Haberle et al. Orphanet Journal of Rare Diseases 2012, 7:32
`
`
`
`http:/ /www.ojrd.com/content/7 /1/32
`
`ORPHANET JOURNAL
`OF RARE DISEASES
`
`REVIEW
`
`Open Access
`
`Suggested guidelines for the diagnosis and
`
`
`
`
`management of urea cycle disorders
`3, Anupam ChakrapaniJohannes Haberle1·, Nathalie Boddaert2, Alberto Burlina
`
`
`
`
`4, Marjorie Dixon5, Martina Huemer6,
`
`Daniela Karall7, Diego Martinelli
`
`8, Pablo Sanjurjo Crespo9, Rene Santer10, Aude SeNais11, Vassili Valayannopou
`los12,
`
`
`Martin Lindner 13*t, Vicente Rubio 14*t and Carlo Dionisi Vid*t
`
`
`Abstract
`Urea cycle disorders (UCDs) are inborn errors of ammonia detoxification/arginine synthesis due to defects affecting the
`
`
`
`
`
`
`catalysts of the Krebs Henseleit cycle (five core enzymes, one activating enzyme and one mitochondrial omithine/
`
`
`
`
`
`
`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 suNivors. 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 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 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 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 UCO patients.
`Urea cycle disorders, UCO, Hyperammonemia, N acetylglutamate synthase, Carbamoylphosphate synthetase
`
`
`
`
`
`Keywords:
`
`
`
`
`
`1, Ornithine transcarbamylase, Ornithine carbamoyl transferase, Argininosuccinate synthetase, Argininosuccinate lyase,
`Arginase 1, Hyperomithinemia hyperammonemia homocitru
`llinuria syndrome
`
`1 cycle enzymes (Figure 1), carbamoylphosphate synthetase
`
`
`
`Introduction
`
`
`(CPSl), ornithine transcarbamylase (OTC), argininosuccinate
`Urea cycle disorders (UCDs) are inborn errors of nitrogen
`
`
`
`
`
`
`
`
`
`detoxification/arginine synthesis due to defects in the urea synthetase (ASS), argininosuccinate lyase (ASL) and arginase
`
`1 (ARGl), leading to respective deficiencies (abbreviated
`• Correspondence: Johannes.Haeberle@kispi.uzh.ch; martin.Ii nd ner@med.uni
`
`
`
`CPSlD, OTCD, ASSD, ASLD and ARGlD; correspondi
`
`heidel berg.de; rubiO@ibv.csic.es; carlo.d ioni sivici@Opbg.net
`ng
`
`#237300, #311250; #215700; #207900;
`MIM numbers,
`tEqual contributors
`
`
`
`#207800 respectively). They also encompass deficiencies of
`
`
`
`
`
`'University Children's Hospital Zurich and Children's Research Centre, Zurich
`8032, Switzerland
`
`
`
`N-acetylglutamate synthase (NAGS) (MIM #237310), asso­
`
`
`13University Children's Hospital, Im Neuenheimer Feld 430, Heidelberg 69120,
`
`
`ciated with lack of the N-acetylglutamate (NAG) essential
`Germany
`
`••institute de Biomedicina de Valencia del Consejo Superior de
`
`activator of CPSl and of the mitochondrial ornithine/citrul­
`
`
`lnvestigaciones Cientfficas (IBV CSIO and Centro de lnvestigaci6n Biomedica
`
`
`line antiporter (ORNTl), causing the hyperornithinemia­
`
`
`en Red para Enfermedades Raras (OBERER), Cl Jaume Roig 11, Valencia
`
`
`hyperammonemia-homocitrullinuria (HHH) syndrome
`46010, Spain
`
`
`
`(MIM #238970). The p revalence of these disorders may
`8Division of Metabolism, Bambino Gesu Children's Hospital, IRCCS, Piazza 5.
`Onofrio 4, Rome I 00165, Italy
`
`(1:8,000-1:44,000 (1-3],
`
`exceed the current estimates
`births
`
`
`Full list of author information is available at the end of the article
`
`Q 2012 Haberle et al; licensee BioMed Cen tral Ltd This is an Open Access article distributed under the terms of the Creative
`0 BioMed Central
`
`rg/licenses/by/2.0), which permits unrestricted use, distribution, and Commons Attribution License (http://creativecommons.o
`
`
`reproduction in any medium, provided the original work is properly cited.
