Supplementary Tables 1 and 2 combined information:

Case28: GT28, Female, 6 years old

DiseaseAssertion: Glanzmann thrombasthenia

FamilyInfo: Not available per supplementary table 1

ParentalGenotype: Parents are not consanguineous, no other genotype information available

CasePresentingHPOs: HP:0000978, HP:0000979, HP:0031364, HP:0000421, HP:0000225, HP:0002239

(Bruising, purpura, ecchymosis, epistaxis, gingival bleeding, gastrointestinal bleeding)

CaseHPOFreeText: Post traumatic hemorrhage

CaseNotHPOs: N/A

CaseNotHPOsFreeText: N/A

ProteinExpression: Showed 10-20% of αIIbβ3 expression measured by flow cytometry

PlateletAggregation: Platelet aggregation showed no response to ADP and collagen but normal response to ristocetin

CasePreviousTesting: All exons from ITGA2B and ITGB3 genes were amplified by PCR using intronic primers. DNA sequencing was determined by Sanger sequencing.

Gene: ITGB3

Variant: NM_000212.2:c.1728del (p.S577Pfs*92) and NM_000212.2:c.892C>T (p.Gln298Ter)

LegacyVariant: N/A

ClinVar: N/A

CAiD: CA915940253 (p.S577Pfs*92) and CA400025098 (p.Gln298Ter)

gnomAD: N/A

PMID: 22513797

Gene: ITGA2B

HGNC: 6138

Case1: (Patient 1) Woman (adult), age not specific, France

DiseaseAssertion: Glanzmann thrombasthenia (also have undergone deep vein thrombosis)

FamilyInfo: N/A

ParentalGenotype: N/A

CasePresentingHPOs: N/A

CaseHPOFreeText: Moderate bleeding according to Table 1

CaseNotHPOs: N/A

ProteinExpression: Trace amounts of residual alphaIIbbeta3 (this is all that was mentioned)

Platelet Aggregation: Absent platelet aggregation (this is all that was mentioned)

CasePreviousTesting: N/A

Gene: ITGB3

Variant: NM_000212.2:c.1448G>A (p.Cys483Tyr)

LegacyVariant: (p.Cys457Tyr)

ClinVar: N/A

CAiD: CA400029324

gnomAD N/A

PreviouslyPublished: 12152649 and 21848888

PMID: 32237906

Case5: Patient HM, gender, age and ethnicity not specified

DiseaseAssertion: Glanzmann thrombasthenia type I

FamilyInfo: N/A

ParentalGenotype: N/A

CasePresentingHPOs: N/A

CaseHPOFreeText: Bleeding symptoms were evaluated by examining available hospital records. Mild bleeders were defined as those who had minor symptoms, such as epistaxis or gum bleeds, and moderate bleeders suffered, in addition, from bleeding complications after surgery and trauma. Severe bleeders were defined as those with a history of spontaneous or life-threatening hemorrhages, such as gastrointestinal bleeding, or who had repeated episodes requiring platelet transfusion. See Table 1.

CaseNotHPOs: N/A

CaseNotHPOsFreeText: N/A

ProteinExpression: See Table 6

PlateletAggregation: See Table 6

CasePreviousTesting: To ascertain the mutations, we sequenced the coding regions of the αIIb and β3 subunits including the exon/intron boundaries

Gene: ITGA2B

Variant: NM_000419.3:c.1878G>C

LegacyVariant: N/A

ClinVar: 2903

CAiD: CA115852

gnomAD: N/A

Case31: 2 mo (at biopsy), Indian

Case 31 (Level 2)

CasePreviousTesting: same as group. found 0.7 nmol/mg protein/18 h propionate incorporation, 1.9 nmol/mg/protein/18 h propionate incorporation with hydroxocobalamin supplementation

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant1: NM_000255.3:c.257C>T (p.Pro86Leu)

ClinVar: 553773

CAID: CA3847150

gnomAD: 0.00009799 (South Asian)

CaseAddInfo: mut- type, homozygous mutations. documented consanguinity.

Case30: 6 wks (at biopsy), Pakistani

Case 30 (Level 2)

CasePreviousTesting: same as group. found 0.8 nmol/mg protein/18 h propionate incorporation, 0.7 nmol/mg/protein/18 h propionate incorporation with hydroxocobalamin supplementation

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant1: NM_000255.3:c.55dupG (p.Val19Glyfs)

ClinVar: 222906

CAID: CA10575888

gnomAD: n/a

CaseAddInfo: mut0 type, homozygous mutations. novel variant.

Case28: 1 y 1 mo (at biopsy), European

Case 28 (Level 2)

CasePreviousTesting: same as group. found 1.1 nmol/mg protein/18 h propionate incorporation, 1.2 nmol/mg/protein/18 h propionate incorporation with hydroxocobalamin supplementation

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant1: NM_000255.3:c.1181dupT (p.Leu394Phefs)

ClinVar: 222930

CAID: CA10575868

gnomAD: n/a

Variant2: NM_000255.3:c.1975C>T (p.Gln659Ter)

ClinVar: 222939

CAID: CA10575859

gnomAD: n/a

CaseAddInfo: mut0 type. both novel mutations. heterozygous mutations, unknown cis or trans.

Case27: no age or ethnicity recorded

Case 27 (Level 2)

CasePreviousTesting: same as group. found 0.4 nmol/mg protein/18 h propionate incorporation, 0.4 nmol/mg/protein/18 h propionate incorporation with hydroxocobalamin supplementation

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant1: NM_000255.3:c.1061delCinsGGA (p.Ser354TrpfsTer20)

ClinVar: n/a

CAID: CA913187308

gnomAD: n/a

Variant2: NM_000255.3:c.1065_1068dupATGG (p.Ser357MetfsTer5)

ClinVar: 280817

CAID: CA10602989

gnomAD: n/a

CaseAddInfo: mut0 type. both novel mutations. heterozygous mutations. Examination of NGS reads determined mutations are in trans

Case26: 7 y (at biopsy), Hispanic

Case 26 (Level 2)

CasePreviousTesting: same as group. found 4.5 nmol/mg protein/18 h propionate incorporation, 7.2 nmol/mg/protein/18 h propionate incorporation with hydroxocobalamin supplementation

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant1: NM_000255.3:c.970G>A (p.Ala324Thr)

ClinVar: 550893

CAID: CA364400785

gnomAD: 0.0001632 (Other)

Variant2: NM_000255.3:c.1846C>T (p.Arg616Cys)

ClinVar: 551499

CAID: CA3846722

gnomAD: 0.00008688 (Latino)

CaseAddInfo: mut- type, heterozygous mutations, unknown cis or trans. Protein change in paper (p.P649L) is different than protein change indicated by clin var and CAID (p.Arg616Cys).

Case29: 3 mo (at biopsy), Chinese

Case 29 (Level 2)

CasePreviousTesting: same as group. found 0.3 nmol/mg protein/18 h propionate incorporation, 0.6 nmol/mg/protein/18 h propionate incorporation with hydroxocobalamin supplementation

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant1: NM_000255.3:c.1280G>A (p.Gly427Asp)

ClinVar: 218990

CAID: CA347873

gnomAD: 0.0004011 (East Asian)

Variant2: Exon 13 deletion

ClinVar: n/a

CAID: n/a

gnomAD: n/a

CaseAddInfo: Variant 2 is novel. Heterozygous mutations in MUT, unknown cis or trans. Additional variant found in TCN2 gene (NM_000355.3:c.509G>A, ClinVar: 341197, CAID: CA10184685, gnomAD: 0.007469 (East Asian)). heterozygous Exon 13 deletion confirmed by microarray-based comparative genomic hybridization.

Case25: 25 y (at biopsy), White

Case 25 (Level 2)

CasePreviousTesting: same as group. found 1.3 nmol/mg protein/18 h propionate incorporation, 1.2 nmol/mg/protein/18 h propionate incorporation with hydroxocobalamin supplementation

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant1: NM_000255.3:c.935G>T (p.Gly312Val)

ClinVar: 218988

CAID: CA347874

gnomAD: n/a

Variant2: NM_000255.3:c.1909G>A (p.Gly637Arg)

ClinVar: n/a

CAID: CA3846712

gnomAD: [0.000008798] (http://insert-your-link-here.com)(European non-Finnish)

CaseAddInfo: mut0 type, heterozygous mutations, unknown cis or trans.

