Case4: Female, 2 wks at initation of follow up, European ancestry

HPOsFreeText: NBS C4-acylcarnitine 2.0 umol/L, Plasma C4-acylcarnitine 1.52 umol/L

CaseAddInfo: no data for urine excretion, unremarkable clinical condition

Case4 (Level 2)

CasePreviousTesting: same as group

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant1: NM_014384.2:c.443C>T (p.Pro148Leu)

ClinVar: n/a

CAID: CA6372945

gnomAD: not in gnomAD

Variant2: NM_014384.2(ACAD8):c.455T>C (p.Met152Thr)

ClinVar: 5355

CAID: CA117433

gnomAD: 0.0001395 (European Non-Finnish)

CaseAddInfo: compound heterozygote, indicated alleles 1 and 2 but not specified maternal and paternal, potential splice site variant of ACAD8 at exon3 (+3A>G)

Case3a: Male, 5d at initiation of follow up, East Indian Ancestry, sibling of Case 3b (family 1)

HPOsFreeText: NBS C4-acylcarnitine 2.4 umol/L, Plasma C4-acylcarnitine 1.14 umol/L, in vitro probe isoC4-acylcarnitine 0.133 umol/ g protein,

NotHPOsFreeText: normal urine isobutyrylglycine ug/mg creatinine

CaseAddInfo: unremarkable clinical condition

Case3a (Level 2)

CasePreviousTesting: same as group

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant: NM_014384.2(ACAD8):c.867C>A (p.His289Gln)

ClinVar: 5358

CAID: CA117441

gnomAD: not in gnomAD

CaseAddInfo: homozygous mutations

Case9: Male, 2 mo at initiation of follow up, Hispanic ancestry

HPOsFreeText: NBS C4-acylcarnitine 1.8 umol/L, Plasma C4-acylcarnitine 3.28, umol/L, in vitro probe isoC4-acylcarnitine 0.074 umol/g protein, urine C4-acylcarnitine 7.86 mmol/mol creatinine, Urine C4/C3 ratio 63

NotHPOsFreeText: normal urine isobutyrylglycine

CaseAddInfo: unremarkable clinical condition

Case9 (Level 2)

CasePreviousTesting: same as group

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant: NM_014384.2(ACAD8):c.233T>C (p.Met78Thr)

ClinVar: 430160

CAID: CA6372883

gnomAD: n/a

CaseAddInfo: homozygous variants, no potential splice site alterations, no data for ACADS (SCAD-deficient) gene variants

Case2: Female, 3 m at initiation of follow up, European ancestry

HPOsFreeText: NBS C4-acylcarnitine 2.5 umol/L, plasma C4-acylcarnitine 10.00 umol/L, isoC4-acylcarnitine 0.120 umol/g protein, in vitro prob C4-acylcarnitine 0.220 umol/ g protein

CaseAddInfo: no data for urine excretion, unremarkable clinical condition

Case2 (Level 2)

CasePreviousTesting: same as group

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant1: NM_014384.2(ACAD8):c.289G>A (p.Gly97Arg)

ClinVar: 281037

CAID: CA6372897

gnomAD: 0.0002175 (East Asian)

Variant2: NM_014384.2(ACAD8):c.455T>C (p.Met152Thr)

ClinVar: 5355

CAID: CA117433

gnomAD: 0.0001395 (European Non-Finnish)

CaseAddInfo: compound heterozygous, potential splice-site alteration on ACAD8 at Exon 3 (+3A>G), homozygous for two common ACADS (SCAD-deficient) gene variants (625G>A/625G>A) which has 22% frequency in general population and correlates with increased accumulation of butyrylcarnitine

Case467 Caucasian

CasePresentingHPOs HP:0003571 (Propionicacidemia)

CaseNotHPOs n/a

CasePreviousTesting none reported

Variant 1. NM_000532.4(PCCB):c.1218_1231delGGGCATCATCCGGCinsTAGAGCACAGGA (p.Gly407Argfs), 2. NM_000532.4:c.1222del (p.Ile408SerfsTer?)

ClinVar 1.38875, 2. n/a

CAID 2. unregistered

gnomAD 1. not in gnomAD

CaseAddInfo parents NOT tested, phase unknown

CasePMIDs n/a

Case# 18804, Spanish, 3yo report

CaseHPOs asymmptomatic

Variant NM_000709.3:c.647C>T, NM_000709.3:c.889C>T

CAID CA9461230(c.647), CA9461294(889)

gnomAD c.647=0.0001332(African), 0.00002463(Overall), c.889=0.00006534(African), 0.00001624(Overall)

VariantEvidence reduced BCKD activity to 30% of normal in patient fibroblasts

CaseAddInfo in trans

Case# 20493, Spanish, 1yo reported

CasePresentingHPOs HP:0008344

CasePreviousTesting categorized into classical and variant phenotypes based on BCKD deficiency assessed by measuring the L-[1-14C] leucine decarboxylation rate by intact cells in absence of BCKD kinase inhibitors (Duran, et al., 1978), plasma amino acid levels, and mode of clinical presentation

CaseMethod1 Direct Sequencing

CaseMethod2 PCR

CaseGenotypingMethod direct sequencing of PCR fragments obtained after amplification of the entire coding sequence of BCKDHA, BCKDHB or DBT genes. sequenced both strands (forward and reverse) of corresponding exons and their intronic flanking sequence for confirmation.

Variant NM_000709.3:c.757G>A, NM_000709.3:c.848G>A, NM_000709.3:c.898G>A

CAID CA9461255(757), [CA406013082(848)&CA406013351(898)]

gnomAD c.757=0.00006681(European(Non-Finnish)), 0.00003236(Overall)

VariantEvidence Patient fibroblasts had <1% of normal BCKD activity

CaseAddInfo parents genotypes confirmed (in trans)

Case# 5888, Spanish, 10mo death

Variant NM_000709.3(BCKDHA):c.940C>T (p.Arg314Ter), NM_000709.3(BCKDHA):c.1037G>A (p.Arg346His)

ClinVar 224072(c.940), 93336(c.1037)

gnomAD c.940=0.000008958(European (Non-Finnish)), 0.000004064(Overall)

CaseAddInfo not confirmed in trans

Case# 16243, Spanish, 5yo reported

CasePresentingHPOs HP:0008344, HP:0001315

CasePreviousTesting categorized into classical and variant phenotypes based on BCKD deficiency assessed by measuring the L-[1-14C] leucine decarboxylation rate by intact cells in absence of BCKD kinase inhibitors (Duran, et al., 1978), plasma amino acid levels, and mode of clinical presentation

CaseMethod1 Direct Sequencing

CaseMethod2 PCR

CaseGenotypingMethod direct sequencing of PCR fragments obtained after amplification of the entire coding sequence of BCKDHA, BCKDHB or DBT genes. sequenced both strands (forward and reverse) of corresponding exons and their intronic flanking sequence for confirmation.

Variant NM_000709.3(BCKDHA):c.117delC (p.Arg40Glyfs), NM_000709.3(BCKDHA):c.117dupC (p.Arg40Glnfs)

ClinVar 93342(del), 166741(dup)

gnomAD del=0.0007494(African), 0.0001696(Overall)

CaseAddInfo in trans

CasePMIDs duplication variant in 8037208

Case23 5yo diagnosis, Anglo-Celtic

CasePresentingHPOs HP:0001083, HP:0001256, HP:0002751, HP:0000766

(Lens dislocation, Mild intellectual disability, Kyphoscoliosis, Abnormality of the sternum)

HPOsFreeText Patient was diagnosed on the basis of dislocated lenses. Treatment began with 100-200mg/day for two weeks followed by an addition of 5mg/day folic acid. Patient was not responsive to the treatment, plasma homocysteine levels went unchanged. Patient was given additional oral betaine(3g twice per day) with a low protein diet

CaseNotHPOs n/a

NotHPOsFreeText n/a

CasePreviousTesting Free homocystine, methionine, cystine, and cysteine-homo-cysteine mixed disulfide levels were measured by amino acid analyzers and total (free plus bound)homocysteine (tHcys) was measured. Exact values were not provided.

CaseMethod1 PCR

CaseMethod2 Sanger

CaseGenotypingMethod PCR amplified then direct sequenced the entire coding region of CBS.

Variant 1. NM_000071.2(CBS):c.430G>A (p.Glu144Lys), 2. IVS1+1G>A, compound heterozygous

ClinVar 1. 122, 2. n/a

CAID 2. need more info

gnomAD 1. MAF=0.00009799(South Asian), no homozygotes, 2. n/a

CaseAddInfo n/a

CasePMIDs n/a

Case13 8yo diagnosis, Anglo-Celtic

CasePresentingHPOs HP:0001083, HP:0000767

(Lens dislocation, Pectus excavatum)

HPOsFreeText borderline intellectual disability. Diagnosis was based on dislocated lenses. Treatment began with 100-200mg/day for two weeks followed by an addition of 5mg/day folic acid. This patient had a partial response to the treatment. Homocystine levels were sharply reduced on pyridoxine and folate treatment, but did not levels did not fall below 20μmol (nv <60μmol) on this treatment alone.

CaseNotHPOs n/a

NotHPOsFreeText n/a

CasePreviousTesting Free homocystine, methionine, cystine, and cysteine-homo-cysteine mixed disulfide levels were measured by amino acid analyzers and total (free plus bound)homocysteine (tHcys) was measured. Exact values were not provided.

CaseMethod1 PCR

CaseMethod2 Sanger

CaseGenotypingMethod PCR amplified then direct sequenced the entire coding region of CBS.

Variant 1. NM_000071.2(CBS):c.430G>A (p.Glu144Lys), 2. NM_000071.2(CBS):c.1111G>A (p.Val371Met), compound heterozygous

ClinVar 1. 122, 2. 212862

gnomAD 1. MAF=0.00009799(South Asian), no homozygotes, 2. MAF= 0.00006219(African), no homozygotes

CaseAddInfo n/a

CasePMIDs n/a

Case27 6yo diagnosis, Anglo-Celtic

CasePresentingHPOs HP:0001083, HP:0001256, HP:0002616, HP:0006280, HP:0002583

(Lens dislocation, Mild intellectual disability, Aortic root dilation, Chronic pancreatitis, Colitis)

HPOsFreeText Patient diagnosed on the basis of presenting marfanoid features. Patient had chronic pancreatitis requiring TPN and colitis requiring colostomy. Final CT scan of patient looking like multi-infarct dementia. Treatment began with 100-200mg/day for two weeks followed by an addition of 5mg/day folic acid. Patient was not responsive to the treatment, plasma homocysteine levels went unchanged. Patient was given additional oral betaine(3g twice per day) with a low protein diet

CaseNotHPOs n/a

NotHPOsFreeText n/a

CasePreviousTesting Free homocystine, methionine, cystine, and cysteine-homo-cysteine mixed disulfide levels were measured by amino acid analyzers and total (free plus bound)homocysteine (tHcys) was measured. Exact values were not provided.

CaseMethod1 PCR

CaseMethod2 Sanger

CaseGenotypingMethod PCR amplified then direct sequenced the entire coding region of CBS.

Variant 1. NM_000071.2:c.684C>A (p.Asn228Lys), 2. IVS11+1G>A, comp het

ClinVar 1. n/a, 2 n/a

CAID 1. CA410600617, 2. not enough info

gnomAD 1. MAF=0.00001759(European(non-Finnish)), no homozygotes, 2. n/a

CaseAddInfo n/a

CasePMIDs n/a

CaseLE12: Female, 23 yrs report

CasePresentingHPOs: HP:0002725 (Systemic lupus erythematosus)

HPOsFreeText: N/A

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Variant 1 homozygous, with variant 2 in-cis with one copy confirmed via parental testing

CasePreviousTesting: Specific plasma MMA levels, urine MMA levels, urine MA levels, and MMA/MA ratio available in Table 2

CaseGenotypingMethod: See Group

Variant1: NM_001243279.3:c.1553C>A (p.Ala518Asp)

CAID: CA8238286, http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA8238286

gnomAD: 0.00006153, https://gnomad.broadinstitute.org/variant/16-89212397-C-A

Variant2: NM_001243279.3:c.473C>T (p.Pro158Leu)

CAID: CA8237662, http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA8237662

gnomAD: 0.0002823, https://gnomad.broadinstitute.org/variant/16-89167562-C-T

Case# GM-649, M

CaseMethod1 Sanger

CaseMethod2 PCR

Variant NM_000709.3:c.284C>A(p.Pro95His)

CAID CA406005099(p.Pro95His)

gnomAD p.Pro95His=0.0003330(African), 0.00002887(Overall)

VariantEvidence total BCKDH activity (4) in fibroblast extracts from the patient (GM649) is < 10% of normal (Table 1) in 2703538

CaseAddInfo examined patient fibroblasts here and in the previous article

CasePMIDs 2703538

Case7: Male, 6m, Chinese

CasePresentingHPOs: HP:0002248, HP:0001290, HP:0008872, HP:0002572 (haematemesis, hypotonia, feeding difficulties in infancy, recurrent vomiting)

HPOsFreeText: (at 19 days) 1.01 C4 umol/L, 0.38 C4/C2, 1.61 C4/C3

NotHPOsFreeText: normal urine isobutyrylglycine

Case7 (Level 2):

CasePreviousTesting: same as group

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant1: NM_014384.2:c.444G>T (p.Pro148=)

ClinVar: n/a

CAID: CA6372946

gnomAD: 0.0002772 (Other)

Variant2: NM_014384.2:c.1176G>T (p.Arg392Ser)

ClinVar: n/a

CAID: CA6373249

gnomAD: 0.0001088 (East Asian)

CaseAddInfo: trans heterozygous, inherited one allele from each parent

Case# Case 5, M, Chinese, 4day onset

CasePresentingHPOs HP:0008344, HP:0040168, HP:0008947, HP:0011968

CasePreviousTesting Tested blood aa levels with liquid chromatography-mass spectrum spectrometry, several branched chain amino acids were overly abundant. Whole exome targeted capture sequencing was applied for the mutation screening of the suspicious patients. A total of 712 genes associated with inherited metabolic diseases were selected and their coding regions were captured.