`Page 1 of30
`
`Horizon Exhibit 2019
`
`Lupin v. Horizon
`IPR2017-01160
`
`

`

`Haberle et al. Orphanet Journal of Rare Diseases 2012, 7:32
`htth/www.ojrd.com/content/7/1 I32
`
`Page20f30
`
`Portal blood
`
`
`
`Mitochondrion
`
`Glutamine
`
`
`
`NAD(P)H
`
`
`
`
`Cytosol
`NH3 + Hco3
`
`Aspartate 4— Oxalacetate
`
`+ 1ATP
`
`Citrulline
`
`Asgarta te czcle
`
`I
`
`Citrulline
`
`Argininosuccinate
`
`Malate
`
`
`
`
`
`
`’
`
`'
`
`»
`
`Urea cycle
`
`Fumarate
`
`Arglnine
`
`+ HgO
`
`
`
`
`
`
`
`Acetyl CoA
`
`+2ATP
`
`mes
`N-Acety|- filo cps1
`L-glutamate
`_
`
`I:ii'
`
`Carbamoyl-
`phosphate
`1
`
`+ Ornithine
`
`ATP, NADPH
`
`“
`
`
`
`L-Glutamate-
`
`EEK]
`
`‘y-semialdehyde
`I
`f
`I
`chemical
`I
`I
`I
`I
`I
`
`A‘-Pyrroline—
`5-carboxylate
`
`* Ornithine
`,
`
`UMp _. Uridine, Uracil
`T
`0MP —* Orotidine
`T
`Proline
`— — — - - - ->-§ >- + -> Orotic acid
`
`Urea
`
`Figure 1 The urea cycle and associated pathways. Non standard abbreviations include: GDH, glutamate dehydrogenase; GL5, glutaminase;
`NAD(P), nicotinamide adenine dinucleotide (phosphate); OAT, ornithine aminotransferase; 0MP, orotidine monophosphate‘; PSCR, pyrroline 5
`carboxylate reductase; PSCS, A1 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 Scarboxylate
`synthetase (MINI #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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`Häberle et al. Orphanet Journal of Rare Diseases 2012, 7:32
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`
`Page 3 of 30
`
`pediatric metabolic specialists (S. Baumgartner [Inns-
`bruck, retired after the first meeting], AB, AC, CDV, S.
`Grünewald, [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.
`Picca, 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 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,
`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 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 case control 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 & 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
`The clinical picture
`The clinical 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 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
`[12,17-19] and the progressive spastic diplegia beginning in
`childhood (or later) in ARG1D and the HHH syndrome,
`
`Page 3 of 30
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`Häberle et al. Orphanet Journal of Rare Diseases 2012, 7:32
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`Page 4 of 30
`
`Table 1 Clinical signs and symptoms of acute and chronic presentations of UCDs, and triggering factors for
`hyperammonemia in UCD patients
`Acute presentation
`(cid:129) Altered level of consciousness (from somnolence and
`lethargy to coma) mimicking encephalitis or drug
`intoxication
`
`Chronic presentation
`(cid:129) Confusion, lethargy, dizziness
`
`(cid:129) Migraine like headaches, tremor, ataxia, dysarthria
`
`(cid:129) Acute encephalopathy (see below)
`
`(cid:129) Asterixis (in adults)
`
`(cid:129) Seizures (generally not isolated but along with an
`altered level of consciousness)
`
`(cid:129) Learning disabilities, neurodevelopmental delay, mental retardation
`
`(cid:129) Ataxia (generally associated with altered consciousness level)
`
`(cid:129) Chorea, cerebral palsy
`
`(cid:129) Stroke like episodes
`
`(cid:129) Transient visual loss
`
`(cid:129) Protracted cortical visual loss
`
`(cid:129) Progressive spastic diplegia or quadriplegia (described in ARG1D
`and HHH syndrome)
`
`(cid:129) Vomiting and progressive poor appetite
`
`(cid:129) Protein aversion, self selected low protein diet
`
`(cid:129) Liver failure
`
`(cid:129) Multiorgan failure
`
`(cid:129) Peripheral circulatory failure
`(cid:129) “Post partum psychosis”
`
`(cid:129) Psychiatric symptoms (hallucinations, paranoia,
`mania, emotional or personality changes)
`
`In neonates:
`
`(cid:129) sepsis like picture, temperature instability