Case24: no age or ethnicity recorded

Case 24 (Level 2)

CasePreviousTesting: same as group. found 0.4 nmol/mg protein/18 h propionate incorporation, 0.4 nmol/mg/protein/18 h propionate incorporation with hydroxocobalamin supplementation

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant1: NM_000255.3:c.795_796insT or NM_000255.3:c.795dupT (p.Met266TyrfsTer7)

ClinVar: n/a

CAID: CA913187322

gnomAD: n/a

Variant2: NM_000255.3:c.1207C>T (p.Arg403Ter)

ClinVar: 167310

CAID: CA234287

gnomAD: 0.0001602 (African)

CaseAddInfo: Variant 1 is novel. Mut0 type, heterozygous mutations, unknown cis or trans.

Case23: 3 mo (at biopsy), French Canadian

Case 23 (Level 2)

CasePreviousTesting: same as group. found 1.4 nmol/mg protein/18 h propionate incorporation, 1.3 nmol/mg/protein/18 h propionate incorporation with hydroxocobalamin supplementation

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant1: NM_000255.3:c.753+3A>G

ClinVar: n/a

CAID: CA3847056

gnomAD: 0.000008890 (European non-Finnish)

Variant2: NM_000255.3:c.1332+1delG

ClinVar: 554656

CAID: CA3846920

gnomAD: 0.000008828 (European non-Finnish)

CaseAddInfo: mut0 type, heterozygous mutations, unknown cis or trans. Variant 1 is novel.

Case22: 28 mo (at biopsy), Korean

Case 22 (Level 2)

CasePreviousTesting: same as group. found 0.8 nmol/mg protein/18 h propionate incorporation, 0.8 nmol/mg/protein/18 h propionate incorporation with hydroxocobalamin supplementation

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant1: NM_000255.3:c.682C>T (p.Arg228Ter)

ClinVar: 203855

CAID: CA312778

gnomAD: 0.0002615 (Latino)

Variant2: NM_000255.3:c.1481T>A (p.Leu494Ter)

ClinVar: 551275

CAID: CA3846864

gnomAD: 0.0001088 (East Asian)

CaseAddInfo: mut0 type, heterozygous mutations, unknown cis or trans

Case21: 2 mo (at biopsy), White

Case 21 (Level 2)

CasePreviousTesting: same as group. found 0.6 nmol/mg protein/18 h propionate incorporation, 0.6 nmol/mg/protein/18 h propionate incorporation with hydroxocobalamin supplementation

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant1: NM_000255.3:c.682C>T (p.Arg228Ter)

ClinVar: 203855

CAID: CA312778

gnomAD: 0.0002615 (Latino)

Variant2: NM_000255.3:c.1287C>G (p.Tyr429Ter)

ClinVar: 554278

CAID: CA364398724

gnomAD: 0.00006483 (European non-Finnish)

CaseAddInfo: mut0 type, heterozygous mutations, unknown cis or trans.

Case20: 5 days (at biopsy), White

Case 20 (Level 2)

CasePreviousTesting: same as group. found 2.1 nmol/mg protein/18 h propionate incorporation, 1.9 nmol/mg/protein/18 h propionate incorporation with hydroxocobalamin supplementation

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant1: NM_000255.3:c.682C>T (p.Arg228Ter)

ClinVar: 203855

CAID: CA312778

gnomAD: 0.0002615 (Latino)

Variant2: NM_000255.3:c.1106G>A (p.Arg369His)

ClinVar: 203846

CAID: CA312764

gnomAD: 0.0001504 (East Asian)

CaseAddInfo: mut0 type, heterozygous mutations, unknown cis or trans.

Patient2: Female, 5.5yo

CasePresentingHPOs: HP:0002572, HP:0001259, HP:0003648, HP:0002919 (episodic vomiting, coma, lacticaciduria, ketonuria)

HPOsFreeText: chronic intermittent IVA, treated with a vegetarian diet and L-carnitine supplements, abnormal excretion of isovalerate metabolites, elevation of isovaleryl glycine in urine, C5-carnitine = 16 µmol/l, birth weight = 2kg, hospitalized at 2yo for vomiting and acidosis. At physical exam at 5.5yo, weight = 18kg, height = 103cm, IQ = 92, and unremarkable systemic examination

CaseNotHPOs:

NotHPOsFreeText:

CaseAddInfo: 4mo at onset, 13mo at diagnosis via clinical presentation, consanguineous family, full-term delivery via Caesarean section due to failure of propagation

CasePMIDs:

CasePreviousTesting: same as group

CaseGenotypingMethod: same as group

Variant1: NM_002225.3:c.1207T>A

CAID: CA391724509, http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA391724509

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

Variant 1: NM_002225.3:c.1207T>A

Variant 2: NM_002225.4(IVD):c.863C>T

VariantEvidence: This variant is located in the turn region next to the alpha helix where hydrogen bonding is favorable, this location along with the presentation of severe phenotypes and biochemical profiles in the patients harboring these variants suggests a genotype-phenotype correlation (fig. 4)

Patient3: Female, 22yo

CasePresentingHPOs: HP:0001249 (intellectual disability)

HPOsFreeText: Blood C5-carnitine (μM) = 14.27, under a protein restricted diet with carnitine and glycine supplementation

CaseNotHPOs:

NotHPOsFreeText:

CaseAddInfo: diagnosed at 3yo after improper treatment

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.1007G>A (p.Cys309Tyr) listed in paper as (p.Cys307Tyr)

CAID: CA391722967, http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA391722967

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

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

CasePresentingHPOs:

HPOsFreeText: Blood C5-carnitine (μM) = 13.47, under a protein restricted diet with carnitine and glycine supplementation, normal development

CaseNotHPOs:

NotHPOsFreeText:

CaseAddInfo: 2mo 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_002225.4: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

Case6 14yo diagnosis, Anglo-Celtic

CasePresentingHPOs HP:0001256, HP:0000098

(Mild intellectual disability, Tall stature)

HPOsFreeText Diagnosis was based on tall height and mild intellectual disability. Plasma free homocystine was reduced from >80μmol to <10μmol (nv <60μmol) in response to treatment with folate and pyridoxine. Treatment began with 100-200mg/day for two weeks followed by an addition of 5mg/day folic acid.

CaseNotHPOs HP:0001083

(Lens dislocation)

NotHPOsFreeText no other seqeulae

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.833T>C (p.Ile278Thr), 2. IVS8+1G>A, compound heterozygous

ClinVar 1. 120, 2. n/a

CAID 2. need more info

gnomAD 1. MAF=0.001432(European(non-Finnish)), no homozygotes, 2. n/a

CaseAddInfo n/a

CasePMIDs n/a

FamilyLabel Family 28

Family28PresentingHPOs n/a

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

FamilyNotHPOs n/a

NotHPOsFreeText no further sequelae

CountryOrigin n/a

Ethnicity Lebanese

Race n/a

Family28PreviousTesting 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.

FamilyMethod1 PCR

FamilyMethod2 Sanger

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

ProbandLabel Case 34

Variant NM_000071.2(CBS):c.19dupC (p.Gln7Profs), homozygous

ClinVar 371345

gnomAD MAF=0.00005473, no homozygotes

FamilyAddInfo n/a

FamilyPMIDs n/a

the Asn541 from allele registry and mutalyzer confused me.

However, there cannot be an Asn542 as there is no Asn542 in the refseq. Asn542 is created when you insert TGAA, that is true, but the HGVS tracks the most immediate change on the refeq which is actually the Asn541.

It was also weird that mutalyzer and allele registry didn't take insTGAA, but only used insA. I wrote out the refseq, the refseq with TGAA inserted and the refseq with just A inserted. Sure enough, Asn541 is the most immediate change and the frameshift is the same "...LRGRP...stop" whether you insert TGAA or just A. Not too surprising since you are still in-frame after adding 3 bases, but cool to check.

interesting!

