During Jan. 2016–Dec. 2019, nine Chinese patients from eight unrelated families were diagnosed with neonatal-onset UCDs by targeted panel sequencing or whole-exome sequencing (WES). Their clinical manifestations, biochemical features, 180-day-age outcomes, and molecular genetic characteristics were reviewed retrospectively. NGS-based tests revealed 7 patients diagnosed with ornithine transcarbamylase deficiency (OTCD) and 2 with carbamoylphosphate synthetase I deficiency (CPS1D). The spectrum of the clinical presentation of nine affected individuals progressed from unspecific symptoms like poor feeding to somnolence, coma, and death. All patients presented with an acute hyperammonemia. The most robust metabolic pattern in OTCD was hyperglutaminemic hyperammonemia with high concentration of urine orotic acid, and it was reported in six patients. Of ten variants found on the
Neonatal-onset UCDs [
The diagnosis of UCDs is generally established by blood tandem mass spectrometry (MS/MS), urine gas chromatography mass spectrometry (GC/MS) [
The overall prevalence of UCDs has recently been determined to be 1 in 35,000 births [
The primary purpose of the study was to summarize the clinical, biochemical, and genetic features and 180-day-age outcome of nine Chinese patients with neonatal-onset UCDs detected by NGS-based tests retrospectively.
This is a retrospective study of clinical course, metabolomic profiling, and genetic findings for 9 patients diagnosed as having a UCD at the corresponding neonatal intensive care units (NICUs) from three tertiary hospitals in China. The institutions included Xinhua Hospital affiliated with Shanghai Jiao Tong University School of Medicine (XH), Children’s Hospital affiliated with Shanghai Jiao Tong University (CH), and Jiaxing Maternity and Child Health Care Hospital (JX). The analysis and publication of data related to the study were approved by the institutional review board at Xinhua Hospital, Shanghai Jiao Tong University School of Medicine (Approval number: XHEC-D-2019-101) with a waiver of consent and authorization.
The diagnosis of UCDs was based on the biochemical findings or molecular analyses of the genes involved in the urea cycle pathway. The phenotypes of the affected neonates were further translated into Human Phenotype Ontology (HPO) [
Data regarding gestational age, birth weight, family history, and other demographic, clinical, biochemical, and related molecular results were extracted from the medical records of the patients. Suspicion of genetic causes was based on clinical evaluation by experienced neonatologists.
Besides the blood ammonia, advanced biochemical investigations carried out for diagnosis of UCDs included blood mass spectrometry profile by MS/MS (API 4000, American Bio-Systems Inc.) using blood filter papers and urine organic acid analysis by GC-MS (Shimadzu Limited, QP2010).
Genomic DNA was extracted from the peripheral whole blood of patients and their parents using a QIAamp Blood DNA Mini Kit (Qiagen GMBH, Hilden, Germany). Four out of five patients from XH underwent targeted exome sequencing (TES) with a specific neonatal metabolism disease 175-gene panel (MyGenostics) (see Supplementary Method for details), and the rest underwent whole-exome sequencing (WES). For WES, the capture probes were those used in GenCap Custom Exome Enrichment Kits (MyGenostics, Beijing, China) and TruSight Rapid Capture Kits (Illumina, Inc., San Diego, CA, USA). Captured libraries were sequenced by an Illumina HiSeq 6000. The Burrows-Wheeler Aligner (BWA, version 0.7.10) was used to align the reads to the human reference genome (GRCh37/hg19). Copy number variations (CNVs) and small variants were identified using VarScan 2 and Genome Analysis Toolkit (GATK) (4.0.10.1).
Detected variants were interpreted and categorized according to the five-tier classification system recommended by the American College of Medical Genetics [
Patients were considered to have a laboratory-confirmed genetic diagnosis if they had a pathogenic variant or likely pathogenic variant detected by a genetic test that explained the patient’s clinical presentation. For CPS1D patients with one variant of uncertain significance (VUS) [
Table
Clinical and laboratory data of the nine patients with neonatal-onset UCDs.