`(cid:129) respiratory distress, hyperventilation
`
`(cid:129) Abdominal pain, vomiting
`
`(cid:129) Failure to thrive
`
`(cid:129) Hepatomegaly, elevated liver enzymes
`
`(cid:129) Psychiatric symptoms: hyperactivity, mood
`alteration, behavioural changes, aggressiveness
`
`(cid:129) Self injurious behaviour
`
`(cid:129) Autism like symptoms
`
`(cid:129) Fragile hair (typical for ASLD)
`
`(cid:129) Dermatitis
`
`(cid:129) Specific neuropsychological phenotype in heterozygous OTC females
`
`(cid:129) Episodic character of signs and symptoms
`
`Potential triggers of hyperammonemic crises in UCD patients
`(cid:129) Infections
`
`(cid:129) Fever
`
`(cid:129) Vomiting
`
`(cid:129) Gastrointestinal or internal bleeding
`
`(cid:129) Decreased energy or protein intake (e.g. fasting pre surgery, major weight loss in neonates)
`
`(cid:129) Catabolism and involution of the uterus during the postpartum period (mostly OTC females)
`
`(cid:129) Chemotherapy, high dose glucocorticoids
`
`(cid:129) Prolonged or intense physical exercise
`
`(cid:129) Surgery under general anesthesia
`
`(cid:129) Unusual protein load (e.g. a barbecue, parenteral nutrition)
`
`(cid:129) Drugs: Mainly valproate and L asparaginase/pegaspargase. Topiramate, carbamazepine, phenobarbitone, phenytoine, primidone,
`furosemide, hydrochlorothiazide and salicylates 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 interventions. Women can first manifest a UCD as
`acute unexplained neurological symptoms in the postpar-
`tum period (reported for CPS1D, 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 CPS1D family)
`
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`Häberle et al. Orphanet Journal of Rare Diseases 2012, 7:32
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`
`Page 5 of 30
`
`[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 (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 and/or secondarily
`decrease ammonia detoxification can cause hyperam-
`monemia and mimic a UCD [16,59-63]. Thus, neo-
`natal hyperammonemia 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
`
`Page 5 of 30
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`

`

`Häberle et al. Orphanet Journal of Rare Diseases 2012, 7:32
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`
`Table 2 Bedside differential diagnosis of inborn errors of metabolism presenting with hyperammonemia
`Parameter
`Condition
`β Oxidation
`defects
`
`UCDs
`
`Organic
`acidurias
`
`Hyperinsulinism
`hyperammonemia
`syndrome
`
`Page 6 of 30
`
`Pyruvate
`carboxylase
`deficiency g
`+
`
`++
`
`+
`
`+
`
`+/
`
`+
`
`+/
`
`(+)d
`
`Acidosis
`Ketonuriaa
`Hypoglycemiab
`" Lactic acidc
`" AST & ALT
`" CPK
`" Uric acid
`# WBC/RBC/Plt
`Weight loss
`
`+ e
`+
`
`+/
`
`+
`
`+
`
`+
`+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.
`a In neonates ketonuria (++ or +++) suggests organic aciduria.
`b Hypoglycemia and hyperammonemia (“pseudo Reye”) can be predominant manifestations of the organic aciduria due to 3 hydroxy 3 methylglutaryl CoA lyase deficiency.
`c Blood lactate >6mmol/L, since lower high lactate levels (2 6mM) may be due to violent crying or to extensive muscle activity.
`d AST & ALT elevations can be found but are not constant in UCDs.
`e Can be absent in neonates.
`f Occurrence only in neonates.
`g 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. ARG1D and
`ASLD can be identified, respectively, by the high plasma
`arginine or the high plasma/urinary argininosuccinate
`(ASA) level. The finding of high plasma citrulline 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
`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. 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 ARG1; still very useful
`in ARG1D [76]),
`
`intestinal mucosa (CPS1, 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 and/or liver tissue
`should be preserved frozen.