FamilyLabel Family 5

Family5PresentingHPOs n/a

HPOsFreeText plasma free homocystine was reduced from >80μmol to <10μmol (nv <60μmol) in response to treatment with folate and pyridoxine. Treatment began with 100-200mg/day for two weeks followed by an addition of 5mg/day folic acid.

FamilyNotHPOs HP:0001249

(Intellectual disability)

NotHPOsFreeText n/a

CountryOrigin n/a

Ethnicity Anglo-Celtic

Race n/a

Family5PreviousTesting 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.

FamilyMethod1 PCR

FamilyMethod2 Sanger

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

ProbandLabel Case 7

Variant 1. NM_000071.2(CBS):c.833T>C (p.Ile278Thr), 2. IVS1+insC, compound heterozygous

ClinVar 1. 120, 2. n/a

CAID 2. need more info

gnomAD 1. MAF=0.001432(European(non-Finnish)), no homozygotes, 2. n/a

FamilyAddInfo n/a

FamilyPMIDs n/a

CaseOS-127_II-1: Male, Birth onset, Germany

CasePresentingHPOs: HP:0001992, HP:0410145 (Organic aciduria, Decreased biotinidase activity)

HPOsFreeText: N/A

CaseNotHPOs: HP:0001263 (Global developmental delay)

NotHPOsFreeText: N/A

CaseAddInfo: Parents are non-consanguineous and healthy; Proband is confirmed homozygous via parental testing

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; Biotinidase deficiency treated via 5 mg biotin/d

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.

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

CaseK1 Female, 12yr report, Bosnian

CasePresentingHPOs HP:0001557, HP:0025116, HP:0001941, HP:0002098, HP:0003270, HP:0002013, HP:0001290, HP:0000750, HP:0011344, HP:0002069, HP:0002305, HP:0001290, HP:0002059, HP:0004881

(Abnormal intrauterine movements, Fetal distress, Acidosis, Respiratory distress, Abdominal distension, Vomiting, Hypotonia, Speech delay, Severe GDD, Grand mal seizures, Athetosis, Hypotonia, Cerebral atrophy, Episodic hypoventilation)

HPOsFreeText born at 42wk gestation. (neonatal, pre-treatment:) apgar score at 1min:7, 5min:8, 10min:8, acidosis, respiratory distress, abdominal distension, vomiting, onset of fits at 2d, resistant to ACD's. (18mo:) introduced to pyridoxine therapy, trial dose/route of 60mg oral, complete cessation of seizures, no apnoea or hypotonia/sleepiness, initial maintenance dose of 800mg. (infancy/childhood:) recurrence of seizures with febrile illness, hypotonia, speech delay/difficulties, severe IQ delay. 1wk after withdrawal from pyridoxine trial at 7y, grand mal seizures recurred(definite PDE).

CaseNotHPOs HP:0000486

(Squint)

NotHPOsFreeText (infancy/childhood:) squint

CasePreviousTesting Administration of pyridoxine relieved patient's seizures. plasma pipecolic acid: 12(nv<2.5μM), plasma AASA: 1.5(nv<0.2μM), urine AASA: 7.5(nv<1 mmol/mol creatinine). Blood lactate: 5.4mM and CSF lactate: 4.4mM (nv <2.5mM). (PMID:17068770:)post treatment testing: Pipecolic acid in plasma 6.7(nv 0.7-2.6mmol/liter), AASA in plasma 0.9(nv<0.2mmol/liter), urine AASA 19(nv<1mmol/molcreatinine).

Variant NM_001182.4(ALDH7A1):c.328C>T (p.Arg110Ter), homozygous

ClinVar 17995

gnomAD MAF=0.0001385(Other), no homozygotes

CaseAddInfo parental consanguinity

CasePMIDs 10894227 (Patient 3...), 17068770 (Patient 8)

FamilyLabel Family S

FamilySPresentingHPOs HP:0002098, HP:0010818, HP:0002280

(Respiratory distress, Generalized tonic seizures, Mega cisterna magna)

HPOsFreeText (neonatal, pre-treatment:) respiratory distress, generalised tonic seizures. (infancy/childhood:) mega cisterna magna

FamilyNotHPOs n/a

CountryOrigin n/a

Ethnicity Arabic

Race n/a

FamilySPreviousTesting Cessation of seizures during pyridoxine therapy

ProbandLabel S1, Female, 15yr report

Variant NM_001182.4(ALDH7A1):c.312+2T>A, homozygous

ClinVar 17997

gnomAD not in gnomAD

FamilyAddInfo parental consanguinity

FamilyPMIDs n/a

Case# Pat3, F, Chinese, 12day onset

CasePresentingHPOs HP:0008344, HP:0002013, HP:0011968, HP:0001250, HP:0001263

CaseNotHPOs HP:0001276, HP:0001279,

CasePreviousTesting tested for serum BCAAs

CaseMethod1 PCR

CaseMethod2 Direct

CaseGenotypingMethod exons and flanking intronic regions of the BCKDHA, BCKDHB and DBT genes were amplified using PCR and sequenced

Variant NM_000709.3:c.178G>T, NM_000709.3:c.1214_1219dup

CAID CA406004676(c.178), CA658820662(c.1214)

CaseAddInfo mutations confirmed in parents by Sanger sequencing, Her elder brother was healthy. She exhibited feeding difficulties and vomiting at 12 days of age. Severe psychomotor retardation was found at the age of 3 months. Cerebral MRI showed bilateral damage to the globus pallidus, basal ganglia, capsula interna and capsula externa. After treatment, clinical improvement was observed. Currently, she is 4 years old and has moderate psychomotor retardation.

Case# Pat1, M, Chinese, 3day onset

CasePresentingHPOs HP:0008344, HP:0008872, HP:0001250, HP:0001276, HP:0001279, HP:0001263

CaseNotHPOs HP:0002013

CaseMethod1 PCR

CaseMethod2 Direct

CaseGenotypingMethod exons and flanking intronic regions of the BCKDHA, BCKDHB and DBT genes were amplified using PCR and sequenced

Variant NM_000709.3:c.853+1delG ?

Case#: 2, female 4 days (onset), 4 y 3 mo (current), Chinese

CasePresentingHPOs: HP: 0008344, HP: 0012088, HP: 0011968, HP: 0001276, HP: 0001941, HP: 0002543, HP: 0011342 (elevated A keto acids in urine, abnormal urinary odor, feeding difficulties, hypertonia, acidosis, abnormal globus pallidus MRI signals, mild global developmental delay)

HPOsFreeText: Poor response to light and sound, abnormal MRI signals in frontal lobes, Blood ammonia: 95 uM, Hemoglobin: 117 g/L, Leucin: 745.01 uM, Valine: 526.40 uM, Isoleucine: 181.06 uM, Alloisoleucine: 69.45 uM

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo:

CasePreviousTesting: n/a

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant: NM_000709.3(BCKDHA): c.659C>T (p.Ala220Val)

ClinVar: 93367

CAID: n/a (because this doesn't need to be included if it is in ClinVar right?)

gnomAD: not in gnomAD

Case#: 7, female, 2 y 5 m (current), Chinese

CasePresentingHPOs: HP: 0008344, HP: 0012751, HP: 0012696 (elevated A keto acids in urine, abnormal signals in cerbral ganglia, abnormal thalamic MRI signal intensity)

HPOsFreeText: leucin: 621.72 uM, valine: 594.15 uM, isoleucine: 225.21 uM, Alloisoleucine: 76.62 uM

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: detected by newborn screening, unsure cis or trans

CasePreviousTesting: same as group

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant: NM_183050.3:c.482T>G (p.Val161Gly), NM_183050.3:c.508C>T (p.Arg170Cys)

ClinVar: not found, 96590

CAID: CA364657700, CA224301

gnomAD: not found, 0.00001759 (European, non-Finnish)