CaseMethod1 Whole Exome

CaseMethod2 Sanger

CaseGenotypingMethod validated whole exome findings with sanger and qPCR.

Variant NM_000056.3:c.863C>A(p.Ala288Asp), ?

CAID CA364660141 (miss), wait for follow up (exon del)

Case# Case 2, M, Chinese, 1day old Age of Onset

CasePresentingHPOs HP:0001276, HP:0000737, HP:0008344

CasePreviousTesting Tested blood aa levels with liquid chromatography-mass spectrum spectrometry, several branched chain amino acids were overly abundant. Whole exome targeted capture sequencing was applied for the mutation screening of the suspicious patients. A total of 712 genes associated with inherited metabolic diseases were selected and their coding regions were captured.

CaseMethod1 Whole Exome

CaseMethod2 Sanger

CaseGenotypingMethod validated whole exome findings with sanger and qPCR.

Variant NM_000056.3:c.523T>C

CAID CA364657780, exon1-7 del waiting on follow up

VariantEvidence in trans with massive deletion

CaseOS-138_II-1: Female, 4 yrs report, Asian

CasePresentingHPOs: HP:0001992, HP:0002373, HP:0001263 (Organic aciduria, Febrile seizure (within the age range of 3 months to 6 years), Global developmental delay)

HPOsFreeText: N/A

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Parents are consanguineous, Proband is homozygous for this variant

CasePreviousTesting: Routine urine screening for organic acidurias by gas chromatography–mass spectrometry showed markedly increased urinary excretion of several N-acetylated amino acids

CaseGenotypingMethod: PCR-amplification and bidirectional sequencing of all exons and exon-intron boundaries was performed on genomic DNA.

Variant: NM_000666.3(ACY1):c.589C>T (p.Arg197Trp)

ClinVar: 18113, https://www.ncbi.nlm.nih.gov/clinvar/variation/18113/

gnomAD: 0.00001764, https://gnomad.broadinstitute.org/variant/3-52021009-C-T

CaseOS-146_II-1: Male, 5 yrs report, Romani

CasePresentingHPOs: HP:0001992, HP:0000954, HP:0000431, HP:0000316, HP:0040194, HP:0004322, HP:0001250, HP:0010819, HP:0032792, HP:0002121, HP:0000752, HP:0002342 (Organic aciduria, Single transverse palmar crease, Wide nasal bridge, Hypertelorism, Increased head circumference, Short stature, Seizure, Atonic seizure, Tonic seizure, Generalized non-motor (absence) seizure, Hyperactivity, Moderate Intellectual disability)

HPOsFreeText: N/A

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Parents are healthy consanguineous cousins, Two cousins and a niece of the mother have epilepsy, Proband is homozygous for this variant

CasePreviousTesting: Routine urine screening for organic acidurias by gas chromatography–mass spectrometry showed markedly increased urinary excretion of several N-acetylated amino acids; Cerebral MRI was normal at the ages of 17 months and 2.5 years

CaseGenotypingMethod: PCR-amplification and bidirectional sequencing of all exons and exon-intron boundaries was performed on genomic DNA.

Variant: NM_000666.3(ACY1):c.1057C>T (p.Arg353Cys)

ClinVar: 18110 https://www.ncbi.nlm.nih.gov/clinvar/variation/18110/

gnomAD: 0.004135 (4 homozygotes), https://gnomad.broadinstitute.org/variant/3-52022837-C-T

CaseSE5: Male, Australia

CasePresentingHPOs: HP:0008353 (Neutral hyperaminoaciduria)

HPOsFreeText: N/A

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseGenotypingMethod: Five loci were investigated for evidence of linkage to Hartnup due to their established role in amino acid transport including SLC3A2, SLC7A8, SLC1A5, SLC6A18, and SLC6A19. These 5 regions were studied with microsattelite markers and produced linkage only between D5S392 and D5S417, the region with SLC6A18 and 19. SLC6A18 did not increase neutral amino acid uptake in X. laevis oocytes so SLC6A19 was investigated and directly sequenced in the families each of which displayed a causal variant.

CasePreviousTesting: SLC1A5 had no causative variants detected.

CaseAddInfo: Asymptomatic except for hyperaminoaciduria

Variant1: NM_001003841.3(SLC6A19):c.517G>A (p.Asp173Asn)

ClinVar: 2019 https://www.ncbi.nlm.nih.gov/clinvar/variation/2019/

gnomAD: 0.003016, https://gnomad.broadinstitute.org/variant/5-1212453-G-A?dataset=gnomad_r2_1

Variant2: NM_001003841.3(SLC6A19):c.718C>T (p.Arg240Ter)

ClinVar: 2020 https://www.ncbi.nlm.nih.gov/clinvar/variation/2020/

gnomAD: 0.0001960, https://gnomad.broadinstitute.org/variant/5-1213632-C-T

CaseF5P: 18 yrs report, India

CasePresentingHPOs: N/A

HPOsFreeText: N/A

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Proband for Family 5, Variant has been shown to abolish canonical splice donor site and lead to a null protein

CasePreviousTesting: Blood phenylalanine before treatment was 1260 μmol/l, Blood tyrosine before treatment was 36 μmol/l

CaseGenotypingMethod: N/A

Variant: NM_000277.3(PAH):c.842+1G>A

ClinVar: 599 https://www.ncbi.nlm.nih.gov/clinvar/variation/599/

gnomAD: 0.00007752, http://gnomad.broadinstitute.org/variant/12-103246592-C-T

CaseF1S: 5 yrs report, India

CasePresentingHPOs: N/A

HPOsFreeText: N/A

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Variant is demonstrated to activate a cryptic splice site and abolish the canon splice site leading to a premature termination and nonsense-mediated decay

CasePreviousTesting: Blood phenylalanine before treatment was 1431 μmol/l, Blood tyrosine before treatment was 35 μmol/l

CaseGenotypingMethod: N/A

Variant: NM_000277.3(PAH):c.169-2A>G

ClinVar: 555212 https://www.ncbi.nlm.nih.gov/clinvar/variation/555212/

gnomAD: 0.00003267, http://gnomad.broadinstitute.org/variant/12-103288698-T-C

CaseF2P: 2 yrs report, India

CasePresentingHPOs: HP:0001010, HP:0000750, HP:0001249, HP:0006801, HP:0004923 (Hypopigmentation of the skin, Delayed speech and language development, Intellectual disability, Brisk deep tendon reflexes, Hyperphenylalaninemia)

HPOsFreeText: N/A

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Family is consanguinous, Parents were confirmed heterozygous, Variant may not undergo NMD however would be missing essential domains and is predicted to be null

CasePreviousTesting: Blood phenylalanine before treatment was 1619 μmol/l, Blood tyrosine before treatment was 60 μmol/l, PKU diagnosis was based on ferric chloride test on patient urine samples and detection of elevated Phenylalanine levels in blood samples by using HPLC, in addition to analysis of clinical features.

CaseGenotypingMethod: Genomic DNA was isolated from blood samples of proband and family members. PCR amplification and sequencing was used to screen for mutations. Each mutation was confirmed by bi-directional DNA sequencing.

Variant: NM_000277.3:c.1177_1178insT (p.Asn393Ilefs*2)

CAID: CA16020958 http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA16020958

gnomAD: Not in gnomAD

CaseST-6: Male, 1 day onset, Morocco

CasePresentingHPOs: HP:0006579, HP:0001249, HP:0001270, HP:0001324, HP:0004322, HP:0000256, HP:0000316, HP:0006236, HP:0001786, HP:0008070, HP:0000708, HP:0002355, HP:0003202, HP:0002071 (Prolonged neonatal jaundice, Intellectual disability, Motor delay, Muscle weakness, Short stature, Macrocephaly, Hypertelorism, Slender metacarpals, Narrow foot, Sparse hair, Behavioral abnormality, Difficulty walking, Skeletal muscle atrophy, Abnormality of extrapyramidal motor function)

HPOsFreeText: N/A

CaseNotHPOs: HP:0001250 (Seizure)

NotHPOsFreeText: N/A

CaseAddInfo: Family is consanguineous, Proband is homozygous for the variant

CasePreviousTesting: See Group

CaseGenotypingMethod: See Group

Variant: NM_001123.3:c.685_687delinsCC (p.Thr229ProfsTer16)

CAID: CA1139768931, http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA1139768931

gnomAD: Not in gnomAD

Case3: Female, Portugal

CasePresentingHPOs: HP:0001290, HP:0003324, HP:0002093, HP:0011968, HP:0001283, HP:0002804, HP:0001558, HP:0001561, HP:0001371 (Generalized hypotonia, Generalized muscle weakness, Respiratory insufficiency, Feeding difficulties, Bulbar weakness, Decreased fetal movement, Polyhydramnios, Contracture)

HPOsFreeText: N/A

CaseNotHPOs: HP:0001627 (Cardiac abnormality)

NotHPOsFreeText: N/A

CaseAddInfo: Severe congenital myopathy, Deceased in neonatal period

CasePreviousTesting: N/A

CaseGenotypingMethod: After LMOD3 was identified as a potential causal gene, bidirectional Sanger sequencing was performed on DNA in genetically unresolved patients using PCR amplification covering 3 exons.

Variant: NM_198271.4:c.154del (p.Met52*)

CAID: CA909810717 http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/allele?hgvs=NM_198271.4%3Ac.154delA

gnomAD: Not in gnomAD

Case12a: Male, Afghanistan

CasePresentingHPOs: HP:0001290, HP:0003324, HP:0002093, HP:0011968, HP:0001283, HP:0002804, HP:0001558, HP:0001622, HP:0001623, HP:0003798 (Generalized hypotonia, Generalized muscle weakness, Respiratory insufficiency, Feeding difficulties, Bulbar weakness, Arthrogryposis, Decreased fetal movement, Preterm delivery, Breech presentation, Nemaline bodies)

HPOsFreeText: Atrophic myofibers

CaseNotHPOs: HP:0001627 (Cardiac abnormality)

NotHPOsFreeText: N/A

CaseAddInfo: Severe congenital myopathy, Deceased in 2 months, Has 2 siblings with same genotype/phenotype

CasePreviousTesting: N/A

CaseGenotypingMethod: After LMOD3 was identified as a potential causal gene, bidirectional Sanger sequencing was performed on DNA in genetically unresolved patients using PCR amplification covering 3 exons.

Variant: NM_198271.4:c.1099_1100delAA (p.Asn367Glnfs*11)

ClinVar: 162218 https://www.ncbi.nlm.nih.gov/clinvar/variation/162218/

gnomAD: South Asian 4.575e-4, Overall 5.625e-5 https://gnomad.broadinstitute.org/variant/3-69168405-GTT-G

Case14a: Female, Australia

CasePresentingHPOs: HP:0001290, HP:0003324, HP:0002093, HP:0011968, HP:0001283, HP:0001561, HP:0001622, HP:0002804, HP:0003798, HP:0001558, HP:0000602 (Generalized hypotonia, Generalized muscle weakness, Respiratory insufficiency, Feeding difficulties, Bulbar weakness, Polyhydramnios, Nemaline bodies, Decreased fetal movements, Ophthalmoplegia)

HPOsFreeText: Atrophic myofibers, Percutaneous endoscopic gastrostomy performed, Nocturnal noninvasive ventilation

CaseNotHPOs: HP:0001627, HP:0002540 (Cardiac abnormality, Inability to walk)

NotHPOsFreeText: N/A

CaseAddInfo: Typical congenital nemaline myopathy, Alive at 10 yrs, Parents are not consanguineous

CasePreviousTesting: N/A

CaseGenotypingMethod: Linkage analysis combined with homozygosity mapping was used to identify candidate chromosomal loci. Whole exome sequencing in this family revealed likely pathogenic mutations in LMOD3 which agreed with the aforementioned analysis and was consistent with autosomal recessive inheritance.

Variant1: NM_198271.4:c.1103_1105del (p.Asn368del)

ClinVar: 162216 https://www.ncbi.nlm.nih.gov/clinvar/variation/162216/

gnomAD: Not in gnomAD

Variant2: NM_198271.4:c.1201C>T (p.Arg401Ter)

ClinVar: 162217 https://www.ncbi.nlm.nih.gov/clinvar/variation/162217/

gnomAD: African 4.137e-5, Overall 7.135e-6 https://gnomad.broadinstitute.org/variant/3-69168305-G-A

Case5: Female, Japan, Asian

CasePresentingHPOs: HP:0001290, HP:0003324, HP:0002093, HP:0011968, HP:0001283, HP:0003798, HP:0001561, HP:0001558, HP:0100309 (Generalized hypotonia, Generalized muscle weakness, Respiratory insufficiency, Feeding difficulties, Bulbar weakness, Nemaline bodies, Polyhydramnios, Decreased fetal movements, Subdural hemorrhage)

HPOsFreeText: Atrophic myofibers

CaseNotHPOs: HP:0001627 (Cardiac abnormality)

NotHPOsFreeText: N/A

CaseAddInfo: Severe congenital myopathy, Alive at 2 months

CasePreviousTesting: N/A

CaseGenotypingMethod: After LMOD3 was identified as a potential causal gene, bidirectional Sanger sequencing was performed on DNA in genetically unresolved patients using PCR amplification covering 3 exons.