Patients | P1 | P2 | P3 | P4 | P5 | P6 | P7 | P8 | P9 |
---|---|---|---|---|---|---|---|---|---|
Gender | M | M | M | M | M | M | F | M | M |
Age at onset (days) | 7 | 7 | 2 | 6 | 2 | 3 | 5 | 1 | 1 |
Age deceased (days) | 170 | 165 | 7 | Survival | 3 | 7 | Survival | 5 | 3 |
Gestation age (weeks) | 40.29 | 38.29 | 39.86 | 38.14 | 36.14 | 36.14 | 37.57 | 41 | 32 |
Birth weight (g) | 4300 | 3900 | 3870 | 3750 | 2370 | 2230 | 3700 | 4080 | 1580 |
Complications during pregnancy | Uneventful | Uneventful | Uneventful | Uneventful | Uneventful | Uneventful | Uneventful | Uneventful | Developed acute hyperammonemia during pregnancy |
Family history | Positive | — | — | — | Positive | Positive | — | — | Positive |
Clinical course | Fulminant | Fulminant | Fulminant | Fulminant | Fulminant | Fulminant | Fulminant | Fulminant | Fulminant |
Clinical features | |||||||||
Seizures (HP:0001250) | Positive | Positive | Positive | Positive | Positive | Positive | — | — | — |
Somnolence (HP:0001262) | Positive | Positive | Positive | Positive | Positive | Positive | Positive | Positive | Positive |
Decreased liver function (HP:0001410) | Positive | — | — | — | — | — | Positive | — | — |
Acute encephalopathy (HP:0006846) | Positive | Positive | Positive | Positive | Positive | Positive | Positive | Positive | Positive |
Coma (HP:0001259) | — | No coma at the first episode, coma at the second episode | Positive | Positive | Positive | Positive | — | — | Positive |
Fever (HP:0001945) | — | Positive | Positive | — | — | — | Positive | — | — |
Hypothermia (HP:0002045) | — | — | — | — | Positive | Positive | — | Positive | Positive |
Cyanosis (HP:0000961) | — | — | — | — | Positive | — | — | — | — |
Neonatal breathing deregulation (HP:0002790) | — | — | Weak, need ventilation | Apnea, need ventilation | Apnea, need ventilation | Weak | — | Tachypnea | Apnea, need ventilation |
Internal hemorrhage (HP:0011029) | — | — | Cerebral | — | — | — | — | — | — |
Feeding difficulties (HP:0011968) | Positive | Positive | Positive | Positive | — | Positive | Positive | Positive | No enteral feeding after birth |
Abdominal distention (HP:0003270) | — | — | — | — | — | — | Positive | — | — |
Arterial blood gas analysis | |||||||||
pH (reference, 7.25-7.45) | 7.38 | 7.42 | 7.4 | 7.43 | 7.45 | 7.45 | 7.41 | 7.47 | 7.21 |
BE (reference, –3 to +3 mmol/L) | -1.8 | -4.8 | 0.1 | -1.3 | -11.4 | -5.9 | -2.1 | 3.8 | -10.1 |
Blood biochemical tests | |||||||||
ALT (reference, 0-38 U/L) | 125↑ | 34 | 25 | 25 | 23 | 5 | 107↑ | 45↑ | 21 |
Lactic acid (reference, 0.7-2.1 mmol/L) | 1.9 | 6.4 | 8.2 | 3 | 7.3 | 7 | 1.5 | 2.4 | 8 |
Glucose (reference, 3.3-6.1 mmol/L) | 4.2 | 5.7 | 2.4 | 3.5 | 1.1 | 3 | 4.8 | 3.8 | 2.9 |
Culture | — | — | — | — | — | — | Staphylococcus haemolyticus | — | — |
Ammonia (reference, 9-30 | |||||||||