Case# P2, 5mon onset, Indian

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

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

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

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

CAID CA658820625

CaseAddInfo consanguineous

Case6: Male, White

CasePresentingHPOs: HP:0012072, HP:0002194, HP:0002788, HP:0001256, HP:0000722, HP:0000750, HP:0002360, HP:0005487, HP:0002057, HP:0000664, HP:0000286, HP:0000494, HP:0000368, HP:0000322, HP:0009778, HP:0004209 (Aciduria, Delayed gross motor development, Recurrent upper respiratory tract infections, Mild intellectual disability, Obsessive-compulsive behavior, Delayed speech and language development, Sleep disturbance, Prominent metopic ridge, Prominent glabella, Synophrys, Epicanthus, Downslanted palpebral fissures, Low-set posteriorly rotated ears, Short philtrum, Short thumb, Clinodactyly of the 5th finger)

HPOsFreeText: N/A

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Parents are non-consanguineous

CasePreviousTesting: Extensive metabolic screening revealed elevated urine AAA (618 mmol/mol creatinine; ref <49) and KAA (348 mmol/mol creatinine; ref not detected) as well as plasma AAA (53 umol/L; ref <12)

CaseGenotypingMethod: All 17 exons and flanking intronic sequences of DHTKD1 were Sanger sequenced.

Variant1: NM_018706.6:c.2185G>A (p.Arg455Gln)

CAID: CA5407838 http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_canonicalid?canonicalid=CA5407838

gnomAD: 0.0002538, https://gnomad.broadinstitute.org/variant/10-12139688-G-A

Variant2: NM_018706.6:c.1159+5G>A

CAID: CA5407761 http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_canonicalid?canonicalid=CA5407761

gnomAD: 0.0001170, https://gnomad.broadinstitute.org/variant/10-12133688-G-A

Case10: Male, Mixed, 6 yrs died

CasePresentingHPOs: HP:0002240, HP:0006579, HP:0003560, HP:0001263, HP:0002376 (Hepatomegaly, Prolonged neonatal jaundice, Muscular dystrophy, Global developmental delay, Developmental regression)

HPOsFreeText: N/A

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Proband is homozygous

CasePreviousTesting: Patient fibroblasts were analyzed for mutations in SLC25A21 and activities of KATII with no abnormalities; Niemann-Pick type C disease was diagnosed via bone marrow investigations; Metabolic screening suggested a defect in lysine metabolism with urine values of AAA and KAA increasing following a lysine challenge (200 mg/kg p.o.)

CaseGenotypingMethod: All 17 exons and flanking intronic sequences of DHTKD1 were Sanger sequenced.

Variant: NM_018706.6:c.700_701delinsGG (p.Leu234Gly)

CAID: CA915940679 http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA915940679

gnomAD: Not in gnomAD

Case#: KM03, F, neonatal (onset), Japanese

CasePresentingHPOs: HP:0001254, HP:0002179, HP:0001276, HP:0001263 (Lethargy, Opisthotonus, Hypertonia, Global developmental delay)

HPOsFreeText: Diagnosed at the time of newborn screening. Originally presented as classical form, but later determined to be the intermittent form.

CaseNotHPOs:

CasePreviousTesting: Lymphoblastoid cells from the patient showed reduced decarboxylation activity: 0.12 nmol/h per mg of protein (~9% of control). The authors previously showed by immunoblot analysis that the level of E2 protein was significantly reduced (PMID 3417306).

CaseMethod1: PCR

CaseMethod2: Sequencing

CaseGenotypingMethod: Total RNA and genomic DNA prepared from lymphoblastoid cells or peripheral leukocytes. RT-PCR was performed with primers in exons 8 and 9 identified an insertion. Gnomic DNA was isolated and sequenced. Confirmed by restriction enzyme digestion.

Variant: NM_001918.3:c.1018-550A>G (homozygous)

ClinVar: 11946

gnomAD: Not in gnomAD

VariantEvidence: The predicted mRNA encoded a truncated protein with 282 amino acids, including 4 novel amino acids at the carboxyl terminus. 265bp fragment that included the 126 bp intronic region was subcloned into an exon trapping vector and transfected into COS-1 cells to confirm the splicing defect.

CaseAddInfo: Consanguineous, parents were heterozygous.

CasePMIDs: 3417306

CaseAD: Male, 5 yrs onset

CasePresentingHPOs: HP:0000750, HP:0002572, HP:0012537, HP:0001944, HP:0001649, HP:0002789, HP:0001252, HP:0001942, HP:0001987, HP:0002919, HP:0012120 (Delayed speech and language development, Episodic vomiting, Food intolerance, Dehydration, Tachycardia, Tachypnea, Muscular hypotonia, Metabolic acidosis, Hyperammonemia, Ketonuria, Methylmalonic aciduria)

HPOsFreeText: Frequent catabolism episodes with vomiting were noticed without decompensation

CaseNotHPOs: HP:0001270, HP:0001919, HP:0011015 (Motor delay, Acute kidney injury, Abnormal blood glucose concentration)

NotHPOsFreeText: Propionyl-CoA carboxylase activity was within normal ranges in fibroblasts

CaseAddInfo: Parents are non-consanguineous with no family history, Dietary restriction and supplementation showed amelioration of symptoms

CasePreviousTesting: Normal ECG and MRI results; Metabolic acidosis (pH = 7.08, bicarbonates = 7.3 mmol/L, normal lactic acid, PCO2 = 25 mmHg) and hyperammmoniemia (100 µmol/L) present; Large increase in plasmatic propionylcarnitine (17.5 µmol/L; N < 0.75), undetectable methylmalonylcarnitine, and high urinary excretion of 3-hydroxypropionate and methylcitrate

CaseGenotypingMethod: Next-generation sequencing in the proband revealed a homozygous nonsense founder variant in the MCEE gene.

Variant: NM_032601.4(MCEE):c.139C>T (p.Arg47Ter)

ClinVar: 2343, http://www.ncbi.nlm.nih.gov/clinvar/variation/2343

gnomAD: 0.0004884, https://gnomad.broadinstitute.org/variant/2-71351575-G-A

Case#: DBT-6

CasePresentingHPOs: Clinical information not available

Variant: NM_001918.3:c.1281+3A>G

CAID: CA658820827

gnomAD: Not in gnomAD

VariantEvidence: Non-canonical splice variant

CaseAddInfo:

CasePMIDs:

Case#: DBT-5

CasePresentingHPOs: Classic form of MSUD

Variant: NM_001918.3:c.1195T>C

CAID: CA341330334

gnomAD: Not in gnomAD

VariantEvidence: No functional evidence

CaseAddInfo:

CasePMIDs:

Case3: Newborn, Hmong (China)

CasePresentingHPOs:

HPOFreeText: Born after normal pregnancy, postnatal course normal except for moderately elevated C5-acylcarnitine found during NBS. Treatment with L-carnitine supplementation.

CaseNotHPOs:

CasePreviousTesting: Urine 2-MBG 55.7 ug/mg creatinine (ref 0.3-7.5); urine IVG 1.2 ug/mg creatinine (ref 0.3-14.3).

CaseGenotypingMethod: PCR and sequencing of entire gene.

Variant: NM_001609.3:c.1165A>G (p.Met389Val) (homozygous)

CAID: CA5730948

gnomAD: East Asian 0.0005513

VariantEvidence: PCR of cDNA using primers in exon 9 and exon 11 showed that exon 10 was missing. Analysis of intron exon junctions flanking exon 10 did not reveal any changes.

CaseAddInfo: Diagnosis confirmed by Western blot of fibroblasts which showed no SBCAD protein.

CasePMIDs:

NonPatientCell: E-Coli (DH5α)

GeneFunction: GO:0006559

GeneAlteration: PAH cDNA was cloned into a pMAL-c2E expression vector encoding an N-terminal MBP tag and an enterokinase cleavage site. PAH mutants were constructed using site-directed mutagenesis and confirmed using DNA sequencing. Both the wild-type and the 10 mutant vectors were transformed into E. coli DH5α via overexpresion of fusion proteins at midexponential phase. The proteins were purified and concentration was determined.

