Haberle et at Orphanet Journai of Rare Diseases 2012, 7:32
`
`httpthwwnjrd.comfcontentflr’l£32
`
`ORPHAN ET JOU RNAL
`OF RARE DISEASES
`
`REVIEW Open Access
`
`Suggested guidelines for the diagnosis and
`management of urea cycle disorders
`
`Johannes Haberlel‘, Nathalie Boddaertz, Alberto Burlinai Anupam Chakrapani‘l, Marjorie Dixong, Martina Huemer°,
`Daniela Karall?r Diego Martinellis, Pablo Sanjurjo Crespo”, Rene Santerm, Aude Servals”. Vassili Valayannopoulos'g,
`Martin Lindner'”, Vicente Rubiol‘” and Carlo Dionisi—Vicia'T
`
`
`Abstract
`
`Urea cycle disorders iUCDsl are inborn errors of ammonia detoxification/arginine synthesis clue 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 #50942) 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 insul‘iicient
`awareness of health care professionals because of disease rarity, These guidelines aim at providing a trans—European
`consensus to: guide practitioners, set standards ofcare and help awareness campaigns. To achieve these goalsr the
`guidelines were developed using a Delphi methodology, by having professionals on UCDs across seven European
`countries to gather all the existing evidence, score it according to the SIGN evidence level system and draw a series of
`statements supported by an assodated 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 ethical issues. Also, it identified knowledge voids that must be filled 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, Hypei‘ammonemia. N—acetylglutamate synthase, Carbamoylphosphate synthetase
`l, Ornithine transcarbamylase, Ornithine carbamoyl transferase, Argininosuccinate synthetase, Argininosuccinate Iyase.
`Arginase l, Hyperornithinemlahyperammonemia-homocitrullinuria syndrome
`
`Introduction
`
`Urea cycle disorders {UCDSJ are inborn errors of nitrogen
`detoxil-icafionr‘arg‘mine synthesis clue to defects in the urea
`" Correspondence; Johannesl Iaeberlertrrkisoiuahch; rnarrinllndnerrtamedunr
`heidelbergde; rLIbiC~@ib'v.csi:.es; Carlodionrsivicigropiugner
`lEoual contributors
`‘Universiiy Children‘s l-losgirai Zurich and Children‘s Research Centre, Eurich
`8032. Switzerland
`"University Children‘s Hospital, rm Neuenheirner reru 430, Herdelberg 69120,
`Germany
`MInstitute de Biomedrcina de Valencra dei Consejo Superior de
`Investigaciones Cientiflcas llBV-CSIC) and Centro de lnvesligacidn Biomedica
`en Red para Enfermedades Raras iClBERERi. G Jaume Roig 1i, Valencia
`46010, Spain
`3Division of Metabolism, Bambino Gesu Children's Hospital. lRCCS, Piazza S.
`Onrrfrip at. Home I 00165, llaly
`Full list of author information is available at the end or the article
`
`cycle enzymes (Figure 1). carbamoylphosphate synthetase 1
`(CPSl), ornithine transcarbamylase (OTC), angininosuccinate
`synthetase (ASS), argininosuccinate lyase (ASL) and arginase
`1
`(ARGI),
`leading to respective deficiencies (abbreviated
`CPSID, OTCD, ASSD, ASLD and ARGlD; corresponding
`MINI numbers, #237300, #311250; #215700; #207900:
`#207800 respectively). They also encompass deficiencies of
`N—acetylglutamate synthase (MAGS) (MIM #237310), asso—
`ciated with lack of the Nracetylglutamate (NAG) essential
`activator of CPSl and of the mitochondrial ornithineicitrul-
`
`line antiporter [ORNTIL causing the hyperornithinemia—
`l'ryperammonemia—homocitrullinuria (Hr-{H}
`syndrome
`(MIM #238970). The prevalence of these disorders may
`exceed the current estimates (1:8.000—1:44,000 births [1—3].
`
`( Bioflled Central
`
`""1 2012 Haberle e: at, llcerizee BloMed Central Ltd This Is an Open Access article distributed Lli‘ldE'I' the terms of the Creative
`Commons Attribution License [l‘rttpanic-era:ivecommnnscrgflicenfiesrhv/ZD} which permits unrestricted use. distribution. and
`reprodrrcrion in any medium, provider! the original work is properly cited.