Peak pretreatment ammonia level | 310↑ | First episode: 126↑; second episode: 290↑ | 1367↑ | 1030↑ | 704.3↑ | 228.6↑ | 311↑ | 1004↑ | 1020↑ |
Final assessment after treatment | 61↑ | First episode: N/A; second episode:219↑ | 1100↑ | 23 | 1258.4↑ | 450.2↑ | 169↑ | 885↑ | 1100↑ |
Blood mass spectrometry profile | |||||||||
Citrulline (reference, 5-40 | 5.953 | First episode: N/A; second episode: 6.112 | 6.261 | 2.71↓ | 3.15↓ | 3.04↓ | 8.776 | 1.58↓ | 27.43 |
Alanine (reference, 70-350 | 213.014 | First episode: N/A; second episode: 362.021↑ | 375.751↑ | 368.22↑ | 411.70↑ | 301.6 | 295.904 | 462.33↑ | 228.41 |
Glutamate (reference, 45-200 | 130.149 | First episode: N/A; second episode: 213.893↑ | 404.649↑ | 652.67↑ | 265.41↑ | 195.73 | 263.168↑ | 185.21 | 145.66 |
Glutamine (reference, 2-35 | 14.047 | First episode: N/A; second episode: 58.012↑ | 97.444↑ | 1577.35↑ | 156.6↑ | 100.62↑ | 14.751↑ | 1104.41↑ | 61.602↑ |
Ornithine (reference, 20-160 | 36.996 | First episode: N/A; second episode: 100.351 | 122.83 | 38.5 | 29.17 | 31.98 | 41.037 | 54.43 | 161.66↑ |
Arginine (reference, 3-50 | 7.606 | First episode: N/A; second episode: 21.207 | 91.894↑ | 6.12 | 20.14 | 9.07 | 20.12 | 5.26 | 97.43↑ |
Urinary organic acids | |||||||||
Urinary orotic acid (reference, 0-2 mmol/L) | Absent | First episode: N/A; second episode: 357.21↑ | 641.56↑ | 352.13↑ | 80.5↑ | 12.5↑ | 0.15 | 0.41 | 1372.66↑ |
Urinary uracil (reference, 0-8 mmol/L) | 4.76 | First episode: N/A; second episode: 35.12↑ | 46.34↑ | 32.03↑ | Absent | 16.3↑ | 0.24 | Absent | 67.11↑ |
Chest X-ray | |||||||||
Pneumonia | — | — | Positive | — | Positive | — | — | — | — |
Pneumorrhagia | — | — | — | — | — | — | — | — | — |
Echocardiography | N/A | N/A | N/A | N/A | N/A | N/A | |||
Ejection fraction | 62% | 59% | 64% | ||||||
Patent ductus arteriosus | Positive | — | — | ||||||
Decision making | — | — | Give up | — | — | Give up | — | Give up | Give up |
↑: elevated, ↓: decreased. N/A: not mentioned.
Five patients (P3, P5, P6, P8, and P9) appeared normal at birth but progressed to seizures or coma within one to three days of age in hospitals. In particular, one preterm boy (P9) presented with tachypnea one hour after birth, deteriorated rapidly, and died at 3 days of age. His mother was a heterozygote for the
The biochemical analysis of each patient is summarized in Table
A total of 10 episodes of acute hyperammonemia were derived from our study. Eight patients (P1, P3, P4, P5, P6, P7, P8, and P9) had one hyperammonemia episode, whereas a male OTCD (P2) had two before 30 days of age. Plasma ammonia level varied among our patients, with the mean value of
Plasma amino acid and urine organic acid analyses were performed in all these patients. Metabolomic finding of OTCD subjects (Table
A definitive molecular diagnosis for every patient in our cohort is provided in Table
Molecular profiles of 9 neonatal-onset UCD patients.
