Haberle et al. Orphanet Journal of Rare Diseases 2012, 7:32
`http://www.ojrd.com/content/7/1/32
`
`ORPHANET JOU RNAL
`OF RARE DISEASES
`
`ae leyel-yi waa
`
`Suggested guidelines for the diagnosis and
`managementof urea cycle disorders
`Johannes Haberle'’, Nathalie Boddaert’, Alberto Burlina’, Anupam Chakrapan'”, Marjorie Dixon”, Martina Huemer”,
`Daniela Karall’, Diego Martinelli®, Pablo Sanjurjo Crespo”, René Santer”, Aude Servais'’, Vassili Valayannopoulos'”,
`Martin Lindner'?’', Vicente Rubio’ and Carlo Dionisi-Vici?’
`
`
`
`Arginase 1, Hyperornithinemia-hyperammonemia-homocitrullinuria syndrome
`
`Abstract
`
`Urea cycle disorders (UCDs) are inborn errors of arnmonia detoxification/arginine synthesis due to defects affecting the
`catalysts of the Krebs-Henseleit cycle (five core enzymes, one activating enzyme and one mitochondrial ornithine/
`citrulline antiporter) with an estimated incidence of 1:8.000. Patients present with hyperammonemia either shortly after
`birth (~50%) or, later at any age, leading to death or to severe neurological handicap in many survivors. Despite the
`existence of effective therapy with alternative pathway therapy and liver transplantation, outcomes remain poor. This
`may be related to underrecognition and delayed diagnosis due to the nonspecific clinical presentation and insufficient
`awareness of health care professionals because of disease rarity. These guidelines aim at providing a trans-European
`consensus to: guide practitioners, set standards of care and help awareness campaigns. To achieve these goals, the
`guidelines were developed using a Delphi methodolagy, by having professionals on UCDs across seven European
`countries to gatherall the existing evidence, score it according to the SIGN evidence level system and draw a series of
`statements supported by an associated level of evidence. The guidelines were revised by external specialist consultants,
`unrelated authorities in the field of UCDs and practicing pediatricians in training. Although the evidence degree did
`hardly ever exceed level C (evidence from non-analytical studies like case reports and series), it was sufficient to guide
`practice on both acute and chronic presentations, address diagnosis, management, monitoring, outcomes, and
`psychosocial and ethicalissues. Also, it identified knowledge voics that must befilled by future research. We believe
`these guidelines will help to: harmonise practice, set common standards and spread good practices with a positive
`impact on the outcomes of UCD patients.
`
`Keywords: Urea cycle disorders, UCD, Hyperammonemia, N-acetylglutamate synthase, Carbarmaylphosphate synthetase
`|, Ornithine transcarbamylase, Ornithine carbamoyl transferase, Argininosuccinate synthetase, Argininasuccinate lyase,
`
`Introduction
`Urea cycle disorders (UCDs) are inborn errors of nitrogen
`detoxification/arginine synthesis due to defects in the urea
`
`* Comespondence: Johannes. Haebenee@kispiuzh.ch; martinJlindner@med.uni-
`heidelberg.de, rubio@iby,csic.es; carla.dienisivici@opbg,net
`"Equal contributors
`‘University Children’s Hospital Zurich and Children's Research Centre, Zurich
`8032, Switzerland
`University Children's Hospital, im Neuenheimer Feld 430, Heidelberg 69120,
`Germany
`“Instituto de Biomedicina de Valencia del Consejo Superior de
`Investigaciones Cientificas (IBV-CSIC) and Centro de Investigacién Biomédica
`en Red para Enferrnedades Raras (CIBERER), C/ Jaume Roig 11, Valencia
`46010, Spain
`8Division of Metabolism, Bambino Gest! Children's Hospital, IRCCS, Piazza 5.