`
`1 °f 3°
`
`Horizon Exhibit 2004
`Horizon Exhibit 2004
`Lupin v. Horizon
`Lupin v. Horizon
`|PR2018-00459
`IPR2018-00459
`
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`Haberle et at Orphanet Journal of Rare Diseases 2012, 7:32
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`Page 2 of 30
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`
`
`Aspartete *— Oxalaceiale
`+ 1 ATP
`
`
`
`ALJD—Jz—sartate c cle
`
`
`
`;;
`
`Citrulllne "
`
`Argininosuccinate
`
`Malate
`
`Portal blood
`
`Mitochondrion
`1
`ii
`Cytosoi
`
`NH3+ HC03
`Glutamine
`NAB P H
`
`entrails)
`Citrulline
`-’
`gluleraie
`
`
`
`
`
`NADI’P)
`Glutamate o-—-'""""—’
`Acetyl mm
`
`
`
`.
`.
`Carbarnoyl-
`
`phosphate 4- Ornlthlne
`‘
`ATP. NADPH
`1'
`
`L'G'i‘ii‘ifi
`
`'y-sem a e y e
`UMP —> Uridine,UraI_::il
`‘1
`1-
`T
`a
`1'
`ens-Tics!
`.
`.
`I
`0MP —* OrotidIne
`-
`1.
`l
`Praline
`II ? Fyrrollne-
`1
`-carboxylate
`:
` a: —————————————-§ r--Iv- -Ir Orotic acid
`
`m
`
`+ 2 ATP
`
`N-Acetyl-
`L-glutamate
`
`Q.
`
`on
`
`.
`
`Ureacycle
`
`urnara e
`F .
`t
`
`-
`-
`°mflhme
`
`Arinine
`H 0
`+ 2
`
`m
`
`Urea
`i
`a
`
`
`
`Figure 1 The urea cycle and associated pathways. Non-Standard aljtileviations iI'Icludez GDH, glutamate dehydrogenase; GL5, gltitamlnase;
`NAUEP‘I, nicotinaI'n-lde adenine din-nucleotide [phosphate]: OAT. omithine aminouansierase; 0MP, orotidine monophosphaie: PSCR. pvnollne-S-
`carboxylate reduciase; PSCS. 11"pynollne-S-carboxylate synthetase, UMP, undine monophosphare.
`
`
`
`for all UCDs jointly) because of unreliable newborn
`screening and underdingnosis of fatal cases. Clinical Fear
`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 [41—7]. Even in partial deficiencies. which have more
`variable clinical presentations and later onset (any age),
`there is increased risk of premature death [5,3]. The dur—
`ation and severity of hyperarnmonemia 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 U035. These guidelines, developed with the Delphi
`methodology are intended to be used by metabolic species
`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 (LPl.
`MlM #222700), deficiencies of pyrroline 5-carboxyiate
`synthetase (MIM #610652) and ornithine aminotransferase
`deficiency (OAT. MIM #258870). despite the fact that they
`may cause hyperatnmunemia.
`
`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, CDV. S.
`Griinewald, [London retired after the first meeting], [H
`[chairman], DK, ML [secretary], DM, PS, W), 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.
`Picca, Rome). liver transplantation (1. de Ville de Goyet,
`Rome), epidemiology (A. Tozzi. Rome) and ethics (C.
`RehmanmSutter. Basel) and a patient group representative
`(S. Hannigan. London]. After further recommendations!
`comments by three highly renowned external reviewers (C.
`Bachmann. Bottmingen; I.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 endorsement to 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,
`httpzl/wmvsignaouk). Systematic literature review en-
`compassing from each disease description until early 2011
`was carried out using mainly Medline, Embase,
`the
`Cochrane Library. MedLinlc. 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 8!. criteria"
`
`1++ High quality meta-analyses, systematic reviews of
`randomized control trials (RCTs), or RCTs with a very
`low risk of bias.
`
`1" Well conducted meta-analyses, systematic reviews of
`RCTs. or RCTs with a low risk of bias.
`
`1' Meta-analyses, systematic reviews or RCTs, or RCTs
`with a high risk of bias.
`2” High quality systematic reviews of caseecontrol or
`cohort studies or high quality case—control or cohort
`
`studies with a very low risk of confounding bias, 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 and a 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 on their level of evidence:
`
`"Grade of recommendation 8: criteria"
`A If level 1 evidence was found (not the case}.
`B Iflevel 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 rer
`ilect 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 wellrinformed, prur
`dent clinical practice.