Patient number | Molecular diagnostic technology | Family member tests | Gene | Nucleotide aberration | Amino acid change | Molecular diagnosis | Inheritance pattern | Zygosity | Parent of origin |
---|---|---|---|---|---|---|---|---|---|
P1 | TES panel | Trio+1 brother | OTC (NM_000531.6) | c.119G>A | p.R40H | Ornithine transcarbamylase deficiency (MIM:311250) | XR | Hemi | Inherited/mother |
P2 | TES panel | Trio | OTC (NM_000531.6) | c.540G>C | p.Q180H | Ornithine transcarbamylase deficiency (MIM:311250) | XR | Hemi | Inherited/mother |
P3 | TES panel | Trio | OTC (NM_000531.6) | p.L59P | Ornithine transcarbamylase deficiency (MIM:311250) | XR | Hemi | Inherited/mother | |
P4 | WES | Trio | OTC (NM_000531.6) | c.803T>C | p.M268T | Ornithine transcarbamylase deficiency (MIM:311250) | XR | Hemi | Inherited/mother |
P5 | WES | Trio+1 sib | OTC (NM_000531.5) | c.626C>T | p.A209V | Ornithine transcarbamylase deficiency (MIM:311250) | XR | Hemi | Inherited/mother |
P6 | WES | Trio+1 sib | OTC (NM_000531.5) | c.626C>T | p.A209V | Ornithine transcarbamylase deficiency (MIM:311250) | XR | Hemi | Inherited/mother |
P7 | WES | Trio | CPS1 (NM_001875.4) | c.2162G>A; | p.R721Q; p.G980S | Carbamoylphosphate synthetase I deficiency (MIM:237300) | AR | Het | Inherited/father+mother |
P8 | WES | Trio | CPS1 (NM_001875.5) | c.3784C>T; | p.R1262 | Carbamoylphosphate synthetase I deficiency (MIM:237300) | AR | Het | Inherited/father+mother |
P9 | TES panel | Trio | OTC (NM_000531.5) | c.583G>A | p.G195R | Ornithine transcarbamylase deficiency (MIM:311250) | XR | Hemi | Inherited/mother |
AR: autosomal recessive inheritance disease; XR: X-linked recessive inheritance disease; Het: heterozygous; Hemi: hemizygous; hom: homozygous; TES: targeted exome sequencing; WES: whole-exome sequencing. Italicized variants were unreported previously.
A total of 7 male patients (P1, P2, P3, P4, P5, P6, and P9) belonging to 6 families were diagnosed with OTCD. Molecular analysis revealed 6 different hemizygotes, all of which were inherited from their mother (Table
Pathogenicity analyses of (a) c.176T>C (p.L59P), (b) c.2938G>A (p.G980S), and (c) c.3734T>A (p.L1245H) by PolyPhen-2.
In silico analysis of (a) p.L59P, (b) p.G980S, and (c) p. L1245H in different species by ClustalX.
3D structure of wild type and mutant type of (a) p.L59P in OTC, (b) p.G980S in CPS1, and (c) p.L1245H in CPS1.
In this study, our NGS assays reported two patients (P7 and P8) carrying two compound heterozygous variants of c.2162G>A (p.R721Q)/c.2938G>A (p.G980S) and c.3734T>A (p.L1245H)/c.3784C>T (p.R1262
During a 180-day follow-up interval, four patients (P1, P2, P4, and P8) in our cohort survived the neonatal period. At the end of the 6 months of age, 2 of the initially surviving patients (P1 and P2) died (Table
There were 4 episodes (P1, the first episode in P2, P7, and P8) of hyperammonemia without coma at the time of admission, and coma was also absent at the time of discharge in majority of them (P1, P2, and P7) (75.00%, 3/4). But three of them (P1, P2, and P8) died before 6 months of age (75.00%, 3/4). However, we found that the patients (the second episode in P2, P3, P4, P5, P6, and P9) were comatose at admission and no coma was present at the time of discharge in only one of them (P4). Five (P2, P3, P5, P6, and P9) died (83.33%, 5/6), all diagnosed with OTCD (Tables
Among 7 patients (P1, P2, P3, P5, P6, P8, and P9) who died, the following severe manifestations/complications of acute hyperammonemia were common, including seizures in 6 patients (P1, P2, P3, P4, P5, and P6), respiratory distress or failure in 5 (P3, P4, P5, P6, and P9), fever in 2 (P2 and P3), liver damage in 2 (P1 and P8), hyperventilation in 1 (P8), and cerebral hemorrhage in 1 patient (P3) individually (Table
In our cohort, we found that four out of five patients (P3, P4, P5, P8, and P9) died, whose peak ammonium level was greater than 500
Eight patients (P1, P3, P4, P5, P6, P7, P8, and P9) were initiated with protein restriction as soon as the onset of the first known hyperammonemia episode occurred, and only one patient (P2) continued protein intake till his second hyperammonemia episode. Six patients (P1, P2, P4, P7, P8, and P9) were treated with intravenous arginine infusion (100–150 mg/kg/day). The provision of adequate calorie was provided for all patients.