`Onofria 4, Rome |-00165, Italy
`Full list of author information is available at the end of the article
`
`cycle enzymes (Figure 1), carbamoylphosphate synthetase 1
`(CPS1), ornithine transcarbamylase (OTC), argininosuccinate
`synthetase (ASS), argininosuccinate lyase (ASL) and arginase
`1
`(ARGI),
`leading to respective deficiencies (abbreviated
`CPS1D, OTCD, ASSD, ASLD and ARG1D; corresponding
`MIM numbers, #237300, #311250; #215700; #207900;
`#207800 respectively). They also encompass deficiencies of
`N-acetylglutamate synthase (NAGS) (MIM #237310), asso-
`ciated with lack of the N-acetylglutamate (NAG) essential
`activator of CPS1 and of the mitochondrial ornithine/citrul-
`line antiporter (ORNT1), causing the hyperornithinemia-
`hyperammonemia-homocitrullinuria (HHH)
`syndrome
`(MIM #238970). The prevalence of these disorders may
`exceed the current estimates (1:8,000-1:44,000 births [1-3],
`
`C BioMed Central
`
`(6 2012 Haberle et aly licensee BloMed Central Ltd, This Is an Open Access article distributed under the terms of the Creative
`Commons Attribution License (httpy/creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
`reproduction in any medium, provided the original work is properly cited,
`
`1 of 30
`
`Horizon Exhibit 2004
`Horizon Exhibit 2004
`Lupin v. Horizon
`Lupin v. Horizon
`IPR2018-00459
`IPR2018-00459
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`

`Haberle et al. Orphanet Journal of Rare Diseases 2012, 7:32
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`
`Page 2 of 30
`
`
`
`Portal blood
`
`Mitochondrion
`:
`Cytosol
`
`Aspartate «————— Oxxalacetate
`
`i
`
`+1ATP
`
`glutarate
`
`NAD(P)
`
`
`
`NH, + HCO, Glutamine
`is Aspartatecycle
`Glutamate———_ ee
`Argininosuccinate
`
`
`Ureacycle
`
`Malate
`
`!
`» Fumarate
`
`
`
`
`
`Acetyl CoA
`
`INAGS
`N-Acetyl- &,
`
`L-glutamate
`
`:
`
`+2ATP
`
`joTC
`
`
`
`
`
`eae + Ornithine
`'
`,
`ATP, NADPH
`
`L-Glutamate-
`
`1¥-sem ialdehyde
`!
`\
`chemical
`ee
`Proline
`: A!-Pyrroline-
`\ 5-carboxylate
` wee ene»ie-—--> Orotic acid
`
`
`
`
`
`Ornithine
`
`Arginine
`HO
`oie
`
`UMP —* Uridine, Uracil
`i
`
`ARG
`
`Urea
`
`
`
`Figure 1 The urea cycle and associated pathways. Non-standard abbreviations include: GDH, glutarnate dehydrogenase; GLS, glutaminase;
`NADIP), nicotinamide adenine dinucleotide (phosphate); OAT, ornithine aminotransferase; OMP, oratidine menophosphate, PSCR, pyrroline-5-
`carboxylate reductase; PSCS, A’ pyrroline-5-carboxylate synthetase; UMP, uridine monophosphate.
`
`for all UCDs jointly) because of unreliable newborn
`screening and underdiagnosis of fatal cases. Clinical fea-
`tures are typical in complete deficiencies, which present
`with hyperammonemic coma a few days after birth with
`~50% mortality [4-7], whereas the survivors experience se-
`vere developmental delay and recurrent hyperammonemic
`crises [4-7]. Even in partial deficiencies, which have more
`variable clinical presentations and later onset (any age),
`there is increased risk of premature death [5,8]. The dur-
`ation and severity of hyperammonemia strongly correlates
`with brain damage [6,9,10]; prompt diagnosis and treat-
`ment of UCD is essential in order to optimise the out-
`come,
`[11]. However, the rarity of UCDs prevents single
`centres or even countries to have all
`the expertise for
`evidence-based management. Therefore, we have devel-
`oped consensus guidelines based on the highest available
`level of evidence, by pooling all the published evidence and
`experience of leading centres from several European coun-
`tries, to help standardise, systematise and harmonise across
`Europe the diagnosis, therapy, procedures and management
`
`of UCDs. These guidelines, developed with the Delphi
`methodology are intended to be used by metabolic specia-
`lists, pediatricians, dietitians, neonatologists, intensive care
`specialists, adult physicians, neurologists, nurses, psycholo-
`gists and pharmacists involved in the care of UCD patients.
`Excluded from these guidelines because of insufficient
`European experience, or of tangential
`relationship with
`UCDs are: citrin deficiency (citrullinemia type 2, MIM
`#605814 and #603471), lysinuric protein intolerance (LPI,
`MIM #222700), deficiencies of pyrroline 5-carboxylate
`synthetase (MIM #610652) and ornithine aminotransferase
`deficiency (OAT, MIM #258870), despite the fact that they
`may cause hyperammonemia.
