Case: patient #10, Male, Argentine

Disease Assertion: UCD/OTCD

Family Info: family history of the disease,

Case Presenting HPOs: Neonatal onset(HP:0003623), Hyperammonemia HP:0001987

Case HPO FreeText:

Case NOT HPOs:

Case NOT HPO Free Text:

Case Previous Testing: The OTC gene mutations were identified using PCR amplification, classical sequencing (Sanger), and multiplex ligation-dependent probe amplification.10,11 Mutations were identified by comparison with the GenBank reference sequence for human OTC (GenBank entries: NG_008471.1, NP 000522.3, NM 000531.5, NC 000023.11) Missense mutations were analyzed using different computational algorithms: CLUSTALW2 (http://www.clustal.org/clustal2/), SIFT (http://blocks.fhcrc.org/sift/SIFT.html),Polyphen2(http://genetics.bwh.harvard.edu/pph/),PoPMuSiC(http://babylone.ulb.ac.be/popmusic/), and SIFT Indel(http://siftdna.org/www/SIFT_indels2.html).

Supplemental Data: Table 1 Notes: died at 6 months and had 2 brothers that died a neonatal stage

Variant: NM_000531.6: c.540+1G>A

ClinVarID: 1458773

CAID: CA412724226

gnomAD: X-38381340-A-T

Gene Name: OTC (ornithine transcarbamylase)

Case: patient#5 , female, Italian

Disease Assertion: UCD/OTCD

Family Info:

Case Presenting HPOs: irritability(HP:0000737), lethargy(HP:0001254), vomiting(HP:0002013), Oriticaciduria (HP:0003218), low plasma citrulline (HP:0003572), Elevated circulating alanine aminotransferase concentration(HP:0031964), Elevated circulating aspartate aminotransferase concentration (HP:0031956), childhood onset (HP:0011463)

Case HPO FreeText:

Case NOT HPOs:

Case NOT HPO Free Text:

Case Previous Testing: Total RNA was isolated from peripheral blood lymphocytes or lymphoblastoid cell lines and from frozen liver biopsy as described in Chomczynski and Sacchi (1987). For each patient two cDNA syntheses were performed: 10mg of total RNA with 800-1000 ng of oligo dT or 500ng of specific primer NR, mapping in the 3’UTR of OTC cDNA Identification of genetic lesions by amplification of the OTC mRNA, expressed in the liver tissue and intestine, from a non-specific tissue like PBL or lymphoblastoid cell lines. Some mutations, particularly those affecting splicing sites, may have a different expression in liver and PBL . In females, including manifesting carriers, this method allows the identification of deletions and gene rearrangements with certainty, but mutations, decreasing mRNA stability, are unlikely to be detected because the normal allele will constitute the majority of the RNA available for RT-PCR and will be preferentially amplified.

Supplemental Data: Case report section Notes: Hepatomegaly. This mutation, previously reported (Reish et al., 1993), has been correlated with a lethal disease form in a male patient, therefore the mild phenotype in our patient could be explained by a not completely unfavorable X-lyonization

Variant: NM_000531.6: c.928G>T(p.Glu310*)

ClinVarID: 97361

CAID: CA224838

gnomAD:

Gene Name: OTC (ornithine transcarbamylase)

Case: 24 yo Laotse patient , female

Disease Assertion: UCD/OTCD

Family Info:

Case Presenting HPOs:Hyperammonemia(HP:0001987), Adult onset (HP:0003581), Elevated circulating alanine aminotransferase concentration(HP:0031964), Elevated circulating aspartate aminotransferase concentration(HP:0031956), oriticaciduria(HP:0003218), Aminoaciduria(HP:0003355, Prolonged prothrombin time(HP:0008151), Cerebral edema(HP:0002181),

Case HPO FreeText:

Case NOT HPOs:

Case NOT HPO Free Text:

Case Previous Testing: " Genomic DNA from the patient was isolated from cultivated fibroblasts. Nine pairs of primers were designed from the published sequence of the OTC gene6,7 to allow amplification of all 10 exons including adjacent intron sequences. Single-strand conformational polymorphism analysis of the polymerase chain reaction–amplified individual exon 8 yielded an unusual migration pattern of exon 9

Supplemental Data: Case report section Notes: Patient had a pregnancy 2 years prior and lost the baby. In this case report, She was a 14weeks pregnant(2nd pregnancy) 24yo . She died during this of severe hyperammonemia 5 days after being administered amino acids through parenteral nutrition. She developed signs of encephalopathy stage 4 with maximal dilated unresponsive pupils, brisk oculocephalic reflex, and severe hyperventilation, requiring mechanical ventilation. MRI revealed revealed diffuse cortical edema with loss of white to gray matter distinction. Increased excretion of Gly, Gln, Ser, Thr, and Lys was found in her urine. Treatment with benzoate was started but didn't save the patient.

