LOW CITRULLINE AS A MARKER FOR THE PROXIMAL UREA CYCLE DEFECTS EXPERIENCE OF THE NEW ENGLAND NEWBORN SCREENING PROGRAM

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1 LOW CITRULLINE AS A MARKER FOR THE PROXIMAL UREA CYCLE DEFECTS EXPERIENCE OF THE NEW ENGLAND NEWBORN SCREENING PROGRAM Inderneel Sahai, MD, FACMG Newborn Screening and Genetic Testing Symposium Oct 2014

2 Acknowledgements New England Newborn Screening Program J Bailey RB Eaton TH Zytkovicz Massachusetts Metabolic Specialists /Centers C Garganta Tufts Medical Center, Boston L Hecht Boston Children s Hospital, Boston HL Levy Boston Children s Hospital, Boston M Martin University of Massachusetts Medical School, Worcester Partner State Collaborators Wendy Smith Maine Medical Center, Portland, ME C Phornphutkul Rhode Island Hospital, Brown University, Providence, RI. L Burke Vermont Regional Genetics Center, Burlington, VT C Ingham Vermont Department of Health. Burlington, VT

3 Urea Cycle Glutamate Acetyl-CoA N-acetylglutamate synthase (NAGS) N acetyl glutamate Nitrogen Pool Ammonia + Carbamoylphosphate synthetase (CPS) Carbamoyl phosphate Aspartate Aspartate-glutamate carrier (Citrin) Ornithine transcarbamylase (OTC) Mitochondria Cytosol Ornithinine Ornithinine Ornithine transporter Aspartate Argininosuccinic synthetase (ASS) Arginase (ARG) Urea Argininosuccinicate Arginine Argininosuccinic lyase (ASL) Fumarate

4 Urea Cycle Disorders Glutamate Acetyl-CoA N-acetylglutamate synthase (NAGS) N acetyl glutamate Nitrogen Pool Ammonia + Carbamoylphosphate synthetase (CPS) Carbamoyl phosphate Aspartate Aspartate-glutamate carrier (Citrin) Ornithine transcarbamylase (OTC) Mitochondria Cytosol Ornithinine Ornithinine Ornithine transporter Aspartate Argininosuccinic synthetase (ASS) Arginase (ARG) Urea Argininosuccinicate Arginine Argininosuccinic lyase (ASL) Fumarate

5 Urea Cycle Disorders Glutamate Acetyl-CoA N-acetylglutamate synthase (NAGS) N acetyl glutamate Nitrogen Pool Ammonia + Carbamoylphosphate synthetase (CPS) Carbamoyl phosphate Aspartate Aspartate-glutamate carrier (Citrin) Ornithine transcarbamylase (OTC) Mitochondria Cytosol Ornithinine Ornithinine Ornithine transporter Aspartate Argininosuccinic synthetase (ASS) Arginase (ARG) Urea Argininosuccinicate Arginine Argininosuccinic lyase (ASL) Fumarate

6 Proximal Urea Cycle Disorders CPS (Carbamoylphosphate synthetase) OTC (Ornithine transcarbamylase) NAGS (N-acetylglutamate synthase) Acute neonatal presentation in 2/3 Milder phenotypes (Late Onset/ Intermittent forms): Progressive neurological disorder. Episodic headaches, ataxia, scotomas, sleep disturbances, behavioral abnormalities, ADD, psychosis. OTC is X-linked (~ 15% females affected) CPS and NAGS autosomal recessive

7 Background NENSP screening for amino acids & acylcarnitines using MS/MS since Feb Targeted analysis with MRM. Amino acids targeted: Arginine, Argininosuccinic acid,, Leucine, Methionine, Ornithine, Phenylalanine, Tyrosine, Valine Low cut-off introduced in 2004

8 (-4.25 SD) (-3.5SD) [Tyrosine x Methionine] Ornithine / (-2.0 SD) 15 (-3.3 SD) Based on analysis of approximately randomly selected newborn screening technically satisfactory specimens collected within 24 hrs to 7 days of birth during Aug 2004 to Aug Although distribution shown is log transformed, values on x-axis reconverted to actual concentrations and ratios. Mean values shown in blue; others shown are 2,3 and 4 SD from the mean

9 Positive Screens for Proximal UCDs < 3uM OR > 3uM to < 3.8 & [Tyrosine x Methionine] <

10 Positive Screen: Categories High Risk Cit < 3. 8 um & /[Tyrosine x Methionine] < & Ornithine / > 15 Moderate Risk Cit < 3. 8 um & /[Tyrosine x Methionine] < OR Cit < 3 um & Ornithine / > 15 Low Risk Cit < 3uM & both ratios in range

11 1.2 Million Screened Aug Aug 2013 Technically satisfactory Collected 24 hrs-7 DOL Positive Screens (Infants) : Initial Specimen 5 Repeat Specimen (Initial Normal)