FunctionalEvidence: Expression of the 10 mutant proteins compared to the wild-type demonstrated that several of these mutants induce protein destabilization and subsequent loss of function. Upon deconvolution analysis, five variants showed significanly distrubed oligomerization and a preponderance to misfold and form high-molecular-weight protein aggregates. Enzyme kinetic parameter analysis demonstrated moderate reduction in PAH activity and severely affected allostery in all but one mutation, indicating the strong possibility of global confirmational changes. Protein confirmation was tested by limited proteolysis with proteinase K with six out of eight tested mutants decreasing the half-life of the protein by >50% and decreased stability in five out of eight. Thermal protein denaturation was analyzed by fluorescence and demonstrated that all of the mutations led to substantial distortion of the protein's confirmation and/or loss of structural integrity compared to the wild-type, especially those in the regulatory domain. Finally, amino acid replacements were shown to disrupt local networks of amino acid interactions and alter the normal oligomeric state and therfore the function of the protein.

FAVariant1: NM_000277.3(PAH):c.1222C>T (p.Arg408Trp)

ClinVar: 577 https://www.ncbi.nlm.nih.gov/clinvar/variation/577/

FAVariant2: NM_000277.3(PAH):c.898G>T (p.Ala300Ser)

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

FAVariant3: NM_000277.3(PAH):c.165T>G (p.Phe55Leu)

ClinVar: 92734 https://www.ncbi.nlm.nih.gov/clinvar/variation/92734/

FAVariant4: NM_000277.3(PAH):c.1249T>C (p.Tyr417His)

ClinVar: 102579 https://www.ncbi.nlm.nih.gov/clinvar/variation/102579/

FAVariant5: NM_000277.3(PAH):c.940C>T (p.Pro314Ser)

ClinVar: 102905 https://www.ncbi.nlm.nih.gov/clinvar/variation/102905/

Case12(Level 2)

CasePreviousTesting: same as group

CaseMethod1: PCR

CaseGenotypingMethod: same as group __

Variant1: NM_015506.2(MMACHC):c.609G > A (p.Trp203Ter)

ClinVar: 30800

CAID: CA259906

gnomAD: 0.0005562 (East Asian)

Variant2: NM_015506.2(MMACHC):c.481C > T (p.Arg161Ter)

ClinVar: 95703

CAID: CA223189

gnomAD: 0.0001241 (African)

CaseAddInfo: proband genotype, unknown cis or trans, no phenotypic info, sibling (fetus) not a carrier

Case 7 (Level 2)

CasePreviousTesting: same as group

CaseMethod1: PCR

CaseGenotypingMethod: same as group

Variant1: NM_015506.2(MMACHC):c.609G > A (p.Trp203Ter)

ClinVar: 30800

CAID: CA259906

gnomAD: 0.0005562 (East Asian)

Variant2: NM_015506.2(MMACHC):c.445_446delTG (p.Cys149Hisfs*32)

ClinVar: 203834

CAID: CA312741

gnomAD: 0.00008509 (Latino)

CaseAddInfo: proband genotype, unknown cis or trans, no phenotypic info, sibling (fetus) carrier for both variants

FamilyGL: N/A

FamilyPresentingHPOs: HP:0012237 (Urocanic aciduria)

HPOsFreeText: N/A

FamilyNotHPOs: HP:0001507, HP:0100543, HP:0010904 (Growth abnormality, Cognitive impairment, Abnormal circulating histidine concentration)

NotHPOsFreeText: N/A

FamilyGenotypingMethod: Genomic DNA was obtained via peripheral blood from the siblings and both parents. PCR and direct sequencing in both forward and reverse directions was used to amplify and sequence the 21 coding exons of UROC1.

FamilyPreviousTesting: Plasma samples from both subjects demonstrated significant elevations in cis-urocanate, trans-urocanate and imidazole propionate; Z-scores for compounds in alternative histidine degradation pathways were found to be within normal limits, including 1-methylhisitidine, imidazole lactate, and 1-methylhistamine; Table 1 shows detailed metabolite levels; Family history was negative for metabolic disorders or any other familial diseases

FamilyAddInfo: Family appears to be asymptomatic, Siblings are confirmed compound heterozygous via parental sequencing (father het for Variant 2, mother het for Variant 1)

Variant1: NM_144639.3:c.356C>G (p.Pro119Arg)

CAID: CA2591587, http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA2591587

gnomAD: 0.0001596, https://gnomad.broadinstitute.org/variant/3-126227314-G-C

Variant2: NM_144639.3:c.907G>C (p.Ala303Pro)

CAID: CA2591263, http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA2591263

gnomAD: 0.00009300, https://gnomad.broadinstitute.org/variant/3-126220119-C-G

Affected: 2

Unaffected: 3

AddSegregationInfo: Not enough affected

Case40 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 84(nv 2–10 mmol/mol creatinine) and 3-OH-glutarate 312(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 R161Q variant destroys an AciI restriction site. Thus, while the patient's DNA fragments would not be cleaved, control fragments would be cleaved. The 848del variant creates a MspI site. This would lead to cleaved patient fragments and uncleaved control fragments.

Variant 1. NM_000159.3(GCDH):c.482G>A (p.Arg161Gln), 2. NM_000159.3(GCDH):c.848delT (p.Leu283Argfs), compound heterozygous

ClinVar 1. 188789, 2. 370672

gnomAD 1. MAF=0.0001384(Other), no homozygotes, 2. MAF= 0.0003331(Other), no homozygotes

CaseAddInfo n/a

CasePMIDs n/a

Case6 unknown sex, Spain

CasePresentingHPOs HP:0000707

(Neurological abnormality)

HPOsFreeText Clinical onset was "insidious," involving slowly progressive psychomotor delay, dystonic postures, lethargy, or macrocephaly. Phenotypes defined as "slight," entailing minimal neurological signs and an otherwise normal life. 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 500(nv 2–10 mmol/mol creatinine) and 3-OH-glutarate 461(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.

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

ClinVar 2083

gnomAD MAF= 0.0004013(African), no homozygotes

CaseAddInfo Sibling (Case 7) presented with same enzyme activity, clinical onset and phenotype. Gluterate was 3200mmol/mol creatinine and 3-OH-glutarate was 843mmol/mol creatinine.

CasePMIDs n/a

Case4 sex unknown, Spain

CasePresentingHPOs HP:0001332

(Dystonia)

HPOsFreeText Clinical onset was "insidious," involving slowly progressive psychomotor delay, dystonic postures, lethargy, or macrocephaly. Phenotypes were "mild," owing to static or improving dystonia, with maintained ability to walk without assistance. 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 HP:0002540

(Non-ambulatory)

NotHPOsFreeText n/a

CasePreviousTesting GC-MS measured glutarate of 4135(nv 2–10 mmol/mol creatinine) and 3-OH-glutarate 752(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 Sibling(Case 5) presented with same enzyme activity, clinical onset and phenotype. Gluterate was 2166mmol/mol creatinine and 3-OH-glutarate was 420mmol/mol creatinine.

CasePMIDs see note on previous studies

Case20 sex unknown, Spain

CasePresentingHPOs HP:0001332

(Dystonia)

HPOsFreeText Clinical onset was "insidious," involving slowly progressive psychomotor delay, dystonic postures, lethargy, or macrocephaly. Phenotypes were "mild," owing to static or improving dystonia, with maintained ability to walk without assistance. 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 HP:0002540

(Non-ambulatory)

NotHPOsFreeText n/a

CasePreviousTesting GC-MS measured glutarate of 3571(nv 2–10 mmol/mol creatinine) and 3-OH-glutarate 232(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 R132Q variant destroys an AciI site. This would lead to uncleaved patient DNA fragments and cleaved control fragments.

Variant 1. NM_000159.3(GCDH):c.395G>A (p.Arg132Gln), 2. NM_000159.3(GCDH):c.1173delG (p.Asn392Metfs), compound heterozygous

ClinVar 1. 377917, 2. 370106

gnomAD 1. not in gnomAD, 2. MAF= 0.00005646(Latino), no homozygotes

CaseAddInfo n/a

CasePMIDs n/a

Case32 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 69(nv 2–10 mmol/mol creatinine) and 3-OH-glutarate 429(nv 2–14 mm/mol creatinine). In patient fibroblasts, %GCD activity was 3.