Variant: NM_000531.6: c.892_893del(p.Trp298Aspfs*15)

ClinVarID: 97348

CAID: CA224820

gnomAD:

Gene Name: OTC (ornithine transcarbamylase)

Case: patient #11, female, 35yo, slovenian

Disease Assertion: UCD/OTCD

Family Info: family history of the disease,

Case Presenting HPOs: Adult onset(HP:0003581), hyperammonemia(HP:0001987), protein avoidance (HP:0002038)

Case HPO FreeText:

Case NOT HPOs:

Case NOT HPO Free Text:

Case Previous Testing: Sequencing was performed at a 3rd-party sequencing center using a standardized seq of procedures following PCR-free WGS library preparation protocol Illumina TrueSeq DNA Nano and sequenced on Illumina NovaSeq 6000 platform with a mean autosomal depth greater than 30×. Variants were interpreted by a medical doctor specialized in the NGS sequencing data analysis and those classified as likely pathogenic or pathogenic according to the ACMG/AMP standards and guidelines were considered for reporting, while variants of uncertain clinical significance, were not considered. Likely pathogenic and pathogenic variants were further evaluated by the referring clinical geneticist and were considered and reported if they were classified as both a likely diagnostic finding and if they were compatible with the clinical presentation of referral.

Supplemental Data: Table 4 and section 3.1(Case description) Notes:

Variant: NM_000531.6: c.540+265G>A

ClinVarID: 449382

CAID: CA658658977

gnomAD:

Gene Name: OTC (ornithine transcarbamylase)

Case: patient #113, Male

Disease Assertion: UCD/OTCD

Family Info:

Case Presenting HPOs: Neonatal onset(HP:0003623), Hyperammonemia HP:0001987

Case HPO FreeText:

Case NOT HPOs:

Case NOT HPO Free Text:

Case Previous Testing: GDNA from blood, cultured skin fibroblasts, liver from patients suspected for otc deficiency was used to amplify all 10 exons and exon/intron boundaries using primers listed in Table 1. The amplified DNA fragments were then screened by single-strand conformational polymorphism (SSCP) and the abnormally migrating DNA fragments were sequenced directly from PCR products (w/o subcloning) to identify the mutation. The amino acid residue substitution created by the mutation is examined using an alignment of 26 OTCase sequences from 23 species.

Supplemental Data: Table 4 Notes:

Variant: NM_000531.6: c.867+1G>A

ClinVarID: 97342

CAID: CA224813

gnomAD:

Gene Name: OTC (ornithine transcarbamylase)

Case: patient #1, female Disease Assertion: UCD/OTCD

Family Info:

Case Presenting HPOs: Hyperammonemia (HP:0001987), Juvenile onset (HP:0003621)

Case HPO FreeText:

Case NOT HPOs:

Case NOT HPO Free Text:

Case Previous Testing: "DNA samples were obtained from either peripheral white blood cells or liver biopsies of probands. Patient 1 total liver RNAs were extracted by the acid-phenol-guanidium method and reverse-transcribed in cDNA by hexanucleotide priming using Superscript 11' (Life Technologies, Cergy-Pantoia, France). First strand cDNA was further PCR-amplified using forward and reverse primers specificons 7 and 9 of the OTC gene, respectively. Sequence comparisons were conducted using the FASTA option of the BISANCE package. Secondary protein conformational changes induced by sequence mutations were predicted using the algorithms of the BISANCE package. Nucleotidic changes potentially altering splice sites were studied using the Senapathy's algorithm from the BISANCE package."

Supplemental Data: TABLE 1, Notes:

Variant: NM_000531.6: c.731_739del(p.Leu244Profs)

ClinVarID: 97307

CAID: CA224765

gnomAD:

Gene Name: OTC (ornithine transcarbamylase)

Case: patient #273, Male

Disease Assertion: UCD/OTCD

Family Info: NA

Case Presenting HPOs: Neonatal onset (HP:0003623)

Case HPO FreeText:

Case NOT HPOs:

Case NOT HPO Free Text:

Case Previous Testing: NA

Supplemental Data: Supplemental table file Notes:

Variant: NM_000531.6: c.818del (p.Glu273Glyfs*16)

ClinVarID: 97338

CAID: CA224807

gnomAD:

Gene Name: OTC (ornithine transcarbamylase)

Case: patient MW

Disease Assertion: UCD/OTCD

Family Info: NA

Case Presenting HPOs: NA

Case HPO FreeText:

Case NOT HPOs:

Case NOT HPO Free Text:

Case Previous Testing: NA

Supplemental Data: NA Notes: mutation might cause exon 8 kipping durring splicing.