`
`Methodology and objectives
`Guidelines development
`Development of these guidelines spanned the time
`period, October 2008 until August 2011 and involved
`one preliminary meeting and four working meetings of
`the guideline development group (GDG),
`formed by
`
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`
`[Inns-
`pediatric metabolic specialists (S. Baumgartner
`bruck, retired after the first meeting], AB, AC, CDY, S.
`Griinewald, [London, retired after the first meeting], JH
`[chairman], DK, ML [secretary], DM, PS, VV), a medical
`biochemist (VR), a psychologist (MH), a specialist meta-
`bolic dietitian (MD), a metabolic specialist caring for
`adult patients (AS) and a neuroradiologist (NB), Each
`meeting was supervised by a moderator (P, Burgard, Hei-
`delberg [first meeting] and RS) who oversaw the discus-
`sion but did not contribute to the content. In the initial
`working meeting the GDG was trained on standardising
`literature evaluation and working groups focusing on
`specific topics were formed. Thereafter GDG members
`discussed and performed systematic literature review and
`drafted
`the guidelines. These drafts were
`further
`reviewed by external specialists on intensive care (L.
`Dupic, Paris), genetics (A. Gal, Hamburg), child neur-
`ology (A. Garcia-Cazorla, Barcelona), nephrology (S.
`Picea, Rome), liver transplantation (J. de Ville de Goyet,
`Rome), epidemiology (A. Tozzi, Rome) and ethics (C.
`Rehmann-Sutter, Basel) and a patient group representative
`(S. Hannigan, London), After further recommendations/
`comments by three highly renowned external reviewers (C.
`Bachmann, Bottmingen;J.V. Leonard, Oxford and H. Ogier,
`Paris), the final version of the guidelines was written and its
`applicability pilot-tested by non-specialist pediatricians in
`training, with subsequent review and revision by the GDG.
`The guidelines will be sent for endorsementto all European
`societies for inherited metabolic diseases.
`
`Systematic literature review and evidence grading
`The guidelines evidence base was collected according to
`the Scottish Intercollegiate Guideline Network (SIGN,
`http://www.sign.ac.uk). Systematic literature review en-
`compassing from each disease description until early 2011
`was carried out using mainly Medline, Embase,
`the
`Cochrane Library, MedLink, and Orphanet. Searches also
`included websites of societies and parents groups for in-
`born errors. Relevant papers were evaluated by at least
`two GDG members before considering conclusions as
`evidence.
`Evidence levels were classified in accordance with the
`
`SIGN methodology:
`
`“Evidence level & criteria"
`1** High quality meta-analyses, systematic reviews of
`randomized control trials (RCTs), or RCTs with a very
`low risk ofbias.
`
`1* Well conducted meta-analyses, systematic reviews of
`RCTs, or RCTs with a low risk ofbias.
`1 Meta-analyses, systematic reviews or RCTs, or RCTs
`with a high risk ofbias.
`2°* High quality systematic reviews of case—control or
`cohort studies or high quality case-control or cohort
`
`studies with a very low risk of confoundingbias, or
`chance and a high probability that the relationship is
`causal.
`2* Well conducted case-control or cohort studies with
`a low risk of confounding, bias, or chance and a
`moderate probability that the relationship is causal.
`2 Case-control or cohort studies with a high risk of
`confounding, bias, or chance anda significant risk that
`the relationship is not causal.
`3 Non-analytic studies, e.g, case reports, case series.
`4 Expert opinion,
`
`Recommendations given in the guidelines are graded
`depending ontheir level of evidence:
`
`"Grade of recommendation & criteria"
`A If level 1 evidence was found (not the case).
`B If level 2 evidence was found.
`
`C If level 3 evidence was found (mainly non-analytical
`studies such as case reports and case series).
`D If level 4 evidence was found (mainly expert opinion).
`
`Disclaimer
`
`These guidelines aim at helping decision making in
`UCD patient care. Although based on the best avail-
`able evidence,
`the recommendations given often re-
`flect only expert opinion and are thus not meant
`to
`be rigidly implemented. Furthermore, although as ex-
`haustive as possible,
`these guidelines cannot
`include
`all possible methods of diagnostic work-up and care
`and may therefore fail
`to mention some acceptable
`and established procedures. Guidelines cannot guaran-
`tee satisfactory diagnosis and outcome in every pa-
`tient. Although helping optimise the care of individual
`patients and assist decision-making by basing clinical
`practice on the existing scientific and medical know-
`ledge,
`they should not substitute well-informed, pru-
`dent clinical practice.