Variant1: NM_198271.4:c.300_304delGACTC (p.Thr101Argfs*4)

CAID: CA913203453 http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA913203453

gnomAD: Not in gnomAD

Variant1: NM_198271.4:c.601_602delGA (p.Asp201Glnfs*9)

CAID: CA913203454 http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA913203454

gnomAD: Not in gnomAD

Patient18: Group 3

CasePresentingHPOs: same as group 3

CasePreviousTesting: same as group 3

CaseGenotypingMethod: same as group 3

Variant1: NM_002225.3:c.1136T>C (p.p.Ile349Thr) **listed in paper as (p.Phe350Ser)

CAID: CA391723904, http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA391723904

gnomAD: n/a

Variant2: IVS3+5G>T no ClinVar or CAID

Patient8: Group 2

CasePresentingHPOs: same as group 2

CasePreviousTesting: same as group 2

CaseGenotypingMethod: same as group 2

Variant1: NM_002225.3(IVD):c.941C>T (p.Ala314Val) listed in paper as c.932C>T (p.Ala282Val)

ClinVar: 100060, https://www.ncbi.nlm.nih.gov/clinvar/variation/100060/

gnomAD: 0.0006507, http://gnomad.broadinstitute.org/variant/15-40707653-C-T

Variant2: NM_002225.3:c.262G>T

CAID: CA391745812, http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA391745812

gnomAD: n/a

Case: Patient C, 11 years, female CaseHPOs: HP:0003235, HP:0011098, Hypermethioninemia, speech dyspraxia

CaseHPOsFreeText: “pronounced hypermethioninaemia on neonatal screening”, cleft lip and palate; Hypermethioninaemia has persisted; unpleasant odor with high blood concentrations of mercaptans; fine nystagmus, dysdiadochokinesis, and increased tendon reflexes led to brain MRI and CT at age 11 years which showed demyelination, calcification of basal ganglia, IQ 87; normal myelination and all symptoms resolved after 12 months treatment with S-adenosylmethionine toluene sulphonate.

CaseNotHPOs: Homocystine was not detected in plasma or urine,normal liver function tests.

CasePreviousTesting: Plasma methionine 600–1,400 umol/L; otherwise normal amino acids. 5-methyl tetrahydrofolate 329, 232 nmol/L (normal 23-170, mean 85+/-41), normalized after 12 months on S-adenosylmethionine toluene sulphonate; S-adenosylmethionine 70, 87 nmol/L (normal 172-450, mean 255 +/-69), improved but still low after 12 months on S-adenosylmethionine toluene sulphonate.

Variant: c.826dupG (homozygous)

ClinVar: N/A

CAID: CA5576692

gnomAD: The highest population minor allele frequency in gnomAD is 0.00003298 (S. Asian); no homozygotes in any population.

VariantEvidence: When the variant was expressed in bacteria, the amount and size of the protein was reduced on Western blot, and there was no detectable MAT1A activity.

CaseAddInfo: The mother is heterozygous for the variant; DNA testing results for the father are not available.

CasePMIDs: 1683972

Points: 2 points. This patient is homozygous for a frameshift variant in MAT1A. When the variant was expressed in bacteria, there was no detectable MAT activity. The highest population minor allele frequency in gnomAD is 0.00003298 (S. Asian); no homozygotes in any population.

CaseAI-1: N/A

CasePresentingHPOs: N/A

HPOsFreeText: Pretreatment phenylalanine level at 330 umol/L

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: N/A

Variant1: NM_000277.3(PAH):c.143T>C (p.Leu48Ser)

ClinVar: 608 https://www.ncbi.nlm.nih.gov/clinvar/variation/608/

gnomAD: 0.001025, https://gnomad.broadinstitute.org/variant/12-102912816-A-G?dataset=gnomad_r3

Variant2: NM_000277.3(PAH):c.898G>T (p.Ala300Ser)

ClinVar: 92751 https://www.ncbi.nlm.nih.gov/clinvar/variation/92751/

gnomAD: 0.006318, https://gnomad.broadinstitute.org/variant/12-102851701-C-A?dataset=gnomad_r3

CaseDI-1: N/A

CasePresentingHPOs: N/A

HPOsFreeText: Pretreatment phenylalanine level at 365 umol/L

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: N/A

Variant1: NM_000277.3(PAH):c.561G>A (p.Trp187Ter)

ClinVar: 102736 https://www.ncbi.nlm.nih.gov/clinvar/variation/102736/

gnomAD: 0.00001760, https://gnomad.broadinstitute.org/variant/12-103249059-C-T

Variant2: NM_000277.3(PAH):c.898G>T (p.Ala300Ser)

ClinVar: 92751 https://www.ncbi.nlm.nih.gov/clinvar/variation/92751/

gnomAD: 0.006318, https://gnomad.broadinstitute.org/variant/12-102851701-C-A?dataset=gnomad_r3

CaseAII-5: N/A

CasePresentingHPOs: N/A

HPOsFreeText: Pretreatment phenylalanine level at 440 umol/L

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: N/A

Variant1: NM_000277.3(PAH):c.165del (p.Phe55fs)

ClinVar: 611 https://www.ncbi.nlm.nih.gov/clinvar/variation/611/

gnomAD: 0.00002639, https://gnomad.broadinstitute.org/variant/12-103306571-CA-C?dataset=gnomad_r3

Variant2: NM_000277.3(PAH):c.898G>T (p.Ala300Ser)

ClinVar: 92751 https://www.ncbi.nlm.nih.gov/clinvar/variation/92751/

gnomAD: 0.006318, https://gnomad.broadinstitute.org/variant/12-102851701-C-A?dataset=gnomad_r3

CaseAII-3: N/A

CasePresentingHPOs: N/A

HPOsFreeText: Pretreatment phenylalanine level at 370 umol/L

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: N/A

Variant1: NM_000277.3(PAH):c.165del (p.Phe55fs)

ClinVar: 611 https://www.ncbi.nlm.nih.gov/clinvar/variation/611/

gnomAD: 0.00002639, https://gnomad.broadinstitute.org/variant/12-103306571-CA-C?dataset=gnomad_r3

Variant2: NM_000277.3(PAH):c.898G>T (p.Ala300Ser)

ClinVar: 92751 https://www.ncbi.nlm.nih.gov/clinvar/variation/92751/

gnomAD: 0.006318, https://gnomad.broadinstitute.org/variant/12-102851701-C-A?dataset=gnomad_r3

Case# PA1

Variant NM_000709.3(BCKDHA):c.347A>G (p.Asp116Gly)

ClinVar 93354

VariantEvidence none

CaseC1: Female, Arab, Israel

CasePresentingHPOs: HP:0010896, HP:0010897, HP:0001638 (Hypersarcosinemia, Hypersarcosinuria, Cardiomyopathy)

HPOsFreeText: Elevated blood Sarcosine (345 μmol/l; reference 0-9), Elevated urine Sarcosine (481 μmol/mmol; reference 0-36)

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Parents are consanguineous and first cousins, sister has the same genotype

CasePreviousTesting: N/A

CaseGenotypingMethod: DNA was extracted from the probands and immediate family. The whole coding region and the exon-intron boundaries of the SARDH gene was then amplified and sequenced.

Variant: NM_007101.3:c.211G>T (p.Val71Phe)

ClinVar: 39448 https://www.ncbi.nlm.nih.gov/clinvar/variation/39448/

gnomAD: Not in gnomAD

Case#: GA3, Turkey, 5 years (onset), gender unknown

CaseHPOs: HP:0012026 (hyperornithinemia)

CaseHPOsFreeText: Presented with visual disturbances at 5 years of age. No further details.

CaseNotHPOs: Neonatal encephalopathy

CasePreviousTesting: No previous DNA testing noted. Plasma ornithine at diagnosis 650umol/L (normal <100)

CaseMethod1: PCR

CaseMethod2: Sequencing

CaseGenotypingMethod: Sequence analysis of the OAT gene from fibroblast or lymphocyte cDNA and genomic DNA. Variant: NM_000274.3(OAT):c.991C>T (p.Arg331Ter) (homozygous)

ClinVar: 56141

gnomAD: The maximum population minor allele frequency in gnomAD is 0.0001446 (Latino); no homozygotes in any population.

Case27 sex unknown, Spain

CasePresentingHPOs HP:0006846, HP:0002540, HP:0001344, HP:0001332

(Acute encephalopathy, Non-ambulatory, Nonverbal, Dystonia)

HPOsFreeText Phenotype was classified as "severe," with passive or static dystonia and an inability to stand up, walk, or speak. Patient may have gone undetected by routine screening as glutarate excretion was normal and 3-hydroxyglutarate was only slightly increased.

CaseNotHPOs n/a

NotHPOsFreeText n/a

CasePreviousTesting GC-MS measured glutarate of 2(nv 2–10 mmol/mol creatinine) and 3-OH-glutarate 18(nv 2–14 mm/mol creatinine). In patient fibroblasts, %GCD activity was 10.

CaseMethod1 SSCP

CaseMethod2 Sanger

CaseGenotypingMethod PCR amplified and SSCP screened 11 exons and intron boundaries of GCDH gene. Confirmed variants with Sanger sequencing and restriction digest. The V400M variant creates an NlaIII site. This would lead to cleaved patient DNA fragments and uncleaved control fragments.

Variant NM_000159.3(GCDH):c.1198G>A (p.Val400Met), homozygous

ClinVar 2088

gnomAD MAF= 0.0002769(Other), no homozygotes

CaseAddInfo n/a

CasePMIDs see note on previous studies

Case38 sex unknown, Spain

CasePresentingHPOs HP:0006846, HP:0002540, HP:0001344, HP:0001332

(Acute encephalopathy, Non-ambulatory, Nonverbal, Dystonia)

HPOsFreeText Phenotype was classified as "severe," with passive or static dystonia and an inability to stand up, walk, or speak. Patient may have gone undetected by routine screens as glutarate excretion was normal and 3-hydroxyglutarate was only slightly increased.

CaseNotHPOs n/a

NotHPOsFreeText n/a

CasePreviousTesting GC-MS measured glutarate of 4(nv 2–10 mmol/mol creatinine) and 3-OH-glutarate 43(nv 2–14 mm/mol creatinine). In patient fibroblasts, %GCD activity was 9. Patient tested negative in glutarylcarnitine blood spot test, even under carnitine supplementation.

CaseMethod1 SSCP

CaseMethod2 Sanger

CaseGenotypingMethod PCR amplified and SSCP screened 11 exons and intron boundaries of GCDH gene. Confirmed variants with Sanger sequencing and restriction digest. The Y155H variant creates a NcoI restriction site. Thus, while the patient's DNA fragments would cleave, control fragments would not be cleaved. The R227P variant creates a NlaIV site. This would lead to cleaved patient DNA fragments and uncleaved control fragments.

Variant 1. NM_000159.3:c.463T>C(p.Tyr155His) , 2. NM_000159.3(GCDH):c.680G>C (p.Arg227Pro), compound heterozygous

ClinVar 1. n/a, 2. 2089

CAID 1. CA404317601

gnomAD 1. MAF=0.000008791(European(non-Finnish)), no homozygotes, 2. MAF= 0.0003566(European(non-Finnish)), no homozygotes

CaseAddInfo n/a

CasePMIDs see notes on previous studies

Case10 sex unknown, Spain

CasePresentingHPOs HP:0006846, HP:0002540, HP:0001344, HP:0001332

(Acute encephalopathy, Non-ambulatory, Nonverbal, Dystonia)

HPOsFreeText Phenotype was classified as "severe," with passive or static dystonia and an inability to stand up, walk, or speak. Diagnosed by routine GC-MS organic acid analysis in urine. There was high excretion of glutarate and 3-hydroxyglutarate with glutarate excretion being higher than that of 3-hydroxyglutarate.

CaseNotHPOs n/a

NotHPOsFreeText n/a

CasePreviousTesting GC-MS measured glutarate of 778(nv 2–10 mmol/mol creatinine) and 3-OH-glutarate 264(nv 2–14 mm/mol creatinine).

CaseMethod1 SSCP

CaseMethod2 Sanger

CaseGenotypingMethod PCR amplified and SSCP screened 11 exons and intron boundaries of GCDH gene. Confirmed variants with Sanger sequencing and restriction digest. The A293T variant, with the addition of a mismatch primer, destroys an HgaI restriction site. Thus, while the patient's DNA fragments would not cleave, control fragments would be cleaved. Likewise, the R402W variant destroys a MspI site. This would lead to uncleaved patient fragments and cleaved control fragments.

Variant 1. NM_000159.3(GCDH):c.877G>A (p.Ala293Thr), 2. NM_000159.3(GCDH):c.1204C>T (p.Arg402Trp), compound heterozygous

ClinVar 1. 2083, 2. 2085

gnomAD 1. MAF=0.0004013(African), no homozygotes, 2. MAF= 0.0007566(European(Finnish)), no homozygotes

CaseAddInfo Sibling (Case 11) was asymptomatic. GC-MS measured glutarate of 2083(nv 2–10 mmol/mol creatinine) and 3-OH-glutarate 262(nv 2–14 mm/mol creatinine)

CasePMIDs n/a

Case3 sex unknown, Spain

CasePresentingHPOs HP:0006846, HP:0002540, HP:0001344, HP:0001332

(Acute encephalopathy, Non-ambulatory, Nonverbal, Dystonia)

HPOsFreeText Phenotype was classified as "severe," with passive or static dystonia and an inability to stand up, walk, or speak. Diagnosed by routine GC-MS organic acid analysis in urine. There was high excretion of glutarate and 3-hydroxyglutarate with glutarate excretion being higher than that of 3-hydroxyglutarate.