`
`Diagnosis
`The clinical picture
`The clinical manifestations of UCDs (Table 1) can occur at
`any age [1246]. with hyperammonemic crises being frer
`quently triggered by catabolic events. protein overload or
`certain drugs. Most symptoms are neurological but nonspe-
`cific. A UCD should be immediately suspected in neonates
`if there are any neurological symptoms or at any age if 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
`[12.17719] and the progressive spastic diplegia beginning in
`childhood (or later) in ARGJD 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 somnoience and
`. Confusion. lethargi'. dizziness
`lethargy to coma! mimicking encephalitis or drug
`intoxication
`
`- Acute encephalopathy [see below)
`- Seizures [generally not isolated but along with an
`altered level of consciousnessl
`
`- Ataxia [generallyr associated with altered consciousness level)
`- Strokelike episodes
`. Transient visual loss
`
`- Vomiting and progressive poor appetite
`- Liver failure
`
`. Multiorgan failure
`. Peripheral circulatory failure
`- ”Postpartum 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
`- Asterixrs (in adults]
`
`- Learning disabilities, neurodevelopmental delay, mental retardation
`
`- Chorea. cerebral palsy
`- Protracted cortical visual
`
`loss
`
`. Progressive spastic diplegia or quadriplegia {described in ARGlD
`and l-ll-ll-l 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-Injurlous behaviour
`
`- Autism-like symptoms
`- Fragile hair (typical for ASLDJ
`. Dermatitis
`
`- Specific rreuropsvt'hological phenotype rn heterozygous OTC Females
`- Episodic character of signs and symptoms
`Potential triggers of hyperammonemic crises in UCD patients
`. Inter-[ions
`- Fever
`
`- Vomiting
`- Gastrointestinal or internal bleeding
`- Decreased energy or protein intake leg. fasting pie 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 tag. a barbecue. parenteral nutrition]
`
`- Drugs: Mainly valproate and L-asparaginaseipegaspargase. Toprtamate, carbamazepine. phenobarbitone. phenytoine, primidone,
` furosel‘rricle hvdrochlorothiazide and salicvlates have also been associated with hyperammonemic decompensation.
`Typical and uncommon signs and symptoms are highlighted in bold- and normal-type, respectively, whereas italic 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 interventicrns Women can first manifest it UCD as
`acute unexplained neurological symptoms in the postpar-
`tum period (reported for CPSID, OTCD, and ASSD [23-
`
`25]). Variability in disease severity is characteristic for
`OTCD heterozygous 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 CPSID 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) [10.4044] 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) [50,511 I, autism-like
`symptoms [5253]. 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 neoe
`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. Ciinical 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 encephaIOpathy 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 halimark for most
`UCDs. The absence of hyperammo nemia 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—
`monernia [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 andfor 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 (CSF) 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 andfor secondarily
`decrease ammonia detoxification can cause hyperam—
`monemia and mimic a UCD [16,59-63}. Thus, fl
`natal hzgerammonemia 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—6711 Lote‘onset
`hygemmmonemia can be triggered by most conditions
`that can also cause neonatal hyperammonemia, by
`chronic liver
`failure,
`exogenous
`intoxications
`(cg.
`amanita phalloides), dmgs (cg. valproic acid). porto—
`cava] shunt and Reye syndrome, by conditions that
`vastly increase either direct ammonia production (cg.
`asparaginase treatment, urease—positive bacteria over-
`growth or genito-urinary infection] or protein catabol»
`ism (e.g. m'yeloma, chemotherapy,
`steroid therapy.
`trauma, gastrointestinal hemorrhage) and when there
`is excessive nitrogen supply (reported in total paren-
`teral nutrition or after glycinevsolution irrigations in
`transurethral prostate resection} [5,17,68-72]. Table ‘3.
`
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`Table 2 Bedside differential diagnosis of inborn errors of metabolism presenting with hyperammonernia
`
`Parameter
`Condition
`
`acidosis
`ltetoriurlaa
`
`Hypoglycemiah
`T Lactic acid‘
`
`UC-Ds
`
`+i—
`-
`
`—
`-
`
`Organic
`acidurias
`
`+°
`+
`
`+i—
`+
`
`B-Otcidation
`Hyperinsulinism-
`defects
`hyperammonemia
`
`syndrome
`_
`- — _.
`—
`
`+f—
`—
`
`_
`
`+
`+i-
`
`+
`-
`
`Pyruvate
`carboxylase
`deficiency 9
`_ _ i
`++
`
`+
`+
`
`n.