Variant: NM_000531.6: c.718-2A>G

ClinVarID: 97303

CAID: CA224761

gnomAD:

Gene Name: OTC (ornithine transcarbamylase)

Case: no patient #ID, female Disease Assertion: UCD/OTCD

Family Info: NA

Case Presenting HPOs: NA

Case HPO FreeText:

Case NOT HPOs:

Case NOT HPO Free Text:

Case Previous Testing: NA

Supplemental Data: TABLE 1, Notes: NA

Variant: NM_000531.6: c.665del(p.(Gly222ValfsTer8)

ClinVarID: 97289

CAID: CA224738

gnomAD:

Gene Name: OTC (ornithine transcarbamylase)

Case: no patient #ID, female Disease Assertion: UCD/OTCD

Family Info: NA

Case Presenting HPOs: NA

Case HPO FreeText:

Case NOT HPOs:

Case NOT HPO Free Text:

Case Previous Testing: NA

Supplemental Data: TABLE 1, Notes: NA

Variant: NM_000531.6: c.663+2T>C

ClinVarID: 97285

CAID: CA224732

gnomAD:

Gene Name: OTC (ornithine transcarbamylase)

Case: no patient #ID, male Disease Assertion: UCD/OTCD

Family Info: NA

Case Presenting HPOs: NA

Case HPO FreeText:

Case NOT HPOs:

Case NOT HPO Free Text:

Case Previous Testing: NA

Supplemental Data: TABLE 1, Notes: NA

Variant: NM_000531.6: c.663+1G>A

ClinVarID: 97283

CAID: CA224730

gnomAD:

Gene Name: OTC (ornithine transcarbamylase)

Case: no patient #ID, female Disease Assertion: UCD/OTCD

Family Info: NA

Case Presenting HPOs: NA

Case HPO FreeText:

Case NOT HPOs:

Case NOT HPO Free Text:

Case Previous Testing: NA

Supplemental Data: TABLE 1, Notes: NA

Variant: NM_000531.6: c.77+1G>T

ClinVarID: 97314

CAID: CA224774

gnomAD:

Gene Name: OTC (ornithine transcarbamylase)

Case: patient #1, male, turkish

Disease Assertion: UCD/OTCD

Family Info: Family history of the disease, Variant found in mother of the patient

Case Presenting HPOs: infantile onset (HP:0003593), coma(HP:0001259), episodic hyperammonemia(HP:0001951), oriticaciduria(HP:0003218)

Case HPO FreeText:

Case NOT HPOs:

Case NOT HPO Free Text:

Case Previous Testing: Liver tissues were used to extract RNA that was later used to synthesize cDNA. The products were compared to healthy controls in order to detect variants. gDNA, in order to determine the size of deletions in patient 3 and 4 , a set of intronic primers presumably flanking the deletions was used and specific primers allowed sequencing of exactly those critical regions(sequencing on paper). To estimate the relevance of the identified intronic variants in terms of their capability to induce splicing, we used a score developed by Shapiro and Senapathy. This splice score offers information about the usage of a certain splice site

Supplemental Data: TABLE 1, Notes: very mild movement disorder, the diagnosis was prenatal so measures were taken from birth,. 2 biopsies were performed and the revealed respectively a 30% and 50 % decrease on OTC activity.

Variant: NM_000531.6: c.867+1126A>G

ClinVarID: 571311

CAID: CA891843643

gnomAD:

Gene Name: OTC (ornithine transcarbamylase)

Case: patient #2, male, Saudi Arabian

Disease Assertion: UCD/OTCD

Family Info: Family history of the disease, Variant found in mother of the patient, Brother died of hyperammonemic crisis

Case Presenting HPOs: intellectual disability (HP:0001249), Neonatal onset (HP:0003623), seizure(HP:0001250), episodic hyperammonemia(HP:0001951), intellectual disability (HP:0001249)

Case HPO FreeText: hyperammonemic encephalopathy

Case NOT HPOs:

Case NOT HPO Free Text:

Case Previous Testing: Liver tissues were used to extract RNA that was later used to synthesize cDNA. The products were compared to healthy controls in order to detect variants. gDNA, in order to determine the size of deletions in patient 3 and 4 , a set of intronic primers presumably flanking the deletions was used and specific primers allowed sequencing of exactly those critical regions(sequencing on paper). To estimate the relevance of the identified intronic variants in terms of their capability to induce splicing, we used a score developed by Shapiro and Senapathy. This splice score offers information about the usage of a certain splice site