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 restriction site. Thus, while the patient's DNA fragments would cleave, control fragments would not be cleaved. 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.1198G>A (p.Val400Met), 2. NM_000159.3(GCDH):c.1204C>T (p.Arg402Trp), compound heterozygous

ClinVar 1. 2088, 2. 2085

gnomAD 1. MAF= 0.0002769(Other), no homozygotes, 2. MAF= 0.0007566(European(Finnish)), no homozygotes

CaseAddInfo n/a

CasePMIDs n/a

Case5 Female, 24yo report, USA, Lumbee

CasePresentingHPOs HP:0001344, HP:0002540, HP:0003150

(Nonverbal, Non-ambulatory, Glutaricaciduria)

HPOsFreeText Urine glutaric acid was highly elevated and 3-hydroxyglutaric acid was elevated. Patient presented with acute metabolic decompensation at 2yo and feeds by mouth. Patient was diet restricted in lysine and tryptophan with per individual adjustments to maintain normal plasma amino acid levels. Treatment also included supplemental carnitine and riboflavin.

CaseNotHPOs n/a

NotHPOsFreeText n/a

CasePreviousTesting n/a

CaseMethod1 PCR

CaseMethod2 Sanger

CaseGenotypingMethod PCR and sanger sequencing was performed on exon 10 of the GCDH gene.

Variant NM_000159.3(GCDH):c.1240G>A (p.Glu414Lys), homozygous

ClinVar 167133

gnomAD MAF=0.0002017(African), no homozygotes

CaseAddInfo nonconsanguineous

CasePMIDs n/a

Case#: Patient 5, Japanese, 8 years old (report) CaseHPOs: Hyperornithinemia

CaseHPOsFreeText: (From Hayasaka et al. 1981; PMID 7260021, Patient 2). First noted visual problems at age 5 years, glasses prescribed for myopia. Evaluation for night blindness at age 8. Best corrected visual acuity was RE 04 with correction of myopia (-8 D), LE 04 with correction of myopia (-10 D). Typical chorioretinal atrophy with scallop margins at the peripheral fundus and an abnormal reflex in the macular area. Concentric constriction of visual fields. Dark adaptation curve was monophasic. ERG was subnormal, and the electro-oculogram was flat. The ciliary processes were normal by cycloscopy. Not responsive to Vitamin B6.

CaseNotHPOsFreeText: Normal lens

CasePreviousTesting: Serum ornithine 12.95 mg/dl (normal 0-36-1-56). OAT activity was not detectable in cell extracts from skin fibroblasts. CaseAddInfo: No consanguinity reported CaseMethod1: PCR

CaseMethod2: Sequence analysis

CaseGenotypingMethod: DGGE and sequence analysis of genomic DNA was carried out to identify the variant on the other allele. Variant: NM_000274.3(OAT):c.1276C>T (p.Arg426Ter)

ClinVar: 177

gnomAD: Maximum population minor allele frequency = 0.00002893 (Latino); no homozygotes in any population.

CaseKLC2: Male, 32 yrs death, Japan, Asian

CasePresentingHPOs: HP:0001249, HP:0002078, HP:0000651, HP:0008353, HP:0000992 (Intellectual disability, Truncal ataxia, Diplopia, Neutral hyperaminoaciduria, Cutaneous photosensitivity)

HPOsFreeText: Monoamino-monocarboxylic aciduria

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Parents are first-cousin consanguineous, Max LOD score is 2.1 for family

CasePreviousTesting: Defects in the intestinal absorption of monoamino-monocarboxylic acids determined by oral loading; Urine analyses confirmed the absence of hyperaminoaciduria in the mother (III-4), father (III-5) and the younger brother (IV-3), Brother of proband

CaseGenotypingMethod: DNA was isolated from peripheral blood samples and from liver samples collected at autopsy. A genome-wide screen with 343 different markers was carried out and linkage analysis performed using homozygosity mapping. 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 a homozygous frameshift mutation that perfectly segregated with the phenotype.

CasePMIDs: 2582682, 11394870

Variant: NM_001003841.3(SLC6A19):c.882_883TG1

ClinVar: 2018 https://ww.ncbi.nlm.nih.gov/clinvar/variation/2018/

gnomAD: 0.000008900, https://gnomad.broadinstitute.org/variant/5-1214174-CTG-C?dataset=gnomad_r2_1

Case4 sex unknown, Netherlands, Dutch

CasePresentingHPOs HP:0001250, HP:0002194

(Seizures, Delayed motor skills)

HPOsFreeText Seizures ceased upon administration of pyridoxine. (PMID:16159904:) 1st seizure at 8do, first administered pyridoxine(and age at trial) 2mo, initial dose:100mg, maintenance dose:100mg/day, mildly affected motor skills. (PMID:17088338:) Definite="recurrent (two or more) seizures of any type that cease within seven days of the administration of oral pyridoxine (usual dose, 30 mg/ kg/day; minimum dose, 15 mg/kg/day; maximum dose, 1000 mg/kg/day) or within 30 minutes of intravenous pyridoxine (usual dose, 100 mg; minimum dose, 50 mg), that recur when pyridoxine supplementation is withdrawn, and that cease again when pyridoxine is given as above"

CaseNotHPOs n/a

NotHPOsFreeText intrauterine seizures

CasePreviousTesting PDS confirmed metabolically: α‐AASA urine 4.0(nv <1mmol/mol cr), α‐AASA plasma 1.1(nv <0.2μmol/l), pipecolic acid urine 0.02(nv 0.55-24.1mmol/mol cr <6mo, 0.01–1.54mmol/mol cr >6 mo) pipecolic plasma 7.0(nv 3.75-10.8μmol/l <1wk, 0.7-2.46μmol/l >1wk)

Variant NM_001182.4(ALDH7A1):c.834G>A (p.Val278=)

ClinVar 18002

gnomAD not in gnomAD(C and not G at that position?)

CaseAddInfo Metabolic testing of sibling(Patient 6, heterozygous for the p.Val278= variant): α‐AASA urine 0.2(nv <1mmol/mol cr), α‐AASA plasma <0.2(nv <0.2μmol/l), pipecolic acid urine 0.02(nv 0.55-24.1mmol/mol cr <6mo, 0.01–1.54mmol/mol cr >6 mo) pipecolic plasma 2.2(nv 3.75-10.8μmol/l <1wk, 0.7-2.46μmol/l >1wk), but was not metabolically confirmed for PDS, development was also normal

CasePMIDs 17088338 , 16159904

CaseF7P: 1 yr 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, Father was confirmed heterozygous, Proband is heterozygous for this mutation with no second pathogenic mutation located

CasePreviousTesting: Blood phenylalanine before treatment was 1105 μmol/l, Blood tyrosine before treatment was 87 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.*144A>G

CAID: CA16020999 http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA16020999

gnomAD: 0.001284, http://gnomad.broadinstitute.org/variant/12-103232809-T-C

Case5: Female, 10m, Chinese, part of Family 1

HPOsFreeText: (at 20 days) 1.38 C4 umol/L, 0.19 C4/C2, 1.79 C4/C3, 2.58 (mmol/mol creatinine) urine isobutyrylglycine

Case5 (Level 2):

CasePreviousTesting: same as group

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant1: NM_014384.2:c.286G>A (p.Gly96Ser)

ClinVar: n/a

CAID: CA6372895

gnomAD: 0.001453 (East Asian)

Variant2: NM_014384.2:c.1092+1G>A

ClinVar: n/a

CAID: CA6373205

gnomAD: 0.00005790 (Latino)

CaseAddInfo: younger sister of patient 6, trans heterozygous, inherited one allele from each parent

Case11a: Male, Sweden

CasePresentingHPOs: HP:0001290, HP:0003324, HP:0002093, HP:0011968, HP:0001283, HP:0001561, HP:0002804 (Generalized hypotonia, Generalized muscle weakness, Respiratory insufficiency, Feeding difficulties, Bulbar weakness, Polyhydramnios, Arthrogryposis)

HPOsFreeText: N/A

CaseNotHPOs: HP:0001627 (Cardiac abnormality)

NotHPOsFreeText: N/A

CaseAddInfo: Severe congenital myopathy, Deceased in 5 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.