CaseNotHPOs n/a

NotHPOsFreeText n/a

CasePreviousTesting GC-MS measured glutarate of 1773(nv 2–10 mmol/mol creatinine) and 3-OH-glutarate 340(nv 2–14 mm/mol creatinine).In patient fibroblasts, %GCD activity was <0.5.

CaseMethod1 SSCP

CaseMethod2 Sanger

CaseGenotypingMethod PCR amplified and SSCP screened 11 exons and intron boundaries of GCDH gene. Confirmed variants with Sanger sequencing and restriction digest. The A293T variant, with the addition of a mismatch primer, destroys an HgaI restriction site. Thus, while the patient's DNA fragments would not cleave, control fragments would be cleaved.

Variant NM_000159.3(GCDH):c.877G>A (p.Ala293Thr), homozygous

ClinVar 2083

gnomAD MAF= 0.0004013(African), no homozygotes

CaseAddInfo n/a

CasePMIDs n/a

Case21 sex unknown, Spain

CasePresentingHPOs HP:0006846, HP:0002540, HP:0001344, HP:0001332

(Acute encephalopathy, Non-ambulatory, Nonverbal, Dystonia)

HPOsFreeText Phenotype was classified as "severe," with passive or static dystonia and an inability to stand up, walk, or speak. Diagnosed by routine GC-MS organic acid analysis in urine. There was high excretion of glutarate and 3-hydroxyglutarate with glutarate excretion being higher than that of 3-hydroxyglutarate.

CaseNotHPOs n/a

NotHPOsFreeText n/a

CasePreviousTesting GC-MS measured glutarate of 204(nv 2–10 mmol/mol creatinine) and 3-OH-glutarate 143(nv 2–14 mm/mol creatinine). In patient fibroblasts, %GCD activity was 2.

CaseMethod1 SSCP

CaseMethod2 Sanger

CaseGenotypingMethod PCR amplified and SSCP screened 11 exons and intron boundaries of GCDH gene. Confirmed variants with Sanger sequencing and restriction digest. The A195T variant destroys a BanI restriction site. Thus, while the patient's DNA fragments would not cleave, control fragments would be cleaved.

Variant 1. NM_000159.3:c.583G>A (p.Ala195Thr), 2. NM_000159.3(GCDH):c.356C>T (p.Ser119Leu), compound heterozygous

ClinVar 1. n/a, 2. 288248

CAID 1. CA404318183

gnomAD 1. not in gnomAD, 2. MAF=0.0001984(Ashkenazi Jewish), no homozygotes

CaseAddInfo n/a

CasePMIDs n/a

Case29 sex unknown, Spain

CasePresentingHPOs HP:0006846, HP:0002540, HP:0001344, HP:0001332

(Acute encephalopathy, Non-ambulatory, Nonverbal, Dystonia)

HPOsFreeText Phenotype was classified as "severe," with passive or static dystonia and an inability to stand up, walk, or speak. Patient may have gone undetected by routine screens as glutarate excretion was normal and 3-hydroxyglutarate was only slightly increased.

CaseNotHPOs n/a

NotHPOsFreeText n/a

CasePreviousTesting GC-MS measured glutarate of 5(nv 2–10 mmol/mol creatinine) and 3-OH-glutarate 114(nv 2–14 mm/mol creatinine).

CaseMethod1 SSCP

CaseMethod2 Sanger

CaseGenotypingMethod PCR amplified and SSCP screened 11 exons and intron boundaries of GCDH gene. Confirmed variants with Sanger sequencing and restriction digest. The V400M variant creates an NlaIII site. This would lead to cleaved patient DNA fragments and uncleaved control fragments.

Variant NM_000159.3(GCDH):c.1198G>A (p.Val400Met), homozygous

ClinVar 2088

gnomAD MAF= 0.0002769(Other), no homozygotes

CaseAddInfo n/a

CasePMIDs n/a

Case18 sex unknown, Spain

CasePresentingHPOs HP:0002540, HP:0001344, HP:0001332

(Non-ambulatory, Nonverbal, Dystonia)

HPOsFreeText Clinical onset was "insidious," involving slowly progressive psychomotor delay, dystonic postures, lethargy, or macrocephaly. Phenotype was classified as "severe," with passive or static dystonia and an inability to stand up, walk, or speak. Diagnosed by routine GC-MS organic acid analysis in urine. There was high excretion of glutarate and 3-hydroxyglutarate with glutarate excretion being higher than that of 3-hydroxyglutarate.

CaseNotHPOs n/a

NotHPOsFreeText n/a

CasePreviousTesting GC-MS measured glutarate of 4097(nv 2–10 mmol/mol creatinine) and 3-OH-glutarate 212(nv 2–14 mm/mol creatinine). In patient fibroblasts, %GCD activity was <0.5.

CaseMethod1 SSCP

CaseMethod2 Sanger

CaseGenotypingMethod PCR amplified and SSCP screened 11 exons and intron boundaries of GCDH gene. Confirmed variants with Sanger sequencing and restriction digest. The R402W variant destroys an MspI restriction site. Thus, while the patient's DNA fragments would not cleave, control fragments would be cleaved.

Variant 1. NM_000159.3(GCDH):c.1204C>T (p.Arg402Trp), 2. NM_000159.3(GCDH):c.1173delG (p.Asn392Metfs), compound heterozygous

ClinVar 1. 2085, 2. 370106

gnomAD 1. MAF=0.0007566(European(Finnish)), no homozygotes, 2. MAF= 0.00005646(Latino), no homozygotes

CaseAddInfo n/a

CasePMIDs n/a

CaseF2: Female, 6 yrs report, Italy

CasePresentingHPOs: HP:0004923 (Hyperphenylalaninemia)

HPOsFreeText: N/A

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Parents do not appear to be tested, DNA sequence analysis "shows the sense sequence expected of compound heterozygotes with two comigrating bands seen"

CasePreviousTesting: Pre-treatment phenylalanine levels determined soon after birth were above 20 mg / 100 ml, Proband has been under treatment since birth and currently tolerates less than 20 mg Phe/kg bodyweight per day

CaseGenotypingMethod: DNA as isolated from leukocytes and haplotype analysis performed. Enzymatic digestion of PCR-amplified DNA fragments was performed and DGGE analysis of all 13 exons and flanking introns were carried out. The resulting samples containing abberant bands were sequenced and cleaved by MaeI and MaeIII for analysis.

Variant1: NM_000277.3(PAH):c.620A>G (p.Asn207Ser)

ClinVar: 102765 https://www.ncbi.nlm.nih.gov/clinvar/variation/102765/

gnomAD: Not in gnomAD

Variant2: NM_000277.3(PAH):c.842C>T (p.Pro281Leu)

ClinVar: 589 https://www.ncbi.nlm.nih.gov/clinvar/variation/589/

gnomAD: 0.0002016, https://gnomad.broadinstitute.org/variant/12-103246593-G-A

CaseF1: Male, 6 yrs report, Italy

CasePresentingHPOs: HP:0004923 (Hyperphenylalaninemia)

HPOsFreeText: N/A

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Parents were confirmed heterozygous, Splicing variant is predicted to lead to a null protein

CasePreviousTesting: Pre-treatment phenylalanine levels determined soon after birth were above 20 mg / 100 ml, Proband has been under treatment since birth and currently tolerates less than 20 mg Phe/kg bodyweight per day

CaseGenotypingMethod: DNA as isolated from leukocytes and haplotype analysis performed. Enzymatic digestion of PCR-amplified DNA fragments was performed and DGGE analysis of all 13 exons and flanking introns were carried out. The resulting samples containing abberant bands were sequenced and cleaved by MaeI and MaeIII for analysis.

Variant1: NM_000277.2(PAH):c.611A>G (p.Tyr204Cys)

ClinVar: 590 https://www.ncbi.nlm.nih.gov/clinvar/variation/590

gnomAD: 0.0001632, https://gnomad.broadinstitute.org/variant/12-103249009-T-C

Variant2: NM_000277.2(PAH):c.442-2A>C

ClinVar: 120276 https://www.ncbi.nlm.nih.gov/clinvar/variation/120276

gnomAD: Not in gnomAD

CaseSA5P: Female, 3 months onset, European

CasePresentingHPOs: HP:0001988, HP:0000825, HP:0001249, HP:0001250 (Recurrent hypoglycemia, Hyperinsulinemic hypoglycemia, Intellectual disability, Seizure)

HPOsFreeText: N/A

CaseNotHPOs: HP:0001987 (Hyperammonemia)

NotHPOsFreeText: N/A

CaseAddInfo: Has father with confirmed variant and similar phenotypes

CasePreviousTesting: Leucine load test showed leucine sensitivity; Protein/leucine restriction and dioxazide was effective at reducing symptoms

CaseGenotypingMethod: Genomic DNA was prepared from all patients via peripheral blood leukocytes. The GLUD1 gene exons 1-13 and the flanking intronic regions were PCR-amplified and directly sequenced. All variants were confirmed via restriction enzyme analysis. Additionally, paternity testing was performed by HLA typing and by southern blot analysis of DNA fragments as well as PCR analysis of STRs.

Variant: NM_005271.5(GLUD1):c.965G>A (p.Arg322His)

ClinVar: 16129, https://www.ncbi.nlm.nih.gov/clinvar/variation/16129/

gnomAD: Not in gnomAD

CaseSA2P: Female, 3 months onset, European

CasePresentingHPOs: HP:0001988, HP:0000825, HP:0001987 (Recurrent hypoglycemia, Hyperinsulinemic hypoglycemia, Hyperammonemia)

HPOsFreeText: N/A

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Proband is confirmed de novo via parental teting and confirmed paternity

CasePreviousTesting: Leucine load test showed leucine sensitivity in some patients; Protein/leucine restriction was effective at reducing symptoms

CaseGenotypingMethod: Genomic DNA was prepared from all patients via peripheral blood leukocytes. The GLUD1 gene exons 1-13 and the flanking intronic regions were PCR-amplified and directly sequenced. All variants were confirmed via restriction enzyme analysis. Additionally, paternity testing was performed by HLA typing and by southern blot analysis of DNA fragments as well as PCR analysis of STRs.

Variant: NM_005271.5(GLUD1):c.965G>A (p.Arg322His)

ClinVar: 16129, https://www.ncbi.nlm.nih.gov/clinvar/variation/16129/

gnomAD: Not in gnomAD

CaseKLE: Male, Japan, Asian

CasePresentingHPOs: HP:0008353 (Neutral hyperaminoaciduria)

HPOsFreeText:

CaseNotHPOs:

NotHPOsFreeText:

CaseAddInfo:

CasePreviousTesting: Identified via neonatal mass screening

CaseGenotypingMethod: Both SLC6A18 and SLC6A19 were investigated for possible pathogenicity, with SLC6A19's pattern of expression supporting it as a primary candidate. Intronic primers were used to bidirectionally sequence each exon and flanking intronic regions, locating two compound heterozygous mutations that perfectly segregate with the phenotype.

Variant1: NM_001003841.3:c.340del (L114fs*114)

CAID: CA916079859 http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA916079859

gnomAD: Not in gnomAD

Variant2: NM_001003841.3:c.682_683delinsTA (p.Thr228Ter)

CAID: CA916079860 http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA916079860

gnomAD: Not in gnomAD

Case#: Patient 3, Lebanon, Arab

CasePresentingHPOs: Intermediate form of MSUD

CasePreviousTesting: Previously diagnosed with MSUD by GC/MS/MS.

CaseMethod1: PCR

CaseMethod2: Sequencing

CaseGenotypingMethod: Genomic DNA extracted, sequencing of BCKDHA, BCKDHB and DBT.

Variant: NM_001918.3:c.1430T>G (homozygous)

ClinVar: 93994

gnomAD: European (Non-Finnish) 8.971e-6, Overall 4.065e-6.

VariantEvidence: Mutation is present in the catalytic domain and would expect a defect in catalytic function (Alamut software analysis).