`_
`+
`—
`H“
`i AST & ALT
`_
`_
`+
`-
`-
`l CPK
`-
`-
`+
`+
`—
`T Uric acid
`_
`_
`_
`+
`_
`lWBCIRBClPIt
`Weight loss + — +' — —
`
`
`
`
`
`In addition to the conditions indicated in the table. mitochondrial oiiidative ohosphorylation defects. citrin deficiency. lysinuric protein intolerance or ornithine
`aminotransferase deficiency can also cause hyperarnmonernia.
`Grade of recommendation, D.
`“ in neonates ketonuria E++ or +++l suggests organic acidurla
`° Hypoglycemia and hyperammonemia :“pseudoiieye”: can be predominant manifestations of the organic aciduria clue to 3-hydr'oity—3-rrlelhqulmaryl-CDA lyase deficiency.
`‘ Blood lactate >5mmon, since lower high lactate levels [zomMi may be due to violent crying or to extensive muscle activity.
`d AST 8i 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 hyperarnmonemia 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 diHerenti-al 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-
`ammonernia is due to an inborn error. ARGID and
`
`ASLD can be identified. respectively, by the high plasma
`arginine or
`the high plasmaiurinary argininosuccinate
`(ASA) level. The finding of high plasma citruiline in the
`absence of ASA is highly suggestive of ASSD. The com—
`bination of hyperammonemia with low plasma citrulline
`and arginine is diagnostic of OTCD when orotic acid is
`increased in the urine, whereas
`it strongly suggests
`CPSlD 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. When the metabol-
`ite pattern is not clear—cut. activity assays of urea cycle
`enzymes in liver (all urea cycle enzymes). red blood cells
`(ASL and ARGl;
`still very useful
`in ARGID [76]).
`
`intestinal mucosa (CPSl. OTC) or fibroblasts (ASS, ASL.
`HHH) can clarify diagnosis, although enzyme assays have
`generally been replaced by genetic testing. Enzyme ana—
`lysis is now mainly reserved for the minority of cases in
`whom genetic 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 UCDs if genetic testing does not identify a spe-
`cific UCD, or if it is not available. In deceased patients
`with a suspicion of UCD, fibroblasts andfor liver 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:/iwwwncbi.nlm.nih.govi
`sltes/entrez?db=omim)
`including citrullinemia type 2
`(SLCZSAJS gene encoding citrin) and the HHH syn
`drome [SLCQSAB gene). Mutation detection has at least
`~80% sensitivity [7?] and permits carrier identification.
`prenatal diagnosis. facilitating pedigree analysis. genetic
`counselling and in some Cases genotype-phenotype
`
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`

`Haberle er of, Orphanet Journal of Rare Diseases 2012, 7:32
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`
`Page I’ of 30
`
`
`
`Gluten-line
`elevated
`
`Cltrulllne
`r
`
`Citrulllne
`n
`
`Cltrulllna
`in
`
`U Arg. Lys, 0
`T U Orotic acid
`
`T ASA
`1" U Orotic acid
`ASL D
`
`Omithlne
`normal
`
`T U gg’éi‘Bacid
`
`*
`
`it:
`OTC D
`
`T T Arg
`final 0
`
`I
`Om can be
`transiently normal
`OAT D
`
`LP]
`
`Glutarnine
`low-normal
`
`
`
`Citrulline
`t
`
`nomA Organic acid TT
`profile
`Acylcamflines T
`
`Citmillne
`TT
`
`U Oral]: acid
`normal
`
`U Drotlc acid
`elevated
`
`omit'iiiikfltigrrifll
`Acidosis
`
`mm”
`M so —
`one - TFO
`HyPDglycemla
`
`"V”g'ywmi“
`will:
`
`T Med: :3"
`A-FF‘. .; actosa
`
`'
`9C: delta-em
`
`MAGS D
`CPS D
`
`Omitt'line
`elevated
`
`t ”M
`Orn. Pro, Arg
`starved
`
`
`
`HHH 5
`
`DAT D
`
`Figure 2 Diagnostic algorithm for neonatal hyperammonemia. Unless indicated, plasma is used for the analytical ClE'lElT‘I'Iii‘IailUnEi. Non-
`standmd abbreviations include A-liP, {I ietoplolein; CIT 2, LlUUlllllE‘lTlld type 2; CPSD. Cl-‘S‘I deiiCIencv; l'li-HA, hvperlHaulinism-hyperammor1emia
`syndrome; HMG. 5-hyclro‘xy-Smethylglutaryi-COA Iyase deficiency; LF‘ir lysinuric protein intolerance; OATD, ornirhine smir'iotransferase deficiency;
`PA. oropionic acidernia; I-‘l'_. Dyruvate (arboxyiase; PSCSD. A] pyrroline S-carboxylate deficiency; THAN. transient hypetdmmonemia oi the newbom;
`TPD, rrsfuncrional protein deficiency, LI, urine. Grade of recommendation, D. " in some patients with late-onset OTC D. plasma citruiline levels ate in
`the lows: part of the normal range.