Variant: NM_198271.4:c.1069G>T (p.Glu357Ter)

ClinVar: 162219 https://www.ncbi.nlm.nih.gov/clinvar/variation/162219/

gnomAD: Not in gnomAD

Case18 Female, 9.9yo report, Ethnicity: Austrian

CasePresentingHPOs HP:0003571, HP:0008872, HP:0001531, HP:0001254, HP:0002789, HP:0002045

(propionicacidemia, feeding difficulties in infancy, failure to thrive in infancy, lethargy, tachypnea, hypothermia)

HPOsFreeText feeding difficulties, failure to thrive, lethargy, tachypnea, hypothermia, exanthema (5do). Scored 55 on Snijders-Omen Test for IQ

CaseNotHPOs n/a

CasePreviousTesting Diagnosed by selective metabolic screening. PCC activity in patient lymphoblasts was 0.4% of control.

Variant NM_000532.4:c.1199-2A>G (Gly400fs), homozygous

ClinVar n/a

CAID CA354648962

gnomAD not in gnomAD

CaseAddInfo parents not consanguineous

CasePMIDs n/a

FamilyPY: White, United Kingdom

FamilyPresentingHPOs: HP:0002131, HP:0001260, HP:0008003, HP:0030453 (Episodic ataxia, Dysarthria, Jerky ocular pursuit movements, Abnormal visual electrophysiology)

HPOsFreeText: N/A

FamilyNotHPOs: HP:0000640, HP:0000648, HP:0003390, HP:0011402, HP:0001272, HP:0002283 (Gaze-evoked nystagmus, Optic atrophy, Sensory axonal neuropathy, Demyelinating sensory neuropathy, Cerebellar atrophy, Global brain atrophy)

NotHPOsFreeText: N/A

FamilyGenotypingMethod: Blood genomic DNA was enriched and whole exome sequenced. Alightment to hg19 and variant filtering was performed with putative pathogenic variants being confirmed via Sanger sequencing.

FamilyPreviousTesting: Underwent brain MRI and CSF examination with oligoclonal band analysis; Negative genetic testing for SCA1, 2, 3, 6, 7, 17 (now known as ATXN1, ATXN2, CACNA1A, ATXN7, and TBP, respectively), DRPLA (now known as ATN1), FA (now known as FXN) and FMR1

FamilyAddInfo: N/A

Variant: NM_004172.5(SLC1A3):c.1496G>A (p.Arg499Gln)

ClinVar: 242994, https://www.ncbi.nlm.nih.gov/clinvar/variation/242994/

gnomAD: 0.0001471, https://gnomad.broadinstitute.org/variant/5-36686238-G-A

Affected: 3

Segregations: 2

AddSegregationInfo: Pedigree in Sup Data, Not enough info provided

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

CasePresentingHPOs HP:0008344, HP:0011968, HP:0001954, HP:0002373

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_000056.4(BCKDHB):c.93_103dupGGCGCGGGGCT (p.Phe35Trpfs), NM_000056.3:c.550del

ClinVar 189101 (dup)

CAID CA658820648 (del)

gnomAD low quality site (dup)

Case11: Male, 3 wk at initiation of follow up, African American ancestry

HPOsFreeText: NBS C4-acylcarnitine 1.4 umol/L, lasma C4 acylcarnitine 1.87 umol/L, in vitro probe isoC4-acylcarnitine 0.053 umol/ g protein

NotHPOsFreeText: normal in vitro probe C4-aclycarnitine, normal urine isobutyrylglycine, normal urine C4-acylcarnitine, normal urine C4/C3 ratio

CaseAddInfo: mild biochemical phenotype observed. Suggested to be at minimum a carrier of IBD deficiency- however presence of promoter mutation, large deletion, or splice-site alteration undetectable by direct gene sequencing cannot be ruled out.

Case11 (Level 2)

CasePreviousTesting: same as group

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant2: NM_014384.2(ACAD8):c.1129G>A (p.Gly377Ser)

ClinVar: 5356

CAID: CA117435

gnomAD: 0.0001551 (European non-Finnish)

CaseAddInfo: heterozygous for mutation

CaseGuC: Male, 5 days onset

CasePresentingHPOs: HP:0200134, HP:0011153, HP:0010850, HP:0011199, HP:0001252, HP:0003487, HP:0003423, HP:0010864, HP:0011344, HP:0002188, HP:0002059 (Epileptic encephalopathy, Focal motor seizure, EEG with spike-wave complexes, EEG with generalized sharp slow waves, Muscular hypotonia, Babinski sign, Thoracolumbar kyphoscoliosis, Severe intellectual disability, Severe global developmental delay, Delayed CNS myelination, Cerebral atrophy)

HPOsFreeText: Multiple seizure types, Bilateral T2 prolongation in caudate heads and putamina

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Variant is confirmed heterozygous de novo

CasePreviousTesting: Investigations include electroencephalography (EEG), head MRI, neurometabolic testing, and chromosome microarray; Individuals with self-limiting benign electroclinical syndromes, such as childhood absence epilepsy (onset > 4 years), were excluded; Head circumference was 44 cm at 4 months (91st percentile); Partial response to ketogenic diet and phenytoin, no response to other antiseizure medications;

CaseGenotypingMethod: Variants within co-expressed candidate genes in Table S1 were extracted from exome sequencing data. Variants were annotated and were verified with Sanger sequencing of the proband and parents.

Variant: NM_004171.4(SLC1A2):c.866C>G (p.Pro289Arg)

ClinVar: 438737, https://www.ncbi.nlm.nih.gov/clinvar/variation/438737/

gnomAD: Not in gnomAD

Case# FIII.4, F, 19 day old diagnosis, Cyprus (Greek Cypriot)

CasePresentingHPOs HP:0010910, HP:0010911, HP:0010913, HP:0001254, HP:0011968, HP:0001290, HP:0001276, HP:0002789, HP:0002353,

HPOcont: Hypervalinemia, Hyperleucinemia, Hyperisoleucinemia, Lethargy, Poor feeding, Hypotonia, Hypertonia, Tachypnea, Abnormal EEG

HPOfreetext: Reduced alloisoleucine and grunting

CasePreviousTesting Tested blood BCAA (AIleu: 0, Ileu: 220 (NR: 26–100), Leu: 3270 (NR: 65–220), Val: 459 (NR: 90–300), urine organic acids (Increased:lactate, 2-keto-glutarate, 2-keto-isocaproic, 2-OH-isovaleric, 2-keto-isovaleric, 2-keto-3-methylvaleric, 4-OH-phenylacetate, 4-OH-phenylpyruvate and BCKD activity (0.03(NR: 0.4–3.4) (Fibroblasts))

CaseMethod1 PCR

CaseMethod2 Direct Sequencing

CaseGenotypingMethod Direct sequencing repeated on proband and father for deletion varaint

Variant (1) NM_000709.3(BCKDHA):c.632C>T (p.Thr211Met) (2) NM_000709.3:c.718delG

ClinVar (1) 93365

CAID (2) register link if I put that in correctly

gnomAD (1) 0.0001163(East Asian), 0.00002043 (Overall)

CaseAddInfo nonconsanguineous

Case1: Female

CasePresentingHPOs: HP:0011343, HP:0012072, HP:0007018 (Moderate global developmental delay, Aciduria, Attention deficit hyperactivity disorder)

HPOsFreeText: N/A

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Parents are non-consanguineous, Unremarkable family history, Variants are parentally confirmed with c.2185 G>A apparently de novo

CasePreviousTesting: At 2 yrs had elevated 2-oxoadipate (range = 10–120 mmol/mol creatinine) and 2-hydroxyadipate (range = 5–40 mmol/mol creatinine) in urine (Reference 0 mmol/mol creatinine); Elevated 2-aminoadipate (120 μmol/l); Moderate protein restriction did not influence disease course and was discontinued, Serial cranial magnetic-resonance-imaging studies showed no morphological abnormalities; Neuropsychological testing at 8 years revealed an IQ of 117; Symptoms improved after implementation of methylphenidate and at 14 is clinically normal; No mutation detected in ODC

CaseGenotypingMethod: Whole exome sequencing was performed to identify mutations responsible for the biochemical abnormality. Exonic sequences were enriched and sequenced with data analysis being used to filter according to inheritance and low allele frequencies in controls. This narrowed down to eight different genes (ADAMTS10, AIRE, C8orf38, DHTKD1, OVOS2, CAMTA2, LPPR3, and RADIL) of which three had been associated with unrelated phenotypes and only one other had predicted pathogenic variants, DHTKD1.