`
`Molecular genetic analysis
`Except
`for OTCD, which is transmitted in the X-
`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.nlm.nih.gov/
`sites/entrez?db=omim)
`including citrullinemia type 2
`(SLC25A15 gene encoding citrin) and the HHH syn-
`drome (SLC25A13 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
`
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`Haberle et aI. Orphanet Journal of Rare Diseases 2012, 732
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`
`Page 7 of 30
`
`Glutamlne
`elevated
`
`Cltrulllne
`
`Cltrqlllne
`
`cttqqlllne
`
`CHEEHIMl
`
`cttrulllne
`TT
`
`U OI'OIIC 86k]
`normal
`
`U Orotlc acld
`elevated
`
`v Arg. Lys, 0m
`T U Arg, Lys, 0
`T u Orotic acid
`LPI
`
`T ASA
`T U Orotic acid
`ASL D
`
`THAN
`
`normal AA AcylcamltlnesT
`
`Organic acld TT
`
`
`
`Organlc acldurlas
`MMA - PA
`Acidosis
`
`FAO detects
`WAD — VLCAD - TPD
`
`Hypogyoemia
`
`ypoglycemla
`HI-HA
`HMG
`
`c" 2
`T Met, Tyr
`A-FP.
`- alactose
`
`PC dellclency
`Lactic acidosis
`
`NAGS D
`CPS D
`
`Omlthlne
`elevated
`
`Ornlthlne
`normal
`
`T U Omtic acid
`ASS D
`
`JyArg
`TAla
`OTC D '
`
`T T Arg
`ARG1 D
`
`Om can be
`transiently normal
`OAT D
`
`HHH S
`T Homocit
`
`OAT D
`
`Figure 2 Diagnostic algorithm for neonatal hyperammonemia. Unless indicated, plasma is used for the analytical determinations Non
`standard abbreviations include A FP, a fetoprotein; CIT 2, citrullinemia type 2; CPSD, CPS] deficiency; HI HA, hyperinsulinism hyperammonemia
`syndrome; HMG, 3 hydroxy 3 methylglutaryl Co/\ Iyase deficiency; LPI, lysinuric protein intolerance; OATD, ornithine aminotransferase deficiency,
`PA, propionic acidemia; PC, pyruvate carboxylase; PSCSD, AI pyrroline S carboxylate deficiency; THAN, transient hyperammonemia of the newborn;
`1P0, trifunctional protein deficiency; U, urine. Grade of recommendation, D. * In some patients with late onset OTCD, plasma citrulline levels are in
`the lower part of the normal range.
`
`correlations [15,27,78], conceivably opening the way to
`future
`therapies
`(e.g.
`nonsense
`read—through
`approaches). DNA, generally from blood,
`is used,
`al—
`though the large number of CPS] exons renders prefer—
`able the utilization of RNA from cultured fibroblasts for
`
`CPSlD studies. For other UCDs RNA analysis (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 some splice—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.
`
`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 vib‘o 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 allele-tracking using chorionic villus
`samples, amniotic fluid cells or cultures thereof [85,86] is
`
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`

`Häberle et al. Orphanet Journal of Rare Diseases 2012, 7:32
`http://www.ojrd.com/content/7/1/32
`
`Page 8 of 30
`
`CPS1D
`
`Table 3 Prenatal testing of UCDs: Recommended analyses
`and sample requirements
`Disorder
`Recommended tests
`Mutation analysis using DNA from CVS or AFCa
`NAGSD
`Mutation analysis using DNA from CVS or AFC
`Enzyme assay in late fetal liver biopsyb
`Mutation analysis using DNA from CVS or AFCc
`Enzyme assay in late fetal liver biopsyb,d
`Mutation analysis using DNA from CVS or AFC
`
`OTCD
`
`ASSD
`
`Citrulline in amniotic fluid
`
`Enzyme assay in intact or cultured CVS or
`in cultured AFC
`
`ASLD
`
`Mutation analysis using DNA from CVS or AFC
`
`Argininosuccinate and its anhydrides in
`amniotic fluid
`
`Enzyme assay in intact or cultured CVS or cultured AFC
`
`ARG1D
`
`Mutation analysis using DNA from CVS
`
`Enzyme assay in fetal blood erythrocytes
`(mid gestation sampling)
`
`HHH syndrome 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.
`a The woman should be informed prior to prenatal testing that in NAGSD the
`phenotype can be normalized completely with life long substitutive therapy.
`b Very limited experience (single patient report) and test not widely available.
`c 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 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 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 #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 enzyme activities in chorionic villi,
`cultured amniotic cells, fetal liver or fetal erythrocytes
`can also 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 ARG1D, 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 se-
`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