`
`Diagnosis
`Theclinical picture
`Theclinical manifestations of UCDs (Table 1) can occur at
`any age [12-16], with hyperammonemic crises being fre-
`quently triggered by catabolic events, protein overload or
`certain drugs. Most symptomsare neurological but nonspe-
`cific. A UCD should be immediately suspected in neonates
`if there are any neurological symptoms orat any ageif there
`is an acute encephalopathy. Hepatic-gastrointestinal and
`psychiatric nonspecific manifestations (Table 1) are second
`in frequency. Only the hair shaft abnormalities with hair
`fragility (trichorrhexis nodosa)
`found mainly in ASLD
`[12,17-19] and the progressive spastic diplegia beginning in
`childhood (or later) in ARGID and the HHH syndrome,
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`Table 1 Clinical signs and symptoms of acute and chronic presentations of UCDs, and triggering factors for
`hyperammonemia in UCD patients
`
`Acute presentation Chronic presentation
`- Altered level of consciousness (from somnolence and
`+ Confusion, lethargy, dizziness
`lethargy to coma) mimicking encephalitis or drug
`intoxication
`
`+ Acute encephalopathy (see below)
`- Seizures (generally not isolated but along with an
`altered level of consciousness)
`
`+ Ataxia (generally associated with altered consciousness level)
`+ Stroke-like episodes
`+ Transient visual loss
`
`+ Vomiting and progressive poor appetite
`+ Liver failure
`
`+ Multiorgan failure
`+ Peripheral circulatory failure
`+ “Post-partum psychosis”
`
`+ Psychiatric symptoms (hallucinations, paranoia,
`mania, emotional or personality changes)
`
`In neonates:
`
`+ sepsis-like picture, temperature instability
`+ respiratory distress, hyperventilation
`
`+ Migraine-like headaches, tremor, ataxia, dysarthria
`» Asterixis (in adults)
`
`- Learning disabilities, neurodevelopmental delay, mental retardation
`
`+ Chorea, cerebral palsy
`+ Protracted cortical visual
`
`loss
`
`+ Progressive spastic diplegia or quadriplegia (described in ARG1D
`and HHH syndrome)
`- Protein aversion, self-selected low-protein diet
`+ Abdominal pain, vomiting
`+ Failure to thrive
`
`+ Hepatomegaly, elevated liver enzymes
`- Psychiatric symptoms: hyperactivity, mood
`alteration, behavioural changes, aggressiveness
`+ Self-injurious behaviour
`
`+ Autism-like symptoms
`- Fragile hair (typical for ASLD)
`+ Dermatitis
`
`« Specific neuropsychalogical phenotype in heterozygous OTC females
`- Episodic character of signs and symptoms
`Potential triggers of hyperammonemic crises in UCD patients
`eIntections
`+ Fever
`
`«Vomiting
`+ Gastrointestinal or internal bleeding
`+ Decreased energy or protein intake (e.g. fasting pre surgery, major weight loss in neonates)
`+ Catabolism and involution of the uterus during the postpartum period (mostly OTC females)
`+ Chemotherapy, high-dose glucocorticoids
`
`+ Prolonged or intense physical exercise
`+ Surgery under general anesthesia
`» Unusual protein load (e.g. a barbecue, parenteral nutrition)
`
`» Drugs: Mainly valproate and L-asparaginase/pegaspargase. [opifamate, carbamazepine, phenobarbitone, phenytoine, primidone,
`furosemide, hydrachlorothiazide and salicylates have also been associated with hyperammonernic decompensation.
`Typical and uncommon signs and symptoms are highlighted in bold- and normal-type, respectively, whereasitalic type marks signs and symptoms reported in
`single patients, Grade of recommendation, D,
`
`frequently without hyperammonemic episodes [20-22], are
`specific manifestations of this group of diseases. Symptoms
`can be subtle, particularly after the neonatal period, and in
`some patients symptomatic episodes can resolve with non-
`specific interventions. Women can first manifest a UCD as
`acute unexplained neurological symptoms in the postpar-
`tumperiod (reported for CPS1D, OTCD, and ASSD [23-
`
`25]). Variability in disease severity is characteristic for
`OTCDheterozygous females (due to lyonization) [11,26],
`but is also found in all UCDs, being mainly attributable to
`differences in the severity of the genetic change [27-30].