Variant1: NM_018706.7(DHTKD1):c.1A>G (p.Met1Val)

ClinVar: 39563 https://www.ncbi.nlm.nih.gov/clinvar/variation/39563/

gnomAD: Not in gnomAD

Variant2: NM_018706.5(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

CaseFI1: Female, Tunisia, 18 months report

CasePresentingHPOs: HP:0001511, HP:0001508, HP:0001263, HP:0001249, HP:0001337, HP:0000325, HP:0000272, HP:0000303, HP:0000411, HP:0007495, HP:0000518, HP:0000963, HP:0000973, HP:0100678, HP:0012785, HP:0004220, HP:0009162, HP:0010107, HP:0002827, HP:0002808, HP:0001252, HP:0008619, HP:0001629, HP:0001684, HP:0001643, HP:0005301, HP:0003572, HP:0005961, HP:0025082, HP:0030774 (Intrauterine growth retardation, Failure to thrive, Global developmental delay, Intellectual disability, Tremor, Triangular face, Malar flattening, Mandibular prognathia, Protruding ear, Prematurely aged appearance, Cataract, Thin skin, Cutis laxa, Premature skin wrinkling, Flexion contracture of finger, Short middle phalanx of the 5th finger, Absent middle phalanx of 5th finger, Short proximal phalanx of hallux, Hip dislocation, Kyphosis, Muscular hypotonia, Bilateral sensorineural hearing impairment, Ventricular septal defect, Secundum atrial septal defect, Patent ductus arteriosus, Persistent left superior vena cava, Low plasma citrulline, Hypoargininemia, Abnormal cutaneous elastic fiber morphology, Mitochondrial swelling)

HPOsFreeText: Weight: 1770 g (< 3rd percentile); length: 39 cm (< 3rd percentile); OFC: 31 cm (< 3rd percentile); Hand and foot malformations; First metacarpal was bowed bilaterally; Serum Ornithine at 23 μmol/l (ref 21–77), Serum Citrulline at 3 μmol/l (ref 27–43), Serum Arginine at 39 μmol/l (ref 49-129), Serum Proline at 105 μmol/l (ref 71-303), Serum Glutamate at 61 μmol/l (ref 8-64), Serum Aspartate at 8 μmol/l (ref <14)

CaseNotHPOs: HP:0002305, HP:0012443, HP:0009832 (Athetosis, Abnormality of brain morphology, Abnormal distal phalanx morphology of finger)

NotHPOsFreeText: Urinary organic acids, oligosaccharides, purines and pyrimidines were normal

CaseAddInfo: Parents are first-degree cousins, Deletion leads to a frameshift and a premature termination codon

CasePreviousTesting: A skin biopsy was taken and amino acid profiles were analyzed from plasma according to standard procedures

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 no result for Exon 15. To confirm if a deletion was responsible, copy number analysis by qPRC was performed with Exon 15 showing a dramatic decrease in signal compared to the others in the affected individual suggesting a homozygous deletion. The parents showed a 50% reduction suggesting a heterozygous phase for the deletion. Different primer combinations allowed for confirmation of the breakpoints with a microdeletion of exon 15 and flanking intronic sequence (1522 bp) with a guanosine insertion.

Variant: NM_002860.4(ALDH18A1):c.1802-2_1924-901del

ClinVar: 156547 https://www.ncbi.nlm.nih.gov/clinvar/variation/156547/

gnomAD: Not in gnomAD

Patient14: Group 3

CasePresentingHPOs: same as group 3

CasePreviousTesting: same as group 3

CaseGenotypingMethod: same as group 3

Variant1: NM_002225.3:c.125T>C

CAID: CA391743774, http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA391743774

gnomAD: n/a

Variant2: IVS2-1G>C no ClinVar or CAID

Case817: Female, 7 years report, Spain

CasePresentingHPOs: HP:0003153, HP:0002345, HP:0002174 (Cystathioninuria, Action tremor, Postural tremor)

HPOsFreeText: Elevated urine cystathionine (1916 umol/g creatine; reference <46), Normal plasma tHcys (3.5 umol/l; reference <7.5), Post treatment normal urine cystathionine (24 umol/g creatine)

CaseNotHPOs: HP:0003345, HP:0001249 (Glutathione synthetase deficiency, Mental retardation)

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

CaseF-22 male, 5do onset, Ethinicity: North Indian, Muslim

CasePresentingHPOs HP:0006846, HP:0001254, HP:0011968, HP:0001250, HP:0001987, HP:0001946, HP:0045045, HP:0003571

(Acute encephalopathy, Lethargy, Poor feeding, Seizures, Hyperammonemia, Ketosis, Elevated acylcarnitines, Propionicacidemia)

HPOsFreeText Serum ammonia 700micromolar

CaseNotHPOs n/a

CasePreviousTesting Also screened for variants in PCCA

Variant NM_000532.4(PCCB):c.183+5G>A

ClinVar 217896

gnomAD MAF= 0.00003328(South Asian), no homozygotes

CaseAddInfo consanguineous, no abnormality in family history noted

CasePMIDs n/a

VariantEvidence: Expression of recombinant GMT was performed in E. coli. Firstly, the wild-type protein showed high solubility compared to the mutant of which only a minor fraction was soluble. Furthermore, enzyme activity was measured and the mutant GMNT lacked any activity while the wild-type exibits activity of >200 pmol/min/ug.

Variant: NM_018960.4:c.296G>A (p.Arg99His)

Case43: Male, Chinese, 2.3y (end of study)

HPOsFreeText: 2y onset, poor neurological outcome

NotHPOsFreeText: not responsive to vitamin B12

Case43 (Level 2)

CasePreviousTesting: NBS

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant1: NM_000255.3:c.2107G>A (p.Gly703Arg)

ClinVar: n/a

CAID: CA364394252

gnomAD: 0.00006152 (African)

CaseAddInfo: one variant, not homozygous.

Case42: Female, Chinese, 2.3y (end of study)

HPOsFreeText: 10 mo onset, poor neurological outcome

NotHPOsFreeText: not responsive to vitamin B12

Case42 (Level 2)

CasePreviousTesting: same as group

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant1: NM_000255.3:c.2080C>T (p.Arg694Trp)

ClinVar: 218996

CAID: CA347907

gnomAD: 0.00006534 (South Asian)

Variant2: NM_000255.3:c.2179C>T (p.Arg727Ter)

ClinVar: 218998

CAID: CA347913

gnomAD: 0.0001504 (East Asian)

CaseAddInfo: heterozygous mutations in trans.

Case41: Male, Chinese, 10.3y (end of study)

HPOsFreeText: 8.8y onset, poor neurological outcome

NotHPOsFreeText: not responsive to vitamin B12

Case41 (Level 2)

CasePreviousTesting: same as group

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant1: NM_000255.3:c.1790T>G (p.Ille597Arg)

ClinVar: 551764

CAID: CA364395663

gnomAD: n/a

Variant2: NM_000255.3:c.2106delA (p.VAl704Ter)

ClinVar: 555523

CAID: CA658821513

gnomAD: n/a

CaseAddInfo: both variants novel. heterozygous mutations in trans.

Family2to4: Maltese

FamilyPresