Case# P2, 5mon onset, Indian

CasePresentingHPOs HP:0002151, HP:0001946, HP:0001942, HP:0001987

CasePreviousTesting Of the negative samples, we include here only those negative for NAGS and CPS1 genes. NAGS testing was done by single-exon amplification and DNA sequencing.24 CPS1 testing was done by RNA sequencing, in most cases using stimulated lymphocytes

CaseGenotypingMethod sequence analysis of coding exons, including flanking intronic regions of CA5A and CA5B, was performed.

Variant NM_001739.1:c.458_459+22del (p.Gly114Alafs*53)

CAID CA658820625

CaseAddInfo consanguineous

Case# P1, 4day onset, Turkish

CasePresentingHPOs HP:0002151, HP:0001946, HP:0001942, HP:0001987

CasePreviousTesting negative for NAGS and CPS1 genes.

CaseGenotypingMethod sequence analysis of coding exons, including flanking intronic regions of CA5A and CA5B, was performed.

Variant NM_001739.1:c.123G>A

CAID CA397038622

VariantEvidence truncated protein

CaseAddInfo consanguineous

Case# P7, 5day onset, Bangladesh

CasePresentingHPOs HP:0002151, HP:0001946, HP:0001942, HP:0001987

CasePreviousTesting Of the negative samples, we include here only those negative for NAGS and CPS1 genes. NAGS testing was done by single-exon amplification and DNA sequencing.24 CPS1 testing was done by RNA sequencing, in most cases using stimulated lymphocytes

CaseGenotypingMethod sequence analysis of coding exons, including flanking intronic regions of CA5A and CA5B, was performed.

Variant NM_001739.1(CA5A):c.721G>A (p.Glu241Lys)

ClinVar 388645

gnomAD 0.002694 (South Asian), 0.0003177 (Overall)

VariantEvidence CA5A variant in Sf9 insect cells exhibited 26.1% of WT enzyme activity (Fig 2d).

CaseAddInfo consanguineous

Case# P4, 4yr onset, Pakistani

CasePresentingHPOs HP:0002151, HP:0001946, HP:0001942, HP:0001987

CasePreviousTesting negative for NAGS and CPS1 genes.

CaseGenotypingMethod sequence analysis of coding exons, including flanking intronic regions of CA5A and CA5B, was performed.

Variant NM_001739.1:c.555+4_555+183del180bp

CAID CA658820661

VariantEvidence intronic splice site

CaseAddInfo consanguineous

Case# F1-2:1, Female, European, 3wk

CasePresentingHPOs HP:0001254, HP:0002789, HP:0001943, HP:0001987, HP:0001950

CasePreviousTesting sequencing and deletion/duplication analysis of known urea cycle defects and primary causes of hyperlactatemia. metabolic analysis of blood (Table 1).

CaseMethod1 Sanger

CaseMethod2 WES

CaseGenotypingMethod Sanger of CA5A

Variant NM_001739.1(CA5A):c.697T>C (p.Ser233Pro)

ClinVar 127087

VariantEvidence COS-7 cells with the p.Ser233Pro substitution showed 20% of the activity of the WT and quickly denatured at body temperature (Figure 1)

CaseAddInfo nonconsanguineous family, WES performed on heterzygous parents and affected siblings.

Case# F2-2:1, 38wk born, Russian

CasePresentingHPOs HP:0001254, HP:0001824, HP:0001943, HP:0001942, HP:0000952, HP:0002789, HP:0002919

CasePreviousTesting molecular analysis of CPS1 and NAGS did no reveal disease-causing mutations. Metabolic analysis (Table 1)

CaseMethod1 Sanger

CaseGenotypingMethod Sanger of all seven CA5A exons

Variant NM_001739.1(CA5A):c.555G>A (p.Lys185=)

ClinVar 127088

gnomAD 0.0004648 (Other), 0.0001554 (Overall)

VariantEvidence RT-PCR amplifying exons 2-7 of CA5A (in proband and WT) generated different sized products. Sanger of smaller band revealed a deletion in exon 4 (Fig. 3).

CaseAddInfo nonconsanguineous, heterozygous parents

Case# F3-2:5, Male, Pakistan, 13mon diagnosis

CasePresentingHPOs HP:0001987, HP:0001942, HP:0001993, HP:0003128

CasePreviousTesting meningo-encephalitis (cultures were negative). Further metabolic investigations are shown in Table 1.

CaseMethod1 Sanger

CaseGenotypingMethod Sanger of CA5A exons

Variant NM_001739.1(CA5A):c.619-3421_774+502del (p.Asp207_Gln258del)

ClinVar 127089

CaseAddInfo consanguineous, heterozygous parents checked with qtPCR