12 Initial Specimens: True Positives Disorder Birth Weight NICU Status Initial Screen Age Risk (in days) (in um) Category Neonatal Clinical Status OTC NO High Seizures & encephalopathy on DOL 3 OTC NO High Seizures & encephalopathy on DOL 3 OTC YES High Seizures & encephalopathy on DOL 3 OTC NO High Seizures & encephalopathy on DOL 3 OTC YES High On treatment from birth in view of family history of OTC. Asymptomatic CPS YES High Hypotonic at birth, seizures on DOL 2. CPS NO High Hypotonic at birth, seizures on DOL 2. NAGS NO High Lethargy noted on awakening on DOL 5. Cardiac arrest shortly thereafter Follow-up Deceased DOL 8 Deceased DOL 6 Deceased DOL 5 Deceased DOL 4 Liver transplant Liver transplant Liver transplant Deceased DOL 5 Repeat Screen Age Risk (in um) Category On Rx High High High High

13 Initial Specimens: False Positives False Positives Birth Weight NICU Status Initial Screen Age Risk (in days) (in um) Category Neonatal Clinical Status FP YES High Pneumothorax, Respiratory distress, Sepsis on DOL 1 Follow-up Amino acids,nh3 Normal. CPS & OTC molecular analysis negative Repeat Screen Age Risk (in um) Category 8 7 Normal FP NO Moderate Asymptomatic. Plasma citrulline persistently low Normal FP NO Moderate Asymptomatic Amino acids,nh3 Normal Low FP NO Moderate Asymptomatic Plasma citrulline low. NH3 normal Developmental delays, hypotonia, hypospadias, pulmonary stenosis. FP YES Moderate Ex 32 weeker. Poor feeding Normal FP YES Moderate Ex 33 weeker. Poor feeding Amino acids,nh3 Normal Normal FP YES Moderate Bowel resection. Immunodeficiency 5 7 Normal FP YES Moderate Respiratory Distress. Hyperbilirubinemia on DOL 1 Amino acids,nh3 Normal. FP YES Moderate Ex 35 weeker. Poor feeding. Infant of diabetic mother. Transient hyperammonemia. Persistent orotic aciduria. OTC molecular analysis negative FP YES Low Ex 32 weeker. Aysmptomatic at 6 months of age. Moved out of state. FP YES Low Ex 35 weeker. Poor feeding. Hyperbilirubinemia & bloody stools Normal Normal Normal FP YES Low Ex 36 weeker. Poor feeding Normal

14 Repeat Specimens All 5 false positives All infants with a h/o bowel resection. On TPN

15 False Negatives OTC Male Initial Screen Birth Weight NICU Status Age (in days) Cit (in um) 2125 YES (-2.55 SD) Ratios C/TM Neonatal Clinical Status Presented in ER after a fall at 11 months of age. H/O mild developmental delays reported. Evaluated, observed and discharged. Returned with change in mental status a week later, with rapid decline into status epilepticus and encephhalopathy from which infant could not be revived. Laboratory work-up revealed hyperammonemia, low plasma citrulline and high concentrations of urinary orotic acid. Molecular analysis revealed a novel mutation. OTC Female Carrier OTC Female 3380 NO (0.88 SD) 2805 YES (-2.56) Normal Infant born before Aug Presented with a h/o developmental delay and hemiperesis at 9 months. Diganostic work-up revealed low plasma citrulline and high concentrations of urinary orotic acid. Retrospective review of newborn screening results performed. A 2nd screen collected at 6 months of age with citrulline value of μm. Normal Female sibling of OTC Case 2. concentration in repeat (DOL 5 days) 6.67 μm. NH3 & Orotic acid normal. Plasma citrulline values in low normal range. Asymptomatic at 2 yrs.

16 Urea Cycle and Interrelated Pathways Mitochondria Glutamine N acetyl glutamate + Acetyl-CoA NAGS NH3 Glutamate NH3 NH3 α-keto glutarate NH3 HCO3 - CPS NH3 Carbamoyl phosphate P5CD P5CS OTC Ornithine OAT Glu- γ- semialdehyde Pyrroline-5- carboxylate Ornithine Pyrroline-5- Cytoplasm carboxylate Proline

17 Additional Disorders Disorder Pyrroline-5- Carboxylate Synthase Deficiency Hyperammonemia- Hyperornithinemia- Homocitrullinuria Birth Weight NICU Status Age (in days) Initial Screen (in um) 2268 NO (-3.2 SD) 3150 NO (-1.1 SD) Ratios Normal O/C Neonatal Clinical Status Hypotonia and "progeroid" features reported at birth. Failure to thrive and developmental delays at 3 months. Plasma amino acids revealed low citrulline and proline. Diagnosis confirmed by sequencing of P5C Synthase deficiency. Presented with gross motor delays and spasticity in lower extremeties at 18 months of age. Chronic mild hyperammonemia identified.

18 Conclusions Low can identify infants with severe proximal urea cycle defects. However these infants presented early in the neonatal period for NBS to prevent mortality in majority. Late onset forms were missed. Adjustments in the cut-off values of citrulline may allow detection of some late onset cases of the proximal UCD and other metabolic disorders such as pyrroline-5-carboxylate synthase.