Patient7: Male, 2yo

CasePresentingHPOs:

HPOsFreeText: Asymptomatic, C5 (DBS) = 1.6 μmol l−1, IVG in urine mmol permol creatinine = 272, IVA incorp (%) = 72, Ammonium (μmol l−1) = 51, pH = 7.39, Protein intake (g kg−1 per day) = 1.5, PDI/IQ in follow-up = 93

CaseNotHPOs: HP:0011968, HP:0002013, HP:0001944, HP:0001254, HP:0001259

(poor feeding, vomiting, dehydration, lethargy, coma)

NotHPOsFreeText: sweaty feet odor

CaseAddInfo: Diagnosed with NBS at 28 days, homozygous

CasePMIDs:

CasePreviousTesting: same as group

CaseGenotypingMethod: same as group

Variant: NM_002225.3:c.706G>A

CAID: CA268780419, http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA268780419

gnomAD: 0.00003187, http://gnomad.broadinstitute.org/variant/15-40705199-G-A

Patient14: Male, 24yo

CasePresentingHPOs: HP:0011968, HP:0002013, HP:0001944, HP:0001254, HP:0001259, HP:0001987, HP:0001941

(poor feeding, vomiting, dehydration, lethargy, coma, hyperammonemia, acidosis)

HPOsFreeText: intermittent isovaleric acidemia, C5 (DBS) = 4.3 μmol l−1, IVA incorp (%) = 12, IVG in urine mmol permol creatinine = 16594, Ammonium (μmol l−1) = 126, pH = 7.17, Protein intake (g kg−1 per day) = 0.7, treated with carnitine and glycine supplements, PDI/IQ in follow-up = 88

CaseNotHPOs:

NotHPOsFreeText: sweaty feet odor

CaseAddInfo: diagnosed via clinical findings at 1yo, hospitalized outside of detection period

CasePMIDs:

CasePreviousTesting: same as group

CaseGenotypingMethod: same as group

Variant1: c.1-?_c.295+? (Delexons 1–3) no ClinVar or CAID

Variant2: NM_002225.4(IVD):c.1184G>A (p.Arg395Gln) listed in paper as (p.Arg395His)

CAID: CA268781747, http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA268781747

ClinVar: 529434, https://www.ncbi.nlm.nih.gov/clinvar/variation/529434/

gnomAD: 0.00001415, http://gnomad.broadinstitute.org/variant/15-40710365-G-A

Patient11: Female, 19yo, sibling to patient 10

CasePresentingHPOs: HP:0011968, HP:0002013, HP:0001944, HP:0001254, HP:0001259

(poor feeding, vomiting, dehydration, lethargy, coma)

HPOsFreeText: intermittent isovaleric acidemia, IVA incorp (%) = 13, IVG in urine mmol permol creatinine = 3488, Ammonium (μmol l−1) = 47 pH = 7.33, Protein intake (g kg−1 per day) = 1.0, treated with carnitine and glycine supplements, PDI/IQ in follow-up = 85, sweaty feet odor

CaseNotHPOs:

NotHPOsFreeText:

CaseAddInfo: Diagnosed via clinical findings at 2yo

CasePMIDs:

CasePreviousTesting: same as group

CaseGenotypingMethod: same as group

Variant1: NM_002225.3(IVD):c.884T>C (p.Leu295Pro)

ClinVar: 555464, https://www.ncbi.nlm.nih.gov/clinvar/variation/555464/

gnomAD: n/a

Variant2: NM_002225.3:c.1211A>G

CAID: CA391724518, http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA391724518

gnomAD: n/a

Patient13 Male, 9yo

CasePresentingHPOs: HP:0011968, HP:0001254, HP:0001259, HP:0001987

(poor feeding, lethargy, coma, hyperammonemia)

HPOsFreeText: acute isovaleric acidemia, C5 (DBS) = 16.3 μmol l−1, IVA incorp (%) = 5.5, IVG in urine mmol permol creatinine = 3352, Ammonium (μmol l−1) = 606, pH = 7.26, Protein intake (g kg−1 per day) = 0.8, treated with carnitine and glycine supplements, PDI/IQ in follow-up = 87, sweaty feet odor

CaseNotHPOs: HP:0002013, HP:0001944 (vomiting, dehydration)

NotHPOsFreeText:

CaseAddInfo: diagnosed via clinical findings at 20 days old, homozygous

CasePMIDs:

CasePreviousTesting: same as group

CaseGenotypingMethod: same as group

Variant: NM_002225.3:c.158G>C listed in clinvar as NM_002225.4(IVD):c.149G>C (p.Arg50Pro)

CAID: CA274129, http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA274129

ClinVar: 188922, https://www.ncbi.nlm.nih.gov/clinvar/variation/188922/

gnomAD: 0.00001591, http://gnomad.broadinstitute.org/variant/15-40699841-G-C

CaseES: Female, 1 yr onset

CasePresentingHPOs: HP:0002312, HP:0002131, HP:0002719, HP:0002078, HP:0002066, HP:0100560, HP:0002345, HP:0008376, HP:0000666, HP:0001348, HP:0002342, HP:0410217, HP:0012237 (Clumsiness, Episodic ataxia, Recurrent infections, Truncal ataxia, Gait ataxia, Upper limb asymmetry, Action tremor, Dysarthic speech, Horizontal nystagmus, Brisk reflexes, Moderate intellectual disability, Reduced blood 5-methyltetrahydrofolate level, Urocanic aciduria)

HPOsFreeText: Urocanis acid levels 158-202 mmol/mol creatinine (reference <10)

CaseNotHPOs: HP:0000496, HP:0001324, HP:0002160, HP:0001903 (Abnormal ocular movements, Muscle weakness, Hyperhomocystinemia, Anemia)

NotHPOsFreeText: N/A

CaseAddInfo: Parents are healthy and nonconsanguineous, Steady improvement after mid-life, Proband is confirmed compound heterozygous by parental confirmation (father heterozygous for Variant 2)

CasePreviousTesting: Cranial MRI and neurophysiological studies, including electroencephalography, electroretinography, visual and brainstem evoked potentials, electromyography, nerve conduction velocities, fundus oculi and cardiological examinations, gave normal results; Cognitive evaluation rated her IQ at 54; Routine laboratory analyses including serum amino transferases, creatinine, urea and other parameters were normal; Screening for inborn errors of metabolism, including plasma and urine amino acids (no histidine accumulation), blood lactate, sialotransferrin isoelectricfocusing, creatine deficiency syndromes, purine and pyrimidine defects, and organic acidurias, gave normal results; Hypothyroidism and coeliac disease were also excluded; Biochemical and/or genetic screening for early onset forms of inherited ataxia gave normal results; Deficiencies in 5-methyltetrahydrofolate, neopterin and biopterin deficiencies were seen, together with an evident reduction in homovanillic acid (HVA) and the HVA:5-hydroxyindolacetic acid ratio; Other parameters determined in cerebrospinal fluid (CSF), such as glucose, proteins, cells and amino acids were normal.

CaseGenotypingMethod: Genomic DNA was obtained from peripheral white blood cells from the proband and her father. Mutation analysis of UROC1 was performed via direct sequencing and PCR amplification of the 20 exons and flanking exon-intronic sequences. In-silico analysis and modeling was performed on the variants.

Variant1: NM_144639.3(UROC1):c.209T>C (p.Leu70Pro)

ClinVar: 408 https://www.ncbi.nlm.nih.gov/clinvar/variation/408/

gnomAD: 0.00005647, https://gnomad.broadinstitute.org/variant/3-126229555-A-G

Variant2: NM_144639.3(UROC1):c.1348C>T (p.Arg450Cys)

ClinVar: 407, https://www.ncbi.nlm.nih.gov/clinvar/variation/407/

gnomAD: 0.00008012, https://gnomad.broadinstitute.org/variant/3-126216984-G-A

CaseFI2: Male, Turkey, Birth onset

CasePresentingHPOs: HP:0009800, HP:0001195, HP:0000773, HP:0003026, HP:0001650, HP:0003561, HP:0001518, HP:0000252, HP:0004887, HP:0001274, HP:0001250, HP:0000411, HP:0007957, HP:0007495, HP:0000963, HP:0000973, HP:0100678, HP:0010648, HP:0001382, HP:0009815, HP:0001181, HP:0010557, HP:0001374, HP:0012330, HP:0030750, HP:0040187 (Maternal diabetes, Single umbilical artery, Short ribs, Short long bone, Aortic valve stenosis, Birth length less than 3rd percentile, Small for gestational age, Microcephaly, Respiratory failure requiring assisted ventilation, Agenesis of corpus callosum, Seizures, Protruding ear, Corneal opacity, Prematurely aged appearance, Thin skin, Cutis laxa, Premature skin wrinkling, Dermal translucency, Joint hypermobility, Aplasia/hypoplasia of the extremities, Adducted thumb, Overlapping fingers, Congenital hip dislocation, Pyelonephritis, Grade III preterm intraventricular hemorrhage, Neonatal sepsis)

HPOsFreeText: Intrauterine growth restriction birth length: 38 cm (< 3rd percentile), birth weight 1370 g (< 3rd percentile) and microcephaly (OFC at birth was 29.5 cm (3rd–10th percentile)). APGAR scores were 5, 9, and 9 after 1, 5, and 10 min, respectively; Typical facial gestalt

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Parents are first-degree cousins that had three healthy daughters and three early abortions

CasePreviousTesting: N/A

CaseGenotypingMethod: Familial DNA was extracted from peripheral blood samples. Direct sequencing of PYCR1 and exons 2-18 of ALDH18A1 was performed in the affected individual with a homozygous single-basepair deletion found predicted to lead to a frameshift and premature stop codon.

Variant: NM_002860.4(ALDH18A1):c.2131del (p.Leu711fs)

ClinVar: 156546 https://www.ncbi.nlm.nih.gov/clinvar/variation/156546/

gnomAD: Not in gnomAD

Case: Patient 4/ BIODEF 263, female, 1 week

CasePresentingHPOs: HP:0008297, HP:0040210, HP:0040206

At 1 week old plasma showed Phe levels at 258 umol/L. Repeat screening at 9 days old showed Phe levels at 900 umol/L. Amino acid quantitation of plasma showd a Phe level of 1200 umol/L. Urine analysis showed low BH4, high neopterin, low biopterin, and the presence of primapterin. A diet of low protein and phenylalanine were begun, and continue with 100 mg/kg of phenylalanine per day. Patient is 6 years old and developing normally.

CaseNOTHPOs:

CasePreviousTesting: n/a 

CaseGenotypingMethod: PCR of exons 2-4 of PCBD1 followed by direct sequencing.

Variant: NM_000281.3(PCBD1):c.292C>T (p.Gln98Ter)

ClinVar ID: 16799

Homozygous

gnomAD: n/a

AddCaseInfo: No information about the family.

VariantEvidence: Expression of recombinant protein in bacteria revealed that both the Q98X variants are present in the insoluble fraction compared to wildtype which is found in the soluble. the little amount retained of each in the soluble fraction showed that they retained activity.

Case: Patient 3/ BIODEF 216, male, Morrocan, 1 month

CasePresentingHPOs: HP:0008297, HP:0040210, HP:0040206

HyperPhe presented a 1 month old with plasma levels of 1850 umol/L. A low Phe diet was initiated for 8 months. Urinary pterin profile showed high neopterin, low biopterin, and elevated primapterin. The diet was ceased at 2 years old, and the child has normal Phe levels (110 umol/L).

CaseNOTHPOs:

CasePreviousTesting: n/a

CaseGenotypingMethod: PCR of exons 2-4 of PCBD1 followed by direct sequencing.

Variant: NM_000281.3(PCBD1):c.289G>A (p.Glu97Lys)

ClinVar ID: 91907

Homozygous

gnomAD: MAF (Euro-NF)= 2.320E-5 in hets only, no homozygous. http://gnomad.broadinstitute.org/variant/10-72643733-C-T

AddCaseInfo: Parents are consanguineous. Older brother (by 17 years) presented with HyperPhe in the neonatal period, but no information on whether he was a carrier of the mutation.

VariantEvidence: Expression of recombinant protein in bacteria revealed that the E97K are present in the insoluble fraction compared to wildtype which is found in the soluble. the little amount retained of each in the soluble fraction showed that they retained activity. Per Ayling et al., 2000 (PMID:10924272), another patient (BIODEF 306) that was homozygous for the same E97K mutation, showed reduced activity of PCBD1 from duodenal mucosa cells compared to wildtype (17% activity), indicating that this mutation severely reduces the activity of PCBD1.

Case764: Female, 4 years report, Spain

CasePresentingHPOs: HP:0011344, HP:0003153, HP:0001257, HP:0003698, HP:0002474 (Severe psychomotor retardation, Cystathioninuria, Spasticity, Difficulty standing, Communication delay)

HPOsFreeText: Elevated urine cystathionine (1611 umol/g creatine; reference <46) and Elevated tHcys in urine (54.3 umol/g creatine; reference <20), Elevated plasma tHcys (20.5 umol/l; reference <7.5), Post treatment normal urine cystathionine (42 umol/g creatine)

CaseNotHPOs: HP:0003345 (Glutathione synthetase deficiency)

NotHPOsFreeText: N/A

CaseAddInfo: Patient is homozygous for this variant

CasePreviousTesting: Karyotype, magnetic resonance imaging, specific genetic tests for neurogenetic diseases and screening for inborn errors of metabolism (lactate, pyruvate, ammonium, uric acid, amino acids, organic acids, glycosaminoglycanes, creatine and guanidinoacetate, purines and pyrimidines, and isoelectric focusing pattern of transferrin), as well as folate, pyridoxine and cobalamine deficiencies, neural tumors and genetically determined homocystinurias, were all ruled out.

CaseGenotypingMethod: All twelve exons of the CTH gene and the intronic flanking sequences of the subjects and their parents was PCR amplified and directly sequenced.

Variant: NM_001902.5:c.200C>T (p.Thr67Ile)

ClinVar: 2939 https://www.ncbi.nlm.nih.gov/clinvar/variation/2939/

gnomAD: Ashkenazi Jewish 9.934e-3, Overall 6.308e-3 https://gnomad.broadinstitute.org/variant/1-70881670-C-T

Case8 Male, 9.8yo report, Ethnicity:Turkish

CasePresentingHPOs HP:0012523, HP:0001508, HP:0002013, HP:0008947, HP:0002789, HP:0002045, HP:0002014, HP:0003571

(propionicacidemia, oral aversion, failure to thrive, vomiting, infantile hypotonia, tachypnea, hypothermia, diarrhea)

HPOsFreeText feeding refusal, failure to thrive, vomiting, hypotonia, tachypnea, hypothermia, diarrhea (4do). 73 IQ on Culture Free Test.

