be elevated ( data not shown ) .
urine s - sulfocysteine ( 44  mol / l ) and s - sulfocysteine : creatinine ratio ( 231  mol / mmol creatinine ) were markedly elevated .
two novel mutations of the mocs2 gene in dna from blood of the patient were predicted to impair mrna synthesis or enzyme activity .
>  t ( p.s140f ) encoded a substitution of phenylalanine for serine and the other , c.501  +  2delt is predicted to disrupt a splice site .
in silico analysis using the crystal structure of bacterial mpt synthase predicted the location of s140 at the end of -stand 6 , which is part of a highly conserved sequence motif forming the active site within the mocs2b ( e. coli moae ) and being involved in binding the c - terminal end of mocs2a ( e. coli moad ) .
the exchange of the polar serine residue to the much larger , hydrophobic phenylalanine presumably influences the stability of the central -sheet of mocs2b and the overall assembly of the heterotetrameric mpt synthase complex .
following expression and purification , the total yield of mocs2b - s140f was much lower than that for wt mocs2b ( fig . 
consistently , circular dichroism spectroscopy revealed an alteration in the protein folding , given that between 210 and 220  nm a more negative signal was recorded , which suggests changes in the content of helical structures in mocs2b - s140f ( fig . 
these might influence either the oligomerization between two mocs2b protomers or the interaction with the small subunit ( mocs2a ) .
therefore , we analyzed the complex formation of mpt synthase by isothermal titration calorimetry using mocs2a and either wt mocs2b or the mocs2b - s140f variant .
while in the presence of wt mocs2a an effective complex formation associated with a saturation of heat release was observed ( fig . 
2c ) , only minor heat release peaks were seen with mocs2b - s140f ( fig . 
2d ) suggesting a severely affected interaction between both subunits , which could not be quantified .
note that wt mocs2b binds mocs2a with a kd  =  0.36    0.047  m ( fig . 
this finding identifies a defective association of both mpt synthase subunits as a major disease - causing mechanism . finally , we determined in vitro mpt synthesis rates as a function of the concentration of small mpt synthase subunit mocs2a . while wt mocs2b showed an effective mpt synthesis ( determined by the oxidation product forma , fig . 
2e ) , mocs2b - s140f was only able to produce low levels of mpt at high concentrations of mocs2a .
the differential diagnosis of mocd includes deficiency of sulfite oxidase and pyridoxine - dependent epilepsy ( struys et al . , 2012 ) .
mocd can be associated with a number of morphological abnormalities of the brain and face .
it is important to emphasize that some of the reported changes can be similar to those seen in neonates with hypoxic  ischemic encephalopathy , so metabolic investigations should not be neglected in cases of presumed intrapartum hypoxia ( topcu et al . , 2001 ) .
investigations should include screening of amino acids in urine , which would identify high levels of s - sulfo - l - cysteine in mocd , associated with high urinary xanthine and low levels of plasma uric acid .
the detection in urine of compound z confirms the diagnosis of mocs2 deficiency , and facilitates decisions about treatment .
exogenous cpmp only works in cases with mocs1 deficiency who can not synthesize cpmp , and could potentially slow or stop the progression of disease ( veldman et al . , 2010 ) , although seizures and  cramped synchronized general movements  have been observed on day 1 in a baby diagnosed prenatally with a mocs1 mutation ( hitzert et al . , 2012 ) , so it is possible that irreversible brain damage occurs prenatally . in this case
, protein expression experiments confirmed that the s140f mutation severely reduces complex formation of mpt synthase .
as some residual activity is detectable in vitro , a milder presentation with low levels of enzyme activity might be considered in this case . in vitro analysis of expression of the allele containing
the splice site mutation was not possible for this patient , although the wild type human sequence at both c.419 and c.501  +  2 is conserved in several species ( table 1 ) ( anonymous , 2014 ) .
biochemical testing for recurrence in a future pregnancy could include measurement of s - sulfo - l - cysteine level or sulfite oxidase activity in chorion villus cells , or of s - sulfo - l - cysteine in amniotic fluid .
mutation analysis reassured the parents that treatment with exogenous cpmp was not indicated , as it has only been shown to help babies with mocs1 mutations .
the child died before the potential benefit of pyridoxine supplementation was published ( struys et al . , 2012 ) .
 backgroundmolybdenum cofactor deficiency ( mocd ) is a severe autosomal recessive neonatal metabolic disease that causes seizures and death or severe brain damage . 
 symptoms , signs and cerebral images can resemble those attributed to intrapartum hypoxia . in humans , 
 molybdenum cofactor ( moco ) has been found to participate in four metabolic reactions : aldehyde dehydrogenase ( or oxidase ) , xanthine oxidoreductase ( or oxidase ) and sulfite oxidase , and some of the components of molybdenum cofactor synthesis participate in amidoxime reductase . a newborn girl developed refractory seizures , opisthotonus , exaggerated startle reflexes and vomiting on the second day of life . 
 treatment included intravenous fluid , glucose supplementation , empiric antibiotic therapy and anticonvulsant medication . 
 her encephalopathy progressed , and she was given palliative care and died aged 1  week . 
 there were no dysmorphic features , including ectopia lentis but ultrasonography revealed a thin corpus callosum.objectivesthe aim of this study is to provide etiology , prognosis and genetic counseling.methodsbiochemical analysis of urine , blood , sanger sequencing of leukocyte dna , and analysis of the effect of the mutation on protein expression.resultsuric acid level was low in blood , and s - sulfo - l - cysteine and xanthine were elevated in urine . 
 compound z was detected in urine . 
 two mocs2 gene mutations were identified : c.501  +  2delt , which disrupts a conserved splice site sequence , and c.419c  >  t ( ps140f ) . 
 protein expression studies confirmed that the p.s140f substitution was pathogenic . 
 the parents were shown to be heterozygous carriers.conclusionsmutation analysis confirmed that the mocd in this family could not be treated with cpmp infusion , and enabled prenatal diagnosis and termination of a subsequent affected pregnancy . 
Introduction
Subject
Methods
Results
Discussion