`However, the same genetic defect can yield both mild and
`severe presentations even in different members of the same
`family (reported for OTCD and for one CPS1D family)
`
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`
`[31-33]. Acute liver failure has been reported as the present-
`ing sign in patients with OTCD, ASSD and HHH syndrome
`[34-39], Although rare, a number of other presentations
`have been reported in UCDs, including stroke-like episodes
`(metabolic strokes) [10,40-44] that may resolve with treat-
`ment, chorea [45], cerebral palsy without hyperammonemia
`or cerebral edema [46,47], episodic transient or protracted
`cortical visual losses [48,49], dermatitis (most probably be-
`cause of treatment-related malnutrition) [50,51], autism-like
`symptoms [52,53], behavioural problems during childhood
`[53] and in postpuberal patients and other episodic psychi-
`atric symptoms that may be the only manifestation [54].
`A careful medical and family history is mandatory
`and should include questions about unexplained neo-
`natal deaths, neurological or psychiatric disorders in
`the family, consanguinity (frequent in all UCDs except
`in OTCD, which is X-linked), evidence of protein
`avoidance in patient and family members and drug in-
`take by the patient.
`
`Statement #1. Grade of recommendation: C
`
`UCDs may present with acute or chronic presentations
`at any age and are often triggered by catabolic events,
`protein load or some drugs. In many cases a precipitat-
`ing factor cannot be identified. Clinical signs and symp-
`toms
`are nonspecific
`and commonly neurological,
`gastrointestinal or psychiatric. It is essential that health-
`care professionals have an awareness of these diseases.
`Key questions should be asked and a detailed family his-
`tory with pedigree is mandatory.
`
`Statement #2. Grade of recommendation: D
`
`UCDs must be included in the differential diagnosis of
`acute unexplained encephalopathy or acute psychiatric
`illness at any age, which must prompt plasma ammonia
`determination.
`
`Laboratory findings
`Hyperammonemia, a nonspecific marker of inadequate
`nitrogen detoxification [55],
`is the hallmark for most
`UCDs. The absence of hyperammonemia in symptomatic
`newborn patients (but not in older patients) renders a
`UCD highly unlikely. Rapid ammonia measurement in
`an emergency setting is crucial since patient outcome
`correlates with the duration and peak level of hyperam-
`monemia [4,6,56]. Respiratory alkalosis in a newborn
`should prompt
`immediate ammonia measurement be-
`cause it
`is present initially in 50% of acute UCDs [5].
`Otherwise the acid—base status is of limited use [57].
`
`Statement #3. Grade of recommendation: C
`
`Ammonia should be determined in an emergency setting
`with results available in 30 minutes.
`
`Statement #4. Grade of recommendation: D
`
`Ammonia should be measured in patients of any age
`presenting 1) an unexplained change in consciousness;
`2) unusual or unexplained neurological illness; 3) liver
`failure; 4) suspected intoxication.
`
`If hyperammonemia is confirmed, determination of
`plasma amino acids, blood or plasma acylcarnitines,
`urinary organic acids and orotic acid should be ur-
`gently requested together with basic laboratory inves-
`tigations, not waiting for the results (which should be
`obtained in <24 h) for treating the patient. When tak-
`ing samples after
`recovery from an acute episode,
`plasma amino acid levels and/or urinary orotic acid
`(measured with a specific method e.g. high perform-
`ance liquid chromatography) can be particularly help-
`ful
`for diagnosis.
`In patients with fatal outcome,
`procurement of anticoagulated blood for DNA isola-
`tion and storage of frozen aliquots of all
`samples
`obtained of plasma, serum, urine and cerebrospinal
`fluid (CSE) is recommended [16,58],
`
`Statement #5. Grade of recommendation: D
`If ammonia is found elevated, further metabolic investi-
`gations should be immediately carried out without delay-
`ing specific treatment.
`
`Differential diagnosis
`The most common misdiagnosis of early onset UCD
`patients is neonatal sepsis. A number of conditions
`that increase ammonia production and/or secondarily
`decrease ammonia detoxification can cause hyperam-
`monemia and mimic a UCD [16,59-63], Thus, meo-
`natal liyperammonemia can be due to UCDs, to other
`inborn errors that cause secondary hyperammonemia,
`to liver failure or to congenital
`infection, Premature
`infants can have transient hyperammonemia, a condi-
`tion which is characterised by a normal blood glutam-
`ine level
`[64] and which is possibly due to ductus
`venosus shunting of portal blood [65-67]. Late-onset
`hyperammonemia can be triggered by most conditions
`that can also cause neonatal hyperammonemia, by
`chronic liver
`failure,
`exogenous
`intoxications
`(e.g.