Supplemental Data: TABLE 1, Patient was severely mentally retarded after the age of 2. Low OTC activity

Variant: NM_000531.6: c.540+265G>A(p.Gln180_Glu181insX4)

ClinVarID: 449382

CAID: CA658658977

gnomAD:

Gene Name: OTC (ornithine transcarbamylase)

Case: no patient ID#, 36yo donor , female

Disease Assertion: UCD/OTCD

Family Info:

Case Presenting HPOs: adult onset (HP:0003581), oriticaciduria (HP:0003218), irritability (HP:0000737), protein avoidance

Case HPO FreeText: hyperammonemic encephalopathy

Case NOT HPOs:

Case NOT HPO Free Text:

Case Previous Testing: N/A

Supplemental Data: TABLE 1, She is vegeterian. The symptoms of OTCD started showing after the patient donated 60% of liver to her sibling. the information reported in this is the biochemical results during hyperammonemic episode following the transplantation. the patient became irritable and confused, and her level of consciousness deteriorated markedly. After hemodialysis the patient recovered.

Variant: NM_000531.6: c.429T>A(p.Tyr143*)

ClinVarID: 1072591

CAID: CA412723166

gnomAD:

Gene Name: OTC (ornithine transcarbamylase)

Case: patient #573, male

Disease Assertion: UCD/OTCD

Family Info:

Case Presenting HPOs:hyperammonemia (HP:0001987), Neonatal onset (HP:0003623)

Case HPO FreeText:

Case NOT HPOs:

Case NOT HPO Free Text:

Case Previous Testing: GDNA extracted from blood leukocytes using the proteinase K/phenol extraction procedure on a model 340 A nucleic acid extractor (Applied Biosystems). 5mg samples of DNA were digested with BamHI, MspI, or TaqI restriction endonuclease, electrophoresed through 1 % agarose gels, and transferred to a nylon membrane by standard procedures. The blots were then hybridized with a radiolabeled full-length cDNA probe for human OTC.

Supplemental Data: TABLE 3,

Variant: NM_000531.6: c.67C>T(p.Arg23*)

ClinVarID:97292

CAID: CA224742

gnomAD:

Gene Name: OTC (ornithine transcarbamylase)

Case: patient SM, male

Disease Assertion: UCD/OTCD

Family Info:

Case Presenting HPOs: Hyperammonemia (HP:0001987), Neonatal onset (HP:0003623)

Case HPO FreeText:

Case NOT HPOs:

Case NOT HPO Free Text:

Case Previous Testing: GDNA extracted from blood leukocytes using the proteinase K/phenol extraction procedure on a model 340 A nucleic acid extractor (Applied Biosystems). 5mg samples of DNA were digested with BamHI, MspI, or TaqI restriction endonuclease, electrophoresed through 1 % agarose gels, and transferred to a nylon membrane by standard procedures. The blots were then hybridized with a radiolabeled full-length cDNA probe for human OTC.

Supplemental Data: TABLE 3,

Variant: NM_000531.6: c.274C>T(p.Arg92*)

ClinVarID:97151

CAID: CA224530

gnomAD:

Gene Name: OTC (ornithine transcarbamylase)

Case: patient #5, female

Disease Assertion: UCD/OTCD

Family Info: N/A

Case Presenting HPOs: Neonatal onset (HP:0003623)

Case HPO FreeText:

Case NOT HPOs:

Case NOT HPO Free Text:

Case Previous Testing: gDNA testing involves PCR amplification of all 10 exons and exon/intron boundaries followed by screening for mutations or sequencing of all fragments, For these patients, confirmation of the diagnosis requires enzymatic assays. No specifications about the test

Supplemental Data: TABLE 1

Variant: NM_000531.6: c.53del (p.His18Profs*20)

ClinVarID: 97239

CAID: CA224671

gnomAD:

Gene Name: OTC (ornithine transcarbamylase)

Case: patient #154, male

Disease Assertion: UCD/OTCD

Family Info: N/A

Case Presenting HPOs: Neonatal onset (HP:0003623)

Case HPO FreeText:

Case NOT HPOs:

Case NOT HPO Free Text:

Case Previous Testing: gDNA testing involves PCR amplification of all 10 exons and exon/intron boundaries followed by screening for mutations or sequencing of all fragments, For these patients, confirmation of the diagnosis requires enzymatic assays. No specifications about the test

Supplemental Data: TABLE 1

Variant: NM_000531.6: c.664-1G>A

ClinVarID: 97288

CAID: CA224811

gnomAD:

Gene Name: OTC (ornithine transcarbamylase)

Case: patient #188, male

Disease Assertion: UCD/OTCD

Family Info: N/A

Case Presenting HPOs: Neonatal onset (HP:0003623)

Case HPO FreeText:

Case NOT HPOs:

Case NOT HPO Free Text:

Case Previous Testing: gDNA testing involves PCR amplification of all 10 exons and exon/intron boundaries followed by screening for mutations or sequencing of all fragments, For these patients, confirmation of the diagnosis requires enzymatic assays. No specifications about the test

Supplemental Data: TABLE 1

Variant: NM_000531.6: c.835C>T(p.Gln279*)

ClinVarID: 97341

CAID: CA224811

gnomAD:

Gene Name: OTC (ornithine transcarbamylase)

Case: patient #213, male

Disease Assertion: UCD/OTCD

Family Info: N/A

Case Presenting HPOs: Neonatal onset (HP:0003623)

Case HPO FreeText:

Case NOT HPOs:

Case NOT HPO Free Text:

Case Previous Testing: gDNA testing involves PCR amplification of all 10 exons and exon/intron boundaries followed by screening for mutations or sequencing of all fragments, For these patients, confirmation of the diagnosis requires enzymatic assays. No specifications about the test

Supplemental Data: TABLE 1

Variant: NM_000531.6: c.962C>A(p.Ser321*)

ClinVarID: 97373

CAID: CA224859

gnomAD:

Gene Name: OTC (ornithine transcarbamylase)

Case: patient #220, male

Disease Assertion: UCD/OTCD

Family Info: N/A

Case Presenting HPOs: Neonatal onset (HP:0003623)

Case HPO FreeText:

Case NOT HPOs:

Case NOT HPO Free Text:

Case Previous Testing: gDNA testing involves PCR amplification of all 10 exons and exon/intron boundaries followed by screening for mutations or sequencing of all fragments, For these patients, confirmation of the diagnosis requires enzymatic assays. No specifications about the test

Supplemental Data: Table 1

Variant: NM_000531.6: c.1005+2T>C

ClinVarID: 97090

CAID: CA224431

gnomAD:

Gene Name: OTC (ornithine transcarbamylase)

Case: patient #303, female

Disease Assertion: UCD/OTCD

Family Info: N/A

Case Presenting HPOs:

Case HPO FreeText:

Case NOT HPOs:

Case NOT HPO Free Text:

Case Previous Testing: No specific functional tests indicated

Supplemental Data: In supplemental data files

Variant: NM_000531.6: c.766G>T(p.256Gly*)

ClinVarID: 870326

CAID: CA412722685

gnomAD:

Gene Name: OTC (ornithine transcarbamylase)

Case: patient #16, female, Japanese

Disease Assertion: UCD/OTCD

Family Info: N/A

Case Presenting HPOs:

Case HPO FreeText:

Case NOT HPOs:

Case NOT HPO Free Text:

Case Previous Testing: The severity of missense mutations was assessed using conservation and solvent accessible area of the replaced amino acid, calculated destabilization of mutant proteins and their SIFT and PolyPhen2 scores

Supplemental Data: Kido_et_al_2022_fgene_Data Sheet 2

Variant: NM_000531.6:c.77+1G>T

ClinVarID: 97314

CAID: CA224774

gnomAD:

Gene Name: OTC (ornithine transcarbamylase)

Case: patient #55, male, Japanese

Disease Assertion: UCD/OTCD

Family Info: N/A

Case Presenting HPOs: Seizure (HP:0001250), Hyperammonemia (HP:0001987), Intellectual disability (HP:0001249)

Case HPO FreeText: abnormal brain MRI and brain waves, acute liver failure, corneal opacity

Case NOT HPOs:

Case NOT HPO Free Text:

Case Previous Testing: The mRNA ref seq were, wherein the “A” nucleotide of the start codon ATG constituted as +1 numbering of the cDNA sequence. Met encoded by the start codon ATG also represented +1 for the amino acid numbering as set forth by the preprotein seq. PolyPhen-2, SIFT, and I-Mutant 3 tools were used for predicting the potential impact of an amino acid alteration in missense mutations on the function of each enzyme.

Supplemental Data: Table 1

Variant: NM_000531.6:c.c.929_931del(p.Glu310Valfs*45)

ClinVarID: 858012

CAID: CA916083888

gnomAD:

Gene Name: OTC (ornithine transcarbamylase)

Case: no patient ID, Female

Disease Assertion: UCD/OTCD

Family Info: N/A

Case Presenting HPOs: HP:0001951, HP: 0001987

Case HPO FreeText: episodic hyperammonemia, hyperammonemia

Case NOT HPOs:

Case NOT HPO Free Text:

Case Previous Testing: "Genomic DNAs were extracted from leukocytes, The ten exons and intron-exon boundaries of the OTC gene were PCR amplified and analyzed by Sanger sequencing on an ABI3100 sequencer. Intragenic deletions/duplications were searched for by Multiple Ligation Probe Dependent Amplification assay. Potential impact of non truncating variants on mRNA and protein was predicted using Splice Site Prediction. OTC variants were split into two groups, “severe” and “mild,” based on their impact on the clinical phenotype and on the OTC protein.

Supplemental Data: Table 3, All nuclear family members were tested but no information about their genotype. the condition to be part of this study was the presence of at least one heterozygous female in the pedigree of the patient.

Variant: NM_000531.6:c.1052delA(p.Lys351Serfs*44)

ClinVarID: 915468

CAID: CA1139667400

gnomAD:

Gene Name: OTC (ornithine transcarbamylase)

Case: no patient ID, Female

Disease Assertion: UCD/OTCD

Family Info: N/A

Case Presenting HPOs: HP:0003623, HP: 0001987

Case HPO FreeText: Neonatal onset, hyperammonemia

Case NOT HPOs:

Case NOT HPO Free Text:

Case Previous Testing: "Genomic DNAs were extracted from leukocytes, The ten exons and intron-exon boundaries of the OTC gene were PCR amplified and analyzed by Sanger sequencing on an ABI3100 sequencer. Intragenic deletions/duplications were searched for by Multiple Ligation Probe Dependent Amplification assay. Potential impact of non truncating variants on mRNA and protein was predicted using Splice Site Prediction. OTC variants were split into two groups, “severe” and “mild,” based on their impact on the clinical phenotype and on the OTC protein.

Supplemental Data: Table 3, All nuclear family members were tested but no information about their genotype. the condition to be part of this study was the presence of at least one heterozygous female in the pedigree of the patient.

Variant: NM_000531.6:c.766G>T(p.Gly256*)

ClinVarID: 870326

CAID: CA412722685

gnomAD:

Gene Name: OTC (ornithine transcarbamylase)

Case: no patient ID, Female

Disease Assertion: UCD/OTCD

Family Info: N/A

Case Presenting HPOs: HP:0001951, HP: 0001987

Case HPO FreeText: episodic hyperammonemia, hyperammonemia

Case NOT HPOs:

Case NOT HPO Free Text:

Case Previous Testing: "Genomic DNAs were extracted from leukocytes, The ten exons and intron-exon boundaries of the OTC gene were PCR amplified and analyzed by Sanger sequencing on an ABI3100 sequencer. Intragenic deletions/duplications were searched for by Multiple Ligation Probe Dependent Amplification assay. Potential impact of non truncating variants on mRNA and protein was predicted using Splice Site Prediction. OTC variants were split into two groups, “severe” and “mild,” based on their impact on the clinical phenotype and on the OTC protein.

Supplemental Data: Table 3, All nuclear family members were tested but no information about their genotype. the condition to be part of this study was the presence of at least one heterozygous female in the pedigree of the patient.

Variant: NM_000531.6:c.568dupA(p.Thr190Asnfs*35)

ClinVarID: 870328

CAID: CA916083887

gnomAD:

Gene Name: OTC (ornithine transcarbamylase)

Case: no patient ID, male

Disease Assertion: UCD/OTCD

Family Info: N/A

Case Presenting HPOs: HP:0003623, HP: 0001987

Case HPO FreeText: Neonatal onset, hyperammonemia

Case NOT HPOs:

Case NOT HPO Free Text:

Case Previous Testing: "Genomic DNAs were extracted from leukocytes, The ten exons and intron-exon boundaries of the OTC gene were PCR amplified and analyzed by Sanger sequencing on an ABI3100 sequencer. Intragenic deletions/duplications were searched for by Multiple Ligation Probe Dependent Amplification assay. Potential impact of non truncating variants on mRNA and protein was predicted using Splice Site Prediction. OTC variants were split into two groups, “severe” and “mild,” based on their impact on the clinical phenotype and on the OTC protein.

Supplemental Data: Table 3, All nuclear family members were tested but no information about their genotype. the condition to be part of this study was the presence of at least one heterozygous female in the pedigree of the patient.

Variant: NM_000531.6:c.217-2A>G(IVS2)

ClinVarID: 915470

CAID: CA412716751

gnomAD:

Gene Name: OTC (ornithine transcarbamylase)

Case: Patient #34, female, Korean

Disease Assertion: UCD/OTCD

Family Info: N/A

Case Presenting HPOs: HP:0011463, HP:0003218, HP: 0001987

Case HPO FreeText: childhood onset, oroticaciduria, hyperammonemia

Case NOT HPOs:

Case NOT HPO Free Text:

Case Previous Testing: "Potential impact of mutations on OTC function and/or folding assessed by multiple alignments of orthologous protein sequences and human OTC and structural data from Protein Data Bank (1C9Y and available orthologs). In M patients, the approximate extent of the deletions assessed by inspection of presence/absence of PCR products. In F patients, the deletions determined by the SALSA multiplex ligation probe amplification (MLPA) KIT P079 OTC (MRC-Holland, Amsterdam, the Netherlands) and the Affymetrix Human SNP 6.0 array (Santa Clara, CA). Sequence spanning 38,211,736 – 38,300,703 bp region on chromosome X (GRCh37) and including OTC was scanned for motifs CCTCCCT, CCTCCTT, CCTCCCTT, CCCCACCCC, CCNCCNTNNCCNC, GGNGGNAGGG and their complements known as being associated with recombination hotspots. Repeats capable of non-B DNA structure formation implicated in double strand breaks (DSBs) were sought by complexity analysis . X-inactivation ratio determined by analysis of methylation status of the human androgen-receptor locus (HUMARA)

Supplemental Data: Table 1&2, This is a manifesting heterozygote. Serum Gln+Glu was considered elevated. The minimum plasma ammonia, orotic acid and Gln+Glu concentrations depends on certain age range: Plasma ammonia: neonates <90μmol/l, other <60μmol/l. Urinary orotic acid: 0–1year <6.6mmol/mol creatinine, 1 – 10 years <3.5 mmol/mol creatinine, over 10 years <2.4 mmol/mol creatinine. Serum glutamate + glutamine: 0 – 1 month 200–1200μmol/l, 1 month–1year 200–1100μmol/l, 1year–18years 200–900μmol/l, over 18years 200–800μmol/l.

Variant: NM_000531.6:c.717+1G>T(IVS7+1G>T)

ClinVarID: 97298

CAID: CA224753

gnomAD:

Gene Name: OTC (ornithine transcarbamylase)

Case: Patient #30, female, Korean

DiseaseAssertion: UCD/OTCD

FamilyInfo: N/A

CasePresentingHPOs: HP:0011463, HP:0003218

CaseHPOFreeText: childhood onset, oroticaciduria,

CaseNOTHPOs:

CaseNOTHPOFreeText:

CasePreviousTesting: "Potential impact of mutations on OTC function and/or folding assessed by multiple alignments of orthologous protein sequences and human OTC and structural data from Protein Data Bank (1C9Y and available orthologs). In M patients, the approximate extent of the deletions assessed by inspection of presence/absence of PCR products. In F patients, the deletions determined by the SALSA multiplex ligation probe amplification (MLPA) KIT P079 OTC (MRC-Holland, Amsterdam, the Netherlands) and the Affymetrix Human SNP 6.0 array (Santa Clara, CA). Sequence spanning 38,211,736 – 38,300,703 bp region on chromosome X (GRCh37) and including OTC was scanned for motifs CCTCCCT, CCTCCTT, CCTCCCTT, CCCCACCCC, CCNCCNTNNCCNC, GGNGGNAGGG and their complements known as being associated with recombination hotspots. Repeats capable of non-B DNA structure formation implicated in double strand breaks (DSBs) were sought by complexity analysis . X-inactivation ratio determined by analysis of methylation status of the human androgen-receptor locus (HUMARA)

Supplemental Data: Table 1&2, Serum Gln+Glu was considered elevated, the minimum plasma ammonia, orotic acid and Gln+Glu concentrations depends on certain age range: Plasma ammonia: neonates <90μmol/l, other <60μmol/l. Urinary orotic acid: 0–1year <6.6mmol/mol creatinine, 1 – 10 years <3.5 mmol/mol creatinine, over 10 years <2.4 mmol/mol creatinine. Serum glutamate + glutamine: 0 – 1 month 200–1200μmol/l, 1 month–1year 200–1100μmol/l, 1year–18years 200–900μmol/l, over 18years 200–800μmol/l.