CaseNotHPOs n/a

CasePreviousTesting Diagnosed by selective metabolic screening

Variant NM_000532.4:c.429+3_6delAAGT (p.Asp125_Lys143del), homozygous

ClinVar n/a

CAID to register

gnomAD not in gnomAD

CaseAddInfo parental consanguinity

CasePMIDs n/a

CaseVC: Male, Birth onset

CasePresentingHPOs: HP:0030082, HP:0002013, HP:0200048, HP:0001250, HP:0005949, HP:0001290, HP:0001662, HP:0008610, HP:0002120 (Abnormal drinking behavior, Vomiting, Cyanotic episode, Seizure, Apneic episodes in infancy, Generalized hypotonia, Bradycardia, Infantile sensorineural hearing impairment, Cerebral cortical atrophy)

HPOsFreeText: Birth weight of 2565 g, Length of 48 cm, Head circumference of 33 cm, Elevated excretion of TMS-derivatives of N-acetylated amino acids

CaseNotHPOs: HP:0001263 (Global developmental delay)

NotHPOsFreeText: N/A

CaseAddInfo: Parents are non-consanguineous, Proband confirmed homozygous via parental testing

CasePreviousTesting: Cerebral MRI showed abnormal signals in the cortico-subcortical zones of the fronto-parietal and temporo-occipital areas consistent with cortical laminar necrosis; Most presenting phenotypes were ameliorated after 6 weeks of age; N-acetylated amino acid excretion shown in Table 1

CaseGenotypingMethod: RT-PCR confirmed expression of aminoacylase I mRNA was significantly lowered in the proband than in controls. Southern blot and nested PCR confirmed the weak band observed was aminoacylase I mRNA and the relative expression levels. Following this, PCR amplification of all 14 coding exons and flanking introns of ACY1 was performed and sequenced.

Variant: NM_000666.3(ACY1):c.1057C>T (p.Arg353Cys)

ClinVar: 18110 https://www.ncbi.nlm.nih.gov/clinvar/variation/18110/

gnomAD: 0.004135 (4 homozygotes), https://gnomad.broadinstitute.org/variant/3-52022837-C-T

CaseAHN16.1: Australia, White

CasePresentingHPOs: HP:0008353 (Neutral hyperaminoaciduria)

HPOsFreeText: N/A

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Variants segregate with the disease

CasePreviousTesting: Hartnup aminoaciduria profile was confirmed by ion exchange high performance liquid chromatography;

CaseGenotypingMethod: All SLC6A19 exons and flanking introns were directly amplified and sequenced from genomic DNA.

Variant1: NM_001003841.3:c.277G>A (p.Gly93Arg)

CAID: CA3182636 http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA3182636

gnomAD: 0.0001195, https://gnomad.broadinstitute.org/variant/5-1208935-G-A

Variant2: NM_001003841.3(SLC6A19):c.517G>A (p.Asp173Asn)

ClinVar: 2019 https://www.ncbi.nlm.nih.gov/clinvar/variation/2019/

gnomAD: 0.003016, https://gnomad.broadinstitute.org/variant/5-1212453-G-A

CaseBE2P3: Male, 9 days onset

CasePresentingHPOs: HP:0001399, HP:0006579, HP:0001396, HP:0004914, HP:0002240, HP:0001290, HP:0007772, HP:0000347, HP:0000218, HP:0000954, HP:0012448, HP:0001684, HP:0003265, HP:0002490, HP:0002151, HP:0003235, HP:0001897, HP:0001272, HP:0002119, HP:0001263, HP:0000733, HP:0001414, HP:0002612 (Hepatic failure, Prolonged neonatal jaundice, Cholestasis, Recurrent infantile hypoglycemia, Hepatomegaly, Generalized hypotonia, Impaired smooth pursuit, Micrognathia, High palate, Single transverse palmar crease, Delayed myelination, Secundum atrial septal defect, Neonatal hyperbilirubinemia, Increased CSF lactate, Increased serum lactate, Hypermethioninemia, Normocytic anemia, Cerebellar atrophy, Ventriculomegaly, Global developmental delay, Stereotypy, Microvesicular hepatic steatosis, Congenital hepatic fibrosis)

HPOsFreeText: Increased bile salts, Biliary stones in gallbladder, Severe intrahepatic extramedullary haematopoiesis

CaseNotHPOs: HP:0001744, HP:0000842, HP:0008163 (Splenomegaly, Hyperinsulinemia, Decreased circulating cortisol level)

NotHPOsFreeText: Abnormally coloured stools

CaseAddInfo: Parents are first cousins

CasePreviousTesting: All patients received ursodeoxycholic acid (600 mg.m−2.d−1) and fat-soluble vitamin supplementation; All patients received an enriched diet with medium chain triglycerides and dextrin maltose; Sanger sequencing of POLG, DGUOK, and TRMU in P3 revealed no pathogenic variants; Mitochondrial RC defect was considered but no anomaly was found in isolated lymphocytes and in primary culture of fibroblasts

CaseGenotypingMethod: Sanger sequencing of the ADK gene revealed a homozygous termination variant. Both parents carried this in a heterozygous state and the younger healthy brother did not carry the mutation

Variant: NM_006721.3:c.519G>A (p.Trp173Ter)

CAID: CA377397711, http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/allele?hgvsOrDescriptor=NM_006721.3%3Ac.519G%3EA

gnomAD: Not in gnomAD

Case2930: Female, 3 weeks diagnosis, United States

CasePresentingHPOs: HP:0003153 (Cystathioninuria)

HPOsFreeText: Elevated total homocysteine (18.2 μM/l; reference 5.1–13.9), Elevated cystathionine (11.8 μM/l; reference 0.04–0.34), Normal total cysteine (246 μmol/l; reference 203–369)

CaseNotHPOs: HP:0001270, HP:0001263, HP:0100543, HP:0030972 (Motor delay, Global developmental delay, Cognitive impairment, Abnormal systemic blood pressure)

NotHPOsFreeText: Unresponsive to B6 treatment

CaseAddInfo: Patient is compound heterozygous for these mutations, Parental testing has confirmed these variants are in trans

CasePreviousTesting: Cystathionine (Cysta), total cysteine (tCys), and total homocysteine (tHcy) panels

CaseGenotypingMethod: PCR amplification of CTH coding regions and exon-intron boundaries was performed. These fragments were sequenced and aligned with a reference to find mutations. Those found were confirmed by restriction digest of 100 controls.

Variant1: NM_001902.5:c.932C>T (p.Thr311Ile)

CAID: CA340839077 https://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/allele?hgvs=NM_001902.5%3Ac.932C%3ET

gnomAD: Not in gnomAD

Variant Too Long

Variant2: NP_001893.2:p.Gly57_Gln196del

ClinVar:

gnomAD:

Case# Patient 1, Female, Japanese, 2 yo diagnosis

CasePresentingHPOs HP:0004923

CasePreviousTesting measuring urinarypteridines and assaying for DHPR activity. The DHPR activity inred blood cells was 6–8% of normal control

CaseMethod1 RG-PCR

Variant NM_000320.2(QDPR):c.106T>C (p.Trp36Arg)

ClinVar 492

VariantEvidence sevenfold higher DHPR activity in WT tranfected fibroblasts than W36T transfected cells(Fig. 1).

Case# Patient 2, male, Japanese, 11 yo diagnosis

CasePresentingHPOs HP:0002521, HP:0004923

CaseMethod1 RG-PCR

Variant NM_000320.2:c.461A>G

CAID CA356444851

VariantEvidence intronic splice variant (Fig. 4)

CaseAddInfo consanguineous parents, mother was heterozygous, patient and 2 brothers are homozygous

CasePMIDs 7444937

Biallelic

Case#:15, 5 months (report), Korean, female

CaseHPOs: HP:0003235, (hypermethioninemia)

CaseNotHPOsFreeText: No neurologic problems or developmental delay; no treatment or special diet.

CaseNotHPOs:

CasePreviousTesting: At 5 months and 25 months, respectively, plasma methionine 300.2, 87.7 umol (normal <50), plasma homocysteine 14.4, 5.4 umol (<15 umol)

CaseMethod1: PCR

CaseMethod2: Sequencing

CaseGenotypingMethod: All nine MAT1A exons (exon I to IX) and exon–intron boundaries were amplified by polymerase chain reaction (PCR) and directly sequenced.

Variant: c.487C>T (p.Arg163Trp) (homozygous)

ClinVar: N/A

CAID: CA5576782

gnomAD: The highest population minor allele frequency in gnomAD is 0.00006152 (African); no homozygoutes in any population.

VariantEvidence: None.

CaseAddInfo: The mother is heterozygous, the father had only the wild type allele in leukocyte DNA. Biparental inheritance was proven by microsatellite analysis suggesting that either the father is mosaic or this is a de novo variant.

CasePMIDs:

Points: 0.25 points. This individual is homozygous for a missense variant in MAT1A. The highest population minor allele frequency in gnomAD is 0.00006152 (African); no homozygotes in any population. The score is reduced because no functional evidence is available to support the deleterious impact of this variant.

Biallelic

Case#: 14, Korean, female, 58 months (report)

CaseHPOs: HP:0003235, (hypermethioninemia)

CaseNotHPOsFreeText: No neurologic problems or developmental delay; no treatment or special diet.

CasePreviousTesting: At 20 days and 58 months, respectively, Plasma methionine 1352.6, 815.2 umol (normal <50), plasma homocysteine 21.1, 19.06 umol (<15 umol)

CaseMethod1: PCR

CaseMethod2: Sequencing

CaseGenotypingMethod: All nine MAT1A exons (exon I to IX) and exon–intron boundaries were amplified by polymerase chain reaction (PCR) and directly sequenced.

Variant: 1) c.895C>T (p.Arg299Cys); 2) c.1005C>G (p.Tyr335X)

ClinVar: 1) 418870 2) N/A

CAID: 2) CA377360418

gnomAD: The highest population minor allele frequency in gnomAD for c.895C>T (p.Arg299Cys) is 0.0001431 (East Asian). c.1005C>G (p.Tyr335X) is not in gnomAD

VariantEvidence: When expressed in E. Coli, the variant resulted in reduction of methionine adenosyltransferase (MAT) and tripolyphosphatase (PPPase) activities to ~15% and ~20% of wild type respectively (Fernandez-Irigoyn et al, 2010, PMID 20675163; Fig 2).

CaseAddInfo: The mother is heterozygous for the nonsense variant, methionine levels are not available.

Points: 1 point. This individual and her sister, who also has hypermethioninemia, are compound heterozygous for a missense variant, p.Arg299Cys, and a nonsense variant in MAT1A (p.Tyr335X). While the nonsense variant occurs in the second to last exon of it is further than 50 bp from the 3’ end of the exon (~80 bp), and therefore should be identified by nonsense-mediated decay. The highest population minor allele frequency in gnomAD for c.895C>T (p.Arg299Cys) is 0.0001431 (East Asian). c.1005C>G (p.Tyr335X) is not in gnomAD. The score is reduced because other causes of hypermethioninemia have not been ruled out.

CaseKDC23: Male, 38 yrs onset, Korea, Asian

CasePresentingHPOs: HP:0002131, HP:0002321, HP:0001260, HP:0000640, HP:0011477, HP:0100543 (Episodic ataxia, Vertigo, Dysarthria, Gaze-evoked nystagmus, Upbeat nystagmus, Cognitive impairment)

HPOsFreeText: N/A

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Proband has no family history of disease

CasePreviousTesting: Negtive response to acetazolamide

CaseGenotypingMethod: Next-generation sequencing was perfomed on all probands with 40 candidate genes and five known EA genes.

Variant: NM_004172.5:c.952A>G (p.Thr318Ala)

CAID: CA3235581, http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA3235581

gnomAD: 0.002656, https://gnomad.broadinstitute.org/variant/5-36679820-A-G NOTE EXTREMELY HIGH gnomAD FREQUENCY

CaseP1 Male, 4.5yr report, Austrian

CasePresentingHPOs HP:0010818, HP:0010851, HP:0001290, HP:0011343, HP:0007359, HP:0001942, HP:0002376, HP:0001290

(Generalized tonic seizures, EEG with burst suppression, Hypotonia, moderate GDD, focal seizures, Metabolic acidosis, Developmental regression, Hypotonia)

HPOsFreeText born 40wk gestation. (neonatal, pre-treatment) apgar scores at 1min:8, 5min:9, 10min: 10, generalized tonic fits at <1d old and burst suppression EEG. (3d:) introduced pyridoxine therapy, 100mg I/v trial dose/route, complete cessation of seizures, apnoea(3d), hypotonia, sleepiness, initial maintenance daily dose of 10 mg/kg. (infancy/childhood:) hypotonia, moderate IQ delay. (4d:) withdrew from pyridoxine trial then focal seizures recurrenced 3 weeks after(definite PDE). (PMID: 0894227) gestational toxicosis, birth weight 3,260g, myoclonic jerks of both arms with hours of birth.Paroxysms of polyspike-wave-activity on EEG, normal cranial imaging,CSF analysis

CaseNotHPOs n/a

CasePreviousTesting Administration of pyridoxine relieved patient's seizures. CSF pipecolic acid: 3.4(nv<0.15μM), urine AASA: 168 before treatment(nv<1 mmol/mol creatinine). Blood lactate: 4.0mM and CSF lactate: 4.2mM (nv <2.5mM). (PMID:17068770:)post treatment testing: Pipecolic acid in plasma 4.7(nv 0.7-2.6mmol/liter), AASA in plasma 2.6(nv<0.2mmol/liter), urine AASA 17(nv<1mmol/molcreatinine). (PMID: 0894227) CSF analysis, organic acids in urine, amino acids in serum, and VLCFAs were normal. Plasma PA at 1st week 49µmol/L(before treatment)(nv 1-3.2), at 1-6mo 14.3µmol/L, 6-12mo 10.1µmol/L, CSF PA at 0-1yr 3.38µmol/L.