Six males (6/7, 85.71%) with OTCD (P1, P2, P3, P5, P6, and P9) died because of hyperammonemic crisis in the neonatal period or after. Among them, three preterm patients (P5, P6, and P9) did not survive the first hyperammonemic episode and died in their first week of life, whereas the others (P1, P2, and P3) died at an older age. Among patients who died, median ammonium levels were similar at baseline (a baseline ammonium level: the last value recorded before treatment) (
A full-term OTC-deficient male (P4) survived all known episodes in our follow-up interval. He presented with serious hyperammonemic encephalopathy at the age of 6 days. His ammonium level remained above 1000
A CPS1-deficient female (P7) was in clinical remission until now. She presented with a sepsis and liver damage besides a high ammonium level at admission. She was provided with a combination of oral sodium benzoate (250 mg/kg/day) and intravenous arginine infusion (150 mg/kg/day) as an alternative pathway therapy immediately after her admission, resulting in a near-normal ammonium level and clinical improvement.
In this study, we presented a detailed clinical course and genetic analysis of nine neonatal Chinese UCD cases. And our results showed that despite early diagnosis and treatment [
Nine patients developed various symptoms within the first 7 days of life. The majority of this cohort (88.89%, 8/9) (P1, P2, P3, P4, P5, P6, P7, and P8) appeared to be healthy at birth except a premature male with OTCD (P9) who manifested tachypnea immediately after birth, whose mother had severe hyperammonemia during pregnancy. Acute encephalopathy and feeding problems (poor feeding in P1, P2, P3, P4, P6, P7, and P8 and no enteral feeding after birth in P5 and P9) were observed in all our patients, followed by breathing problems at the time of hospitalization. In the present study, phenotypic homogeneity of the same mutation was observed. The two male siblings (P5 and P6) with the same mutation in the
Once a UCD is suspected, the diagnosis should be confirmed rapidly by a specialized metabolic laboratory, such as blood ammonia, blood amino acids, and urine organic acids. In this study, hyperammonemia of different levels was observed in all patients before the first week after birth. However, the concurrent low plasma citrulline was found only in three OTCD patients (P4, P5, and P6). The low level of urinary orotic acid was observed in one CPS1D patient (P7). So we cannot distinguish from different types of UCD due to similar intermediary metabolites or normal levels of some amino acids by mass spectrometry.
Molecular genetic testing is the primary method of diagnostic confirmation for all UCDs. As the use of NGS becomes standard practice, we expect that disease-causing mutations will be rapidly found in neonates who have severe hyperammonemia. It is reported that conventional Sanger sequencing cannot detect variants in about ~20% of OTCD patients [
Finally, our results reflected the poor prognosis for neonatal-onset UCDs in China, compared with other populations [
In this study, we presented the detailed clinical features and genetic analysis of nine patients with neonatal-onset UCDs with poor 180-day-age outcomes and discovered three novel pathogenic variants in one OTCD and two CPS1D by NGS. Our results demonstrated that although biochemical phenotypes during the neonatal period were better than clinical profiles to predict OTCD, a molecular analysis was the gold standard to confirm the diagnosis, indicating that it is indispensable to expand NBS by MS/MS for some UCDs.
The data used to support the findings of this study are included within the article.
The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article.
Tianwen Zhu was responsible for the conceptualization, methodology, formal analysis, writing, reviewing, and editing. Qingnv Zhou, Huafei Huang, Li Ma, and Tianwen Zhu were responsible for investigation and data curation. Qingnv Zhou and Huafei Huang were responsible for writing—original draft preparation.
We express our deep gratitude to the patients and the families for their willingness and cooperation in the study.
Supplementary Methods: a section about the list of a specific neonatal metabolism disease 175-gene panel (designed by MyGenostics, Beijing, China) for 4 patients in our cohort.