Variant: NM_000531.6:c.491C>G(p.Ser164*)

ClinVarID: 97220

CAID: CA224642

gnomAD:

GeneName: OTC (ornithine transcarbamylase)

Case: Patient #42, female, Korean

DiseaseAssertion: UCD/OTCD

FamilyInfo: N/A

CasePresentingHPOs: HP:0011463, HP:0001987, HP:0003218

CaseHPOFreeText: Childhood onset, hyperammonemia, oroticaciduria

CaseNOTHPOs:

CaseNOTHPOFreeText:

CasePreviousTesting: Genomic DNA was extracted from peripheral blood leukocytes. A total of 10 coding exons and exon–intron boundaries of the OTC gene were amplified by PCR with customized primers. PCR products were directly sequenced with the same primers . Sequencing results were compared with the established human OTC sequences(NM_000531.5). Multiplex ligation-dependent probe amplification analysis was performed for patients in whom no OTC mutations were identified by direct sequencing using the OTC MLPA kit.

Supplemental Data: Table 1, no range was given for blood ammonia concentration, range given in the tables for glutamine and urine orotate is slightly different than the one in the results paragraphs.

Variant: NM_000531.6:c.958C>T(p.Arg320*)

ClinVarID: 97371

CAID:CA285809

gnomAD:

GeneName: OTC (ornithine transcarbamylase)

Case: Patient #30, male, Chinese

DiseaseAssertion: UCD/OTCD

FamilyInfo: no family history of the disease

CasePresentingHPOs: HP:0003593, HP:0001987, HP:0003218

CaseHPOFreeText: infantile onset, hyperammonemia, oroticaciduria

CaseNOTHPOs:

CaseNOTHPOFreeText: No neurological damage

CasePreviousTesting: gDNA extracted from peripheral blood leukocytes. PCR all coding exons and exon–intron boundaries of the OTC gene using 9 pairs of synthetic oligonucleotide primers, and the primer sequences and annealing temperature. PCR products were then purified and bidirectionally sequenced. The library was sequenced using Illumina HiSeq4000 and generated 150 bp paired-end reads. Data analysis was performed as previously described [Sun Y, Hu G, Liu H, Zhang X, Huang Z, Yan H, et al. Further delineation of the phenotype of truncating KMT2A mutations: the extended Wiedemann–Steiner syndrome. Am J Med Genet A. 2017;173:510–4.]. Multiplex ligation-dependent probe amplification analysis was performed for samples in which Sanger sequencing or WES failed to detect any disease-causing mutation.

SupplementalData: Table 3, drug treatment(L-arginine, L-Citrulline, sodium benzoate, and sodium phenylbutyrate)

Variant: NM_000531.6:c.929_931del(p.Glu310Valfs*45)

ClinVarID: 858012

CAID:CA916083888

gnomAD:

GeneName: OTC (ornithine transcarbamylase)

Case: Patient #59, female, Chinese

DiseaseAssertion: UCD/OTCD

FamilyInfo: variant in proband's mother and father

CasePresentingHPOs: HP:0003621, HP:0001987, HP:0003218, HP:0003217

CaseHPOFreeText:juvenile onset, hyperammonemia , oroticaciduria, hyperglutaminemia

CaseNOTHPOs: N/A

CaseNOTHPOFreeText: No neurological damage

CasePreviousTesting: gDNA extracted from peripheral blood leukocytes. PCR all coding exons and exon–intron boundaries of the OTC gene using 9 pairs of synthetic oligonucleotide primers, and the primer sequences and annealing temperature. PCR products were then purified and bidirectionally sequenced. The library was sequenced using Illumina HiSeq4000 and generated 150 bp paired-end reads. Data analysis was performed as previously described [Sun Y, Hu G, Liu H, Zhang X, Huang Z, Yan H, et al. Further delineation of the phenotype of truncating KMT2A mutations: the extended Wiedemann–Steiner syndrome. Am J Med Genet A. 2017;173:510–4.]. Multiplex ligation-dependent probe amplification analysis was performed for samples in which Sanger sequencing or WES failed to detect any disease-causing mutation.

SupplementalData: Table 3, drug treatment(L-arginine, L-Citrulline, sodium benzoate, and sodium phenylbutyrate)

Variant: NM_000531.6:c.664-1G>A

ClinVarID: 97288

CAID: CA224737

gnomAD:

GeneName: OTC (ornithine transcarbamylase)

HGNCID:

https://www.ncbi.nlm.nih.gov/clinvar/variation/3586/

https://www.ncbi.nlm.nih.gov/clinvar/variation/92258/

https://www.ncbi.nlm.nih.gov/clinvar/RCV000211437/