`amanita phalloides), drugs (e.g. valproic acid), porto-
`caval shunt and Reye syndrome, by conditions that
`vastly increase either direct ammonia production (e.g.
`asparaginase treatment, urease-positive bacteria over-
`growth or genito-urinary infection) or protein catabol-
`ism (e.g. myeloma, chemotherapy,
`steroid therapy,
`trauma, gastrointestinal hemorrhage) and when there
`is excessive nitrogen supply (reported in total paren-
`teral nutrition or after glycine-solution irrigations in
`transurethral prostate resection)
`[5,17,68-72]. Table 2
`
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`
`Table 2 Bedside differential diagnosis of inborn errors of metabolism presenting with hyperammonemia
`
`Parameter
`Condition
`B-Oxidation
`Hyperinsulinism-
`ucDs
`Organic
`Pyruvate
`defects
`hyperammonemia
`acidurias
`carboxylase
`
`syndrome
`deficiency 9
`oe - 4
`-
`+
`
`Acidosis
`Ketonuria®
`
`4l-
`-
`
`+°
`+
`
`+/-
`-
`
`Hypoglycemia”
`} Lactic acid®
`
`-
`-
`
`+=
`+
`
`+
`+/-
`
`+
`-
`
`+
`+
`
`4f-
`-
`-
`co
`| AST & ALT
`-
`-
`-
`-
`T cPK
`-
`-
`~
`+
`-
`| Uric acid
`-
`-
`-
`+
`-
`| WBC/RBC/PIt
`Weightloss + - +f - -
`
`
`
`
`
`In addition to the conditions indicated in the table, mitochondrial oxidative phosphorylation defects, citrin deficiency, lysinuric protein intolerance or ornithine
`aminotransferase deficiency can also cause hyperammonemia,
`Grade of recommendation, D.
`* In neonates ketonuria (++ or +++) suggests organic aciduria,
`® Hypoglycemia and hyperammonemia(pseudo-Reye") can be predominant manifestations of the organic aciduria due to 3-hydroxy-3-methylglutary-Coa lyase deficiency,
`* Blood lactate >6mmol/L, since lower high lactate levels (2-6mM) may be dueto violent crying or to extensive muscle activity.
`4 AST & ALT elevations can be found but are not constant in UCDs.
`* Can be absent in neonates.
`' Occurrence only in neonates.
`9 Only type B is associated with hyperammonemia but not types A and C.
`
`lists errors of metabolism leading to hyperammone-
`mia, guiding bedside differentiation.
`
`Statement #6. Grade of recommendation: C
`
`In newborns with clinical distress where sepsis is sus-
`pected, hyperammonemia must always form part of the
`initial differential diagnosis.
`
`Standard clinical and analytical procedures generally
`differentiate between hyperammonemia due to inborn
`errors and that due to other conditions such as liver fail-
`
`ure [1,16,73-75]. The algorithm given in Figure 2 guides
`the identification of the specific defect when the hyper-
`ammonemia is due to an inborn error. ARGID and
`ASLD canbeidentified, respectively, by the high plasma
`arginine or
`the high plasma/urinary argininosuccinate
`(ASA)level. The finding of high plasmacitrulline in the
`absence of ASAis highly suggestive of ASSD. The com-
`bination of hyperammonemia with low plasmacitrulline
`and arginine is diagnostic of OTCD when orotic acid is
`increased in the urine, whereas
`it strongly suggests
`CPS1D or NAGS deficiency (NAGSD) when urinary oro-
`tic acid is low. The finding of high plasma ornithine and
`hyperammonemia, (these two traits can also be found in
`OAT deficiency) with high urinary homocitrulline is
`characteristic of the HHH syndrome. Whenthe metabol-
`ite pattern is not clear-cut, activity assays of urea cycle
`enzymesin liver (all urea cycle enzymes), red blood cells
`(ASL and ARGI;
`still very useful
`in ARGID [76}),
`
`intestinal mucosa (CPS1, OTC) or fibroblasts (ASS, ASL,
`HHH)canclarify diagnosis, although enzyme assays have
`generally been replaced by genetic testing. Enzyme ana-
`lysis is now mainly reserved for the minority of cases in
`whomgenetic analysis fails to identify a specific UCD
`(see below).