Variant NM_001182.4(ALDH7A1):c.1279G>C (p.Glu427Gln), homozygous

ClinVar 17994

gnomAD MAF= 0.0006123(European(Non-Finnish)), no homozygotes

CaseAddInfo n/a

CasePMIDs 10894227 (Patient 2), 17068770 (Patient 11)

Patient1: Male, 6yo

CasePresentingHPOs:

HPOsFreeText: Blood C5-carnitine (μM) = 18.96, under a protein restricted diet with carnitine and glycine supplementation, normal development

CaseNotHPOs:

NotHPOsFreeText:

CaseAddInfo: 10days old and asymptomatic at diagnosis

CasePMIDs:

CasePreviousTesting: Blood amino acids and acylcarnitine analyzed with ESI-MS/MS, urine organic acid analysis with GC/MS

GroupMethod1: PCR (primers in table 2)

GroupMethod2: Direct sequencing

CaseGenotypingMethod: genomic DNA extracted from peripheral blood leukocytes, IVD exons amplified via PCR, then products were purified and underwent direct sequencing for variant analysis

Variant1: NM_002225.3:c.386A>G (p.Tyr99Cys) listed in paper as (p.His100Arg)

CAID: CA391716511, http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA391716511

gnomAD: n/a

Variant2: NM_002225.4(IVD):c.1199A>G (p.Tyr400Cys) listed in paper as (p.Tyr371Cys)

ClinVar: 371390, https://www.ncbi.nlm.nih.gov/clinvar/variation/371390/

gnomAD: n/a

Patient5-1: Female, 2.5yo, sibling to patient 5-2

CasePresentingHPOs:

HPOsFreeText: under a protein restricted diet with carnitine and glycine supplementation, normal development

CaseNotHPOs:

NotHPOsFreeText:

CaseAddInfo: 2mo diagnosis

CasePMIDs:

CasePreviousTesting: Blood amino acids and acylcarnitine analyzed with ESI-MS/MS, urine organic acid analysis with GC/MS

GroupMethod1: PCR (primers in table 2)

GroupMethod2: Direct sequencing

CaseGenotypingMethod: genomic DNA extracted from peripheral blood leukocytes, IVD exons amplified via PCR, then products were purified and underwent direct sequencing for variant analysis

Variant1: NM_002225.4:c.347C>T (p.Ser89Phe) listed in paper as (p.Ser87Phe)

CAID: CA391716394, http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA391716394

gnomAD: n/a

Variant2: NM_002225.4(IVD):c.1199A>G (p.Tyr400Cys) listed in paper as (p.Tyr371Cys)

ClinVar: 371390, https://www.ncbi.nlm.nih.gov/clinvar/variation/371390/

gnomAD: n/a

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# Patient 6, Female, 8mo diagnosis

Variant NM_000320.2:c.148G>A (p.G23D)

CAID CA2868208

gnomAD 0.001332 (African)

CaseGM01454: Male, 7 years report, Asian, China

CasePresentingHPOs: HP:0003153 (Cystathioninuria)

HPOsFreeText: N/A

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Proband is a homozygote for this variant.

CasePreviousTesting: N/A

CaseGenotypingMethod: Primers were developed and the CTH exons 1-12 and exon-intron boundaries were PCR amplified and sequenced. All sequence variations were verified by restriction enzyme analysis and/or repeat sequencing.

Variant: CTH, 2-BP DEL, 940CT AKA: NM_001902.5:c.784_785del (p.Leu262Thrfs*21)

ClinVar: 2937 https://www.ncbi.nlm.nih.gov/clinvar/variation/2937/

gnomAD: East Asian 1.088e-4, Overall 7.960e-6 https://gnomad.broadinstitute.org/variant/1-70897822-ACT-A

Case14 age/gender unknown, Caucasian, Bosnia

CasePresentingHPOs HP:0001250

(Seizures)

HPOsFreeText seizure onset in neonatal period from minutes after birth until 3 weeks of age. Definite PDE: formal pyridoxine withdrawal had resulted in reoccurrence of seizures that again responded to pyridoxine.

CaseNotHPOs n/a

CasePreviousTesting post treatment testing: Pipecolic acid in plasma 2.8(nv 0.7-2.6µmol/liter), AASA in plasma 0.5(nv<0.2µmol/liter), urine AASA 1.6(nv<1mmol/molcreatinine). Normal urine PA after pyridoxine treatment.

Variant 1. NM_001182.4:c.605G>T (p.Gly202Val ), 2. NM_001182.4:c.1184T>G (p.Val395Gly), compound heterozygous

ClinVar n/a

CAID 1. CA360731073, 2. CA360723766

gnomAD neither in gnomAD

CaseAddInfo n/a

CasePMIDs n/a

Case13 age/gender unknown, Caucasian, Germany

CasePresentingHPOs HP:0001250

(Seizures)

HPOsFreeText seizure onset in neonatal period from minutes after birth until 3 weeks of age. Possible PDE: no trial of pyridoxine withdrawal performed and no antecedent affected.

CaseNotHPOs n/a

CasePreviousTesting pre-treatment testing: Pipecolic acid in plasma 53(nv 1-3.2µmol/liter), PA in urine 410µmol/mmol creati-nine(nv 0.55–24.1), AASA in plasma 14(nv<0.2µmol/liter), urine AASA 342(nv<1mmol/molcreatinine).

Variant NM_001182.4(ALDH7A1):c.1279G>C (p.Glu427Gln), homozygous

ClinVar 17994

gnomAD not in gnomAD

CaseAddInfo n/a

CasePMIDs n/a

Case1 age/gender unknown, Caucasian, German

CasePresentingHPOs HP:0001250

(Seizures)

HPOsFreeText seizure onset in neonatal period from minutes after birth until 3 weeks of age. Possible PDE: no trial of pyridoxine withdrawal performed and no antecedent affected

CaseNotHPOs n/a

CasePreviousTesting post treatment testing: Pipecolic acid in plasma 16(nv 0.4-4.9µmol/liter), AASA in plasma 3.9(nv<0.2µmol/liter), urine AASA 62(nv<1mmol/molcreatinine). Normal urine PA after pyridoxine treatment.

Variant 1. NM_001182.4(ALDH7A1):c.902A>T (p.Asn301Ile), 2. NM_001182.4(ALDH7A1):c.1279G>C (p.Glu427Gln), compound heterozygous

ClinVar 1. 18001, 2. 17994

gnomAD neither in gnomAD

CaseAddInfo n/a

CasePMIDs n/a

Case12 age/gender unknown, Caucasian, Italy

CasePresentingHPOs HP:0001250

(Seizures)

HPOsFreeText seizure onset in neonatal period from minutes after birth until 3 weeks of age. Definite PDE: formal pyridoxine withdrawal had resulted in reoccurrence of seizures that again responded to pyridoxine.

CaseNotHPOs n/a

CasePreviousTesting post treatment testing: Pipecolic acid in plasma 5.4(nv 0.7-2.6µmol/liter), AASA in plasma 9.6(nv<0.2µmol/liter), urine AASA 24(nv<1mmol/molcreatinine). Normal urine PA after pyridoxine treatment.

Variant NM_001182.4:c.1003C>T (p.Arg335Ter), homozygous

ClinVar n/a

CAID CA126889823

gnomAD MAF= 0.0001307(South Asian), no homozygotes

CaseAddInfo n/a

CasePMIDs n/a

Patient4: Male, 2.5yo

CasePresentingHPOs: HP:0002033, HP:0001254, HP:0001987, HP:0001873, HP:0002013 (poor sucking, lethargy, hyperammonaemia, thrombocytopenia, vomiting)

HPOsFreeText: acute neonatal IVA, treated with IVA special formula along with sodium benzoate and glycine (benzoate and glycine discontinued after 1yr), elevated isovalerylglycine, C5-carnitine = 17.4 µmol/l, birth weight = 3kg, able to sit at 6mo, walked at 13mo, began to babble at 17mo, did not have bladder/bowel control until 2.5yo, current weight = 14kg, current height = 86.5cm

CaseNotHPOs:

NotHPOsFreeText:

CaseAddInfo: symptom onset at 5do, diagnosis at 30do via clinical presentation, consanguineous family

CasePMIDs:

CasePreviousTesting: same as group

CaseGenotypingMethod: same as group

Variant1: NM_002225.4(IVD):c.863C>T (p.Ala288Val) listed in paper as c.872C>T (p.Ala291Val)

ClinVar: 281076, https://www.ncbi.nlm.nih.gov/clinvar/variation/281076/

gnomAD: n/a

Variant2: NC_000015.9:g.40699940T>C (NM_002225.4(IVD):c.234+14C=)

CAID: CA147572, http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/allele?hgvs=NC_000015.9%3Ag.40699940T%3EC

ClinVar: 94053, https://www.ncbi.nlm.nih.gov/clinvar/variation/94053/

gnomAD: 0.6830, https://gnomad.broadinstitute.org/variant/15-40699940-T-C

Patient1: Male, 4yo, Twin A, Family A (fig. 2)

CasePresentingHPOs:

HPOsFreeText: acute neonatal IVA, treated with IVA special formula, elevated isovalerylglycine in urine organic acids, C5-carnitine = 6.15 µmol/l, at 2yo stopped formula and switched to vegetarian diet with L-carnitine supplements, admitted every 3-4mo due to metabolic crisis, at 4yo began formula again along with vegetarian diet and L-carnitine supplements, sat alone at 6mo, walked alone at 1yo, spoke first word at 1yo, unremarkable systemic examination at 4yo

CaseNotHPOs:

NotHPOsFreeText:

CaseAddInfo: diagnosed at 3 days old via NBS (positive family history and 3 sibling deaths), consanguineous family

CasePMIDs: PMID: 24516753

CasePreviousTesting: same as group

CaseGenotypingMethod: same as group

Variant1: NM_002225.4(IVD):c.1184G>A (p.Arg395Gln) listed in paper as c.1193G>A (p.R398Q)

ClinVar: 529434, https://www.ncbi.nlm.nih.gov/clinvar/variation/529434/

gnomAD: n/a

Variant2: NC_000015.9:g.40699940T>C (NM_002225.4(IVD):c.234+14C=)

CAID: CA147572, http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/allele?hgvs=NC_000015.9%3Ag.40699940T%3EC

ClinVar: 94053, https://www.ncbi.nlm.nih.gov/clinvar/variation/94053/

gnomAD: 0.6830, https://gnomad.broadinstitute.org/variant/15-40699940-T-C

CaseTS: Male, Poland

CasePresentingHPOs: HP:0000256, HP:0001263, HP:0000729, HP:0001992 (Macrocephaly, Global developmental delay, Autistic behavior, Organic aciduria)

HPOsFreeText: Apgar score of 9

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Parents are non-consanguineous though grandfathers were from neighboring villages, Older brother is healthy, Proband is confirmed homozygous via parental testing

CasePreviousTesting: Biochemical tests included urinary organic acid analysis, amino acid analysis, transaminases, transferrin isoforms, phosphoribosylaminoimidazole-succinocarboxamide (SAICAr), and D-arabinitol; Urinary organic acids analysis by GC-MS was performed repeatedly and revealed N-acetylated derivatives of methionine, glutamic acid, alanine, and glycine in large amounts, as well as leucine, valine, and isoleucine in smaller amounts with brother and parent analysis being unremarkable

CaseGenotypingMethod: PCR amplification and bidirectional sequencing of all exons and exon-intron boundaries was performed on genomic DNA.

Variant: NM_000666.3(ACY1):c.1057C>T (p.Arg353Cys)

ClinVar: 18110 https://www.ncbi.nlm.nih.gov/clinvar/variation/18110/

gnomAD: 0.004135 (4 homozygotes), https://gnomad.broadinstitute.org/variant/3-52022837-C-T

CaseOS-120_II-1: Female, Birth onset, German/Italian

CasePresentingHPOs: HP:0001992, HP:0011343 (Organic aciduria, Moderate global developmental delay)

HPOsFreeText: N/A

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Younger brother is unaffected; Proband is confirmed compound heterozygous as mother is heterozygous for R353C but father is unavailable

CasePreviousTesting: GS-MS analysis identified urinary organic acids dominated by N-aceylated AAs, supported by NMR spectroscopy of urine samples with serum samples being negative; Values of excreted AAs are in Table 1; Neuropediatric evaluation performed

CaseGenotypingMethod: Genomic DNA was extracted from blood samples or after EBV transformation of peripheral blood lymphocytes. Exons and exon-intron boundaries were amplified and bidirectionally sequenced.

Variant1: NM_000666.3(ACY1):c.699A>C (p.Glu233Asp)

ClinVar: 18111, https://www.ncbi.nlm.nih.gov/clinvar/variation/18111/

gnomAD: 0.0001846, https://gnomad.broadinstitute.org/variant/3-52021204-A-C

Variant2: NM_000666.3(ACY1):c.1057C>T (p.Arg353Cys)

ClinVar: 18110, https://www.ncbi.nlm.nih.gov/clinvar/variation/18110/

gnomAD: 0.004135 (4 homozygotes), https://gnomad.broadinstitute.org/variant/3-52022837-C-T

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.

CaseF-2 male, 14mo onset, Ethnicity: North Indian, Hindu

CasePresentingHPOs HP:0001263, HP:0003571

(Developmental delay, Propionicacidemia)

HPOsFreeText normal plasma amino acids, acylcarnitines 13.9(normal <3.6nmol/mL), normal development by 6.5 years

CaseNotHPOs HP:0001942, HP:0001987

(Metabolic acidosis, Hyperammonemia)

CasePreviousTesting Also screened for variants in PCCA

Variant NM_000532.4(PCCB):c.1210G>A (p.Glu404Lys)

ClinVar 217891

gnomAD MAF= 0.00003266(South Asian), no homozygotes

CaseAddInfo nonconsanguineous, no abnormality in family history noted

CasePMIDs n/a

Case1: Newborn

CasePresentingHPOs:

HPOFreeText: Identified by NBS and is healthy with normal development.