`
`
`correlations [1527,78], conceivably opening the way to
`future
`therapies
`(eg.
`nonsense
`read—through
`approaches). DNA, generally from blood,
`is used, al-
`though the large number of CPSI exons renders prefer—
`able the utilization of RNA from cultured fibroblasts for
`
`CPSID studies. For other [1035 RNA analysis (from liver
`in the case of OTCDJ is only carried out when DNA arias
`lysis is negative [79-81]. Prognostic judgements on the
`disease—causing nature of missense mutations [the most
`frequent ones) and of some splice—site mutations are diffi—
`cult if not backed by in vitro expression studies of the mur
`taut 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,
`
`Statement #9. Grade of recommendation: D
`
`Mutation analysis has some pitfalls and limitations, includ—
`ing the difficulty in establishing the pathogenic potential of
`a missense mutation. In vitro protein expression studies
`and in silico analyses based on sequence conservation and
`protein structure can help infer pathogenic potential but
`are not part of routine clinical management.
`
`Prenatal testing
`Prenatal investigations in UCDs are available in many coun-
`tries and may enable pregnancy termination of 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 allelertracking using chm-ionic villus
`samples, araniotic lluid Cells or cultures thereof [35.86] is
`
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`

`Haberle er of. Orphaneljoumoi of Rare Diseases mi 2, 7:32
`httprfr‘wvvwojrd.comicontentr‘WlI32
`
`Page 8 of 30
`
`Table 3 Prenatal testing of UCDs: Recommended analyses
`and sample requirements
`Disorder
`Recommended tests
`
`NAGSD
`CPS l D
`
`Mutation analysis using DNA from CVE- or AFC"
`Mutation analysis using DNA lrom CV5 or AFC
`
`OTCD
`
`ASSD
`
`ASLD
`
`Enzyme assay in late fetal liver biopsy”
`Mutation analysis using DNA horn CV5 or AFCc
`
`Enzyme assay in late fetal liver biopsyEm
`Mutation analysis using DNA from CVS or AFC
`Citrulline in amniotic fluid
`
`Enzyme assay in intact or cultured CV5 or
`in cultured AFC
`
`Mutation analysis using DNA from CVS or AFC
`Argininosuctinate and its anhydrides in
`amniotic fluid
`
`ARGID
`
`Enzyme assay in intact or cultured CV5 or cultured AFC
`Mutation analysis using DNA from CVS
`Enzyme assay in fetal blood erythrocytes
`[mid‘gestation sampling]
`HHH ‘iyr'ltlfuf'lll‘ Mutation analysis using DNA from CVS or AFC
`Enzyme assay in cva or titlttrred AFC
`First choices are given in bold-type. CV5. chorionic villus sampling. AFC,
`amniotic fluid cells. Grade of recommendation, D.
`a The woman should be informed prior to prenatal testing that in NAGSD the
`phenotype can be normalized completely with lite-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.
`d Informative in males but interpretation not clear in females due to
`lyonization-caused K-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 ASLD prenatal diagnosis [86-88].
`
`Statement #10. Grade of recommendation: D
`
`Prenatal testing requires joint careful counselling by clin-
`ical geneticists and metabolic specialists.
`
`Statement #1 i. 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 enzyme activities in chorionic villi,
`cultured amniotic cells, fetal
`liver or fetal erythrocytes
`can also be used.
`
`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
`ASSDl 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 for see
`were 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
`ARGID and HHH is. unknown. since in these diseases
`
`arginine and ornithine levels. respectively, may be “01”
`mal in the first days of life [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. CPSlD and OTCD can—
`not currently be recommended.
`
`Statement #13. Grade of recommendation: C
`
`Newborn screening for ASSD, ASLD, a