`
`Statement #7. Grade of recommendation: D
`
`Genetic testing is the method of first choice to confirm
`the diagnosis. Liver tissue,
`intestinal mucosa, erythro-
`cytes and fibroblasts can be used for enzyme activity
`assays in UCDsif genetic testing does not identify a spe-
`cific UCD,or if it is not available. In deceased patients
`with a suspicion of UCD, fibroblasts and/orliver tissue
`should be preserved frozen.
`
`Molecular genetic analysis
`transmitted in the X-
`Except
`for OTCD, which is
`chromosome, UCDs exhibit autosomal recessive inherit-
`ance [12-16]. Mutations in the corresponding genes
`(homonymous with the enzymes) have been identified in
`patients of all UCDs (see http://www.ncbi.nim.nih.gov/
`sites/entrez?db=omim)
`including citrullinemia type 2
`(SLC25A15 gene encoding citrin) and the HHH syn-
`drome (SEC25A13 gene). Mutation detection has at least
`~80%sensitivity [77] and permits carrier identification,
`prenatal diagnosis, facilitating pedigree analysis, genetic
`counselling and in some
`cases genotype-phenotype
`
`6 of 30
`
`6 of 30
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`

`

`Haberle et al. Orphanet Journal of Rare Diseases 2012, 7:32
`http://www.ojrd.com/content/7/1/32
`
`Page 7 of 30
`
`
`
`Glutamine
`low-normal
`
`
`
`Glutamine
`elevated
`
`
`
`Citrulline Citrulline|)CitrullineCitrulline
`T
`+
`u
`
`T ASA
`+
`THAN
`
`
`
`normal Aa,||Ofganic acid tT Citrulline||U Orotic acid U Arg, Lys, On||TU Orotic acidU Orotic acid
`
`
`profile elevated||ty orotic acidtf nennel ASLD
`
`
`LPI
`
`pipeectomy; Ornithine||OrnithineHypoglycemial NAGS D
`
`
`
`Acidesis
`eat
`cPpso
`elevated
`normal
`TO roacid
`FAOdefects
`\ P5CsD
`MCAD —VLGAD - TP
`Orn, Pro, Arg
`Hypoglycemia
`starved
`hyperNH,
`
`; na
`oTeB
`
`4
`
`
`
`+ in 7ye
`A-FP, galactose
`
`
`
`\
`PC deiiciency
`Lactic acidosis
`
`f
`
`HHH =
`
`r
`
`oaTD
`
`TT Arg
`ARGID
`
`—
`Om canbe
`transiently normal
`OATD
`
`Figure 2 Diagnostic algorithm for neonatal hyperammonemia.|nless indicated, plasma js used for the analytical determinations, Nan-
`standard abbreviations include: A-FP, a fetoprotein; CIT 2, citrullinemia type 2; CPSD, CPS1 deficiency; HI-HA, hyperinsulinism-hyperammonemia
`syndrome: HMG, 3-hydroxy-3-methylglutaryl-CoA lyase deficiency; LP|, lysinuric protein intolerance; OATD, ornithine aminotansterase deficiency;
`PA, propionic acidernia; PC, pyruvate carboxylase, PSCSD, A’ pyrroline 5-carboxylate deficiency; THAN, transient hyperammmonemia of the newborn;
`TPD,trifunctional protein deficiency; U, urine. Grade of recommendation, D. * in some patients with late-onset OTCD,plasmacitrulline levels are in
`the lower part of the normal range.
`
`correlations [15,27,78], conceivably opening the way to
`future nonsense—read-throughtherapies (eg. Mutation analysis has somepitfalls and limitations, includ-
`
`
`
`
`approaches). DNA, generally from blood,
`is used, al-
`ing the difficulty in establishing the pathogenic potential of
`though the large number of CPS/J exons renders prefer-
`a missense mutation. /m vitro protein expression studies
`able the utilization of RNA from cultured fibroblasts for
`and in silico analyses based an sequence conservation and
`protein structure can help infer pathogenic potential but
`are not part of routine clinical management.
`
`Statement #9. Grade of recommendation: D
`
`CPS1D studies. For other UCDs RNAanalysis (from liver
`in the case of OTCD)is only carried out when DNA ana-
`lysis is negative [79-81]. Prognostic judgements on the
`disease-causing nature of missense mutations (the most
`frequent ones) and of somesplice-site mutations are diffi-
`cult if not backed by in vitro expression studies of the mu-
`tant protein.
`
`Statement #8. Grade of recommendation: C
`
`Mutation analysis is the method of choice for definitive
`diagnosis of UCDs, to help with genetic counselling and
`in some instances indicate the prognosis.