CaseNotHPOs:

CasePreviousTesting: DBS C5 carnitine 1.2, 2.5, 1.1 umol/L (ref <0.46); plasma alloisoleucine 2.6 umol/L (ref <2.1); urine C5 carnitine 84.4, 58.1, 64.8 umol/mmol creatine (ref <3.0); urine 2-EHA 36, 31, 22 U (ref <5); urine 2-MBG 36.3, 29.3, 35.2 (ref <2.3); urine 2-methylbutyric acid conjugates as R-isomer 45%, 40% (ref 43-56%).

CaseGenotypingMethod: PCR amplification and sequencing of genomic DNA (entire coding region) from patient and available family members.

Variant: 1. NM_001609.3:c.1165A>G (p.Met389Val), 2. NM_001609.3:c.848A>G (p.Tyr283Cys)

CAID: 1. CA5730948; 2. CA5730830

gnomAD: 1. East Asian 0.0005513; 2. East Asian 0.0007068

VariantEvidence:

CaseAddInfo: In trans

CasePMIDs:

Case1: Female, Mixed, Birth onset

CasePresentingHPOs: HP:0001508, HP:0000252, HP:0002076, HP:0002018, HP:0002013, HP:0011342, HP:0000750, HP:0001270, HP:0007018 (Failure to thrive, Microcephaly, Migraine, Nausea, Emesis, Mild global developmental delay, Delayed speech and language development, Motor delay, Attention deficit hyperactivity disorder)

HPOsFreeText: Seizure-like episodes, Persistent head tilting, Symptoms impreoved over time

CaseNotHPOs: HP:0001252 (Muscular hypotonia)

NotHPOsFreeText: No brain MRI abnormalities

CaseAddInfo: Parents are non-consanguineous

CasePreviousTesting: Born at full term after a pregnancy complicated by maternal hypertension with no postnatal complications; Elevated levels of plasma 2-aminoadipate (37 μmol/L, nl < 4) with elevated 2-ketoadipate (434 mmol/mol creatinine, nl < 2) and 2-hydroxyadipate (28 mmol/mol creatinine, nl < 2) in urine; Electroencephalogram, magnetic resonance imaging, and magnetic resonance angiogram of the brain were unremarkable

CaseGenotypingMethod: Whole exome sequencing was performed as a trio with both parents. Identified DHTKD1 mutations were evaluated for possible effects and filtered for frequencies and predicted pathogenicity. No other candidate genes were found during the WES analysis for either patient sequenced.

Variant1: NM_018706.6:c.2143C>T (p.Arg715Cys)

ClinVar: 242503 https://www.ncbi.nlm.nih.gov/clinvar/variation/242503/

gnomAD: 0.0002007, https://gnomad.broadinstitute.org/variant/10-12150003-C-T

Variant2: NM_018706.6(DHTKD1):c.2185G>A (p.Gly729Arg)

ClinVar: 39564 https://www.ncbi.nlm.nih.gov/clinvar/variation/39564/

gnomAD: 0.002748, https://gnomad.broadinstitute.org/variant/10-12154929-G-A

Case4 Female, 7yo diagnosis, Korean

CasePresentingHPOs HP:0001083, HP:0001249, HP:0003235

(Ectopia lentis, Intellectual disability, Increased blood methionine)

HPOsFreeText Marfan syndrome, vascular occlusion, increased pre-treatment blood homocysteine and post-treatment total homocysteine, patient treated with low-methionine diet, vitamin B6, folate, vitamin B12, and betaine after diagnosis

CaseNotHPOs n/a

NotHPOsFreeText n/a

CasePreviousTesting pretreatment/posttreatment methionine: 24/575 μM(nv 7-47), pretreatment/posttreatment: 34/0 μM(nv 0-5), posttreatment total homocysteine 194 μM(nv 5-15)

CaseMethod1 PCR

CaseMethod2 Sanger

CaseGenotypingMethod PCR amplified all 16 coding exons of CBS gene, then direct sequenced

Variant 1. NM_000071.2(CBS):c.1006C>T (p.Arg336Cys), 2. NM_000071.2(CBS):c.1058C>T (p.Thr353Met), compound heterozygous

ClinVar 1. 92423, 2. 131

gnomAD 1. MAF=0.00004417(European(non-Finnish)), no homozygotes, 2. MAF=0.0002916(African), no homozygotes

CaseAddInfo R336C variant was paternally inherited, T353M variant was maternally inherited

CasePMIDs n/a

CasePA008 10mo report, Taiwan

CasePresentingHPOs HP:0001987, HP:0001943, HP:0011342, HP:0003571

(Hyperammonemia, Hypoglycemia, mild global developmental delay, propionicacidemia)

HPOsFreeText hyperammonemia and hypoglycemia for a few days after birth. Showed mild developmental delay at 10 months old

CaseNotHPOs n/a

CasePreviousTesting coding regions and flanking areas of PCCA also analyzed with PCR and Sanger

Variant 1. NM_000532.4(PCCB):c.1301C>T (p.Ala434Val), 2. NM_000532.4:c.-4156_183+3713del

ClinVar 1. 554490, 2. n/a

CAID 2. n/a

gnomAD 1. MAF=0.0003510(East Asian), no homozygotes, 2. n/a

CaseAddInfo n/a

CasePMIDs n/a

Case2: European

CasePresentingHPOs:

HPOFreeText: Identified by NBS with elevated C5-carnitine levels. Asymptomatic up to 3 year follow up. Levels of C5-carnitine decreased with time.

CaseNotHPOs:

CasePreviousTesting: Followup C5 carnitine 1.89 nM/ml (ref <0.63). Additional testing was performed on patients identified by NBS: all either had elevation of 2-methylbutyrlglycine in urine (with no intermediates consistent with isovaleric acidemia), or increase in 2-methylbutyrylcarnitine in culture from fibroblasts.

CaseGenotypingMethod: Bidirectional sequence analysis of genomic DNA.

Variant: 1. NM_001609.3(ACADSB):c.443C>T (p.Thr148Ile) 2. NM_001609.3(ACADSB):c.1159G>A (p.Glu387Lys)

ClinVar: 1. 9202 2. 9203

gnomAD: 1. South Asian 0.002678 2. Latino 0.0004797

VariantEvidence: 1. Decreases ACADSB enzymatic activity to 2.8% of wild type in vitro 2. In vitro expression of c.1159G>A in E. coli shows no protein on Western blot, reduced enzymatic activity to 2% and defects in protein folding and stabilit

CaseAddInfo: No info on cis or trans. Also heterozygous for p.R13K polymorphism.

CasePMIDs:

Case4:

CasePresentingHPOs:

HPOFreeText: Identified by NBS with elevated C5-carnitine levels. Asymptomatic up to 3 year follow up. Levels of C5-carnitine decreased with time.

CaseNotHPOs:

CasePreviousTesting: Followup C5 carnitine 2.58 nM/ml (ref <0.63). Additional testing was performed on patients identified by NBS: all either had elevation of 2-methylbutyrlglycine in urine (with no intermediates consistent with isovaleric acidemia), or increase in 2-methylbutyrylcarnitine in culture from fibroblasts.

CaseGenotypingMethod: Bidirectional sequence analysis of genomic DNA.

Variant: NM_001609.3(ACADSB):c.303+1G>A (homozygous)

ClinVar: 203367

gnomAD: Other 0.0005549

VariantEvidence: Canonical splice site variant. Discrupts canonical splicing signal of intron 3.

CaseAddInfo:

CasePMIDs:

CaseII-2: Female, Japanese, 4 years death

CasePresentingHPOs: HP:0001290, HP:0001263, HP:0002453, HP:0004372, HP:0001942, HP:0045011, HP:0002339, HP:0002138, HP:0002795, HP:0007695, HP:0008000, HP:0001635, HP:0001942 (Generalized hypotonia, Developmental delay, Abnormality of the globus pallidus, Lowered consciousness, Metabolic acidosis, Decreased urine bicarbonate concentration, Abnormality of the caudate nucleus, Subarachnoid hemorrhage, Abnormal respiration, Abnormal pupillary light reflex, Decreased corneal reflex, Heart failure, Metabolic acidosis)

HPOsFreeText: Her birth weight was 2714 g, She measured 44 cm (− 2.6 SD) in length, Had an occipitofrontal circumference of 32.5 cm (− 0.5 SD), Could hold her head up at 5 months and roll over at 10 months, Febrile illness at 15 months, Required ventilator support, Absence of brain activity, Auditory brainstem response audiometry showed no response

CaseNotHPOs: HP:0009782, HP:0001250, HP:0001332 (Aplasia/Hypoplasia of the biceps, Seizures, Dystonia)

NotHPOsFreeText:

CaseAddInfo: Parents are nonconsanguineous, Older sister of patient II-2 who is also homozygous for the mutation and has the disorder, Parents are confirmed heterozygous for the mutation

CasePreviousTesting: Lactate and pyruvate levels in arterial blood (16.5 mg/dL and 1.48 mg/dL, respectively; normal range: 5–20 mg/dL and 0.3–0.9 mg/dL, respectively) and cerebrospinal liquor (13.5 mg/dL and 1.12 mg/dL, respectively; normal range: 9.1–18.8 mg/dL and 0.26–1.32 mg/dL, respectively) were almost normal, Arterial blood ketone bodies, pH, pCO2, and base excess were (8700 μmol/L; normal range: < 130 μmol/L), 7.051, 6.7 mm Hg, and − 26.9 mEq/L, respectively, CSF lactate at 1.5 (< 2.0 mmol/L reference)

CaseGenotypingMethod: Whole exome analysis was performed on the parents and the proband. Afterwards, genomic DNA was extracted from peripheral blood leukocytes and targeted sequences were amplified and sequenced. All SNVs and Indels were evaluated and filtered to confirm the mutations. Afterwards, after identifying the exon 4 mutation, a DNA segment was amplified via PCR and sequenced to confirm these findings.

Variant: NM_014362.3:c.287C>A (p.Ala96Asp)

CAID: CA349896230 http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/allele?hgvs=NM_014362.3%3Ac.287C%3EA

gnomAD: Not in gnomAD

Case1: Male, 3 months onset, Pakistan,

CasePresentingHPOs: HP:0002376, HP:0008947, HP:0001336, HP:0002197, HP:0002490, HP:0012240, HP:0002572, HP:0000737, HP:0002360, HP:0002453, HP:0002352, HP:0002283, HP:0001332, HP:0002871, HP:0000648, HP:0000365, HP:0000252 (Developmental regression, Infantile hypotonia, Myoclonic jerks, Generalized seizures, Increased CSF lactate, Increased muscle lipid droplets, Episodic vomiting, Irritability, Sleep disturbance, Abnormality of the globus pallidus, Leukoencephalopathy, Global brain atrophy, Dystonia, Central apnea, Optic atrophy, Hearing loss, Microcephaly)

HPOsFreeText: Born at term weighing 3.2 kg, Loss of smile

CaseNotHPOs: HP:0002151, HP:0003200, HP:0003688, HP:0010663, HP:0006846 (Increased serum lactate, Ragged-red fibres, Cytochrome C oxidase-negative muscle fibers, Abnormality of thalamus morphology, Acute encephalopathy)

NotHPOsFreeText: N/A

CaseAddInfo: Parents were distantly related, Died at 3 years, Has a younger brother with the same variant, Has a family history of neurological/neuromuscular disorders, Variant was confirmed by parental testing

CasePreviousTesting: At 8 months Nissen fundoplication was performed because of persistent vomiting, Mildly elevated CSF lactate at 2.2 mmol/l (reference range <2 mmol/l) at 8 months, with a normal venous blood lactate of 1.7 mmol/l (reference range < 1.8 mmol/l), Complex I at 0.068 (Reference; 0.104-0.268 ratio to citrate synthase activity), Complexes II + III at 0.010 (0.040-0.204 ratio to citrate synthase activity), Complex IV (COX) at 0.010 (0.014-0.034 ratio to citrate synthase activity)

CaseGenotypingMethod: Genomic DNA was extracted from skin fibroblasts. Exons and flanking intronic sequences of HIBCH was sequenced after PCR amplification. Sequence data was compared to the reference sequence and filtered for variants.

Variant: NM_014362.3:c.950G>A (p.Gly317Glu)

ClinVar: 187864 https://www.ncbi.nlm.nih.gov/clinvar/variation/187864/

gnomAD: Not in gnomAD

Case#: KM24, M, 7 months (Dx), Japanese

CasePresentingHPOs: HP:0001945, HP:0001250, HP:0002013, HP:0002014, HP:0001254 (Fever, Seizures,Vomiting, Diarrhea, Lethargy)

HPOsFreeText: Newborn screening for MSUD was negative. Diagnosis of intermittent MSUD was established by blood and urine analysis after an illness.

CaseNotHPOs: HP:0001263 (Global development delay)

CasePreviousTesting: Lymphoblastoid cells from the patient showed reduced decarboxylation activity (~20% of control).

CaseMethod1: PCR

CaseMethod2: Sequencing

CaseGenotypingMethod: cDNAs from the patient were amplified, subcloned and sequenced. Confirmed by restriction enzyme digestion.

Variant: NM_001918.3(DBT):c.1448G>T (p.Ter483Leu) (homozygous)

ClinVar: 11947

gnomAD: Not in gnomAD

VariantEvidence: The variant adds 7 amino acids at the carboxyl terminus of the protein. No functional evidence.

CaseAddInfo: Consanguineous, parents and 2 siblings were heterozygous.