`
`7 of 30
`
`Prenatal testing
`Prenatal investigations in UCDsare available in many coun-
`tries and may enable pregnancy terminationof affected foe-
`tuses. These may also be indicated in milder UCDs or for
`NAGSD (which has substitutive therapy) for psychological
`reasons and to prepare for perinatal management [82-84].
`Among the techniques
`that can be used (Table 3),
`mutation- or disease allele-tracking using chorionic villus
`samples, amniotic fluid cells or cultures thereof [85,86] is
`
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`

`

`Haberle et al. Orphanet Journal of Rare Diseases 2012, 7:32
`http://www.ojrd.com/content/7/1/32
`
`Page 8 of 30
`
`Table 3 Prenatal testing of UCDs: Recommended analyses
`and sample requirements
`
`Disorder
`Recommended tests
`NAGSD
`CPSID
`
`Mutation analysis using DNA from CVSor AFC
`Mutation analysis using DNA from CVS or AFC
`
`OTCD
`
`ASSD
`
`ASLD
`
`Enzymeassay inlatefetal liver biopsy”
`Mutation analysis using DNA from CVS or AFC“
`
`Enzymeassay in late fetal liver biopsy”®
`Mutation analysis using DNA from CVS or AFC
`Citrulline in amniotic fluid
`
`Enzyme assay in intact or cultured CVS or
`in cultured AFC
`
`Mutation analysis using DNA from CVS or AFC
`Argininosuccinate and its anhydrides in
`amniotic fluid
`
`ARGID
`
`Enzyme assay in intact or cultured CVS or cultured AFC
`Mutation analysis using DNA from CVS.
`Enzyme assayin fetal blood erythrocytes
`(mid-gestation sampling)
`Helsyndrome Mutation analysis using DNA from CVS or AFC
`Enzyme assay in. CVS or cultured AFC
`First choices are given in bold-type. CVS, chorionic villus sampling. AFC,
`amniotic fluid cells. Grade of recommendation, D.
`* The womanshould be informed prior to prenatal testing that in NAGSD the
`phenotype can be normalized completely with life-long substitutive therapy.
`© Very limited experience (single patient report) and test not widely available.
`© The presence of one mutation in a female fetus cannot predict the
`phenotype given the effect of lyonization.
`4 Informative in males but interpretation not clear in females due to
`lyonization-caused X-mosaicism.
`
`the method of choice since it gives rapid and clear-cut
`results relatively early on, with little fetal risk. Amniotic fluid
`citrulline and ASA determinations are also suitable for re-
`
`spective ASSD and ASLDprenatal diagnosis [86-88].
`
`Statement #10. Grade of recommendation: D
`
`Prenatal testing requires joint careful counselling by clin-
`ical geneticists and metabolic specialists.
`
`Statement #11. Grade of recommendation: C-D
`
`Molecular genetic analysis is the preferred prenatal test-
`ing method for all UCDs. Investigations of metabolites in
`amniotic fluid and of enzymeactivities in chorionic villi,
`cultured amniotic cells, fetal
`liver or fetal erythrocytes
`canalso be used.
`
`Newborn screening (NBS)
`UCD patients manifesting severe neonatal hyperammo-
`nemia benefit little from NBS or even from early diagno-
`sis, because of their poor prognosis [89-91] although the
`family would benefit from knowing the diagnosis. How-
`ever, NAGSD, CPS1D and OTCD are generally not
`
`screened for, given the instability of glutamine and the
`low specificity and sensitivity for detection of decreases
`in the citrulline level [92]. The benefits of screening for
`ASSD, ASLD, and ARGID, carried out
`in most US
`states, Taiwan and Australia by assessing respectively
`citrulline, ASA and arginine levels in dried blood spots,
`have not yet been formally evaluated. Although forse-
`vere ASSD and ASLD there are few false positives and
`no false negatives [93-95], ASLD screening was aban-
`doned in Austria because of the high rate of positive
`newborns
`probably
`having
`partial
`deficiency but
`remaining asymptomatic [96]. The sensitivity of NBS for
`ARGI1D and HHH is unknown, since in these diseases
`arginine and ornithine levels, respectively, may be nor-
`mal in the first days oflife [97]. A further difficulty with
`HHH is the potential production of ornithine by red cell
`arginase as the blood spot dries.
`
`Statement #12. Grade of recommendation: D
`
`Newborn screening for NAGSD, CPS1D and OTCDcan-
`not currently be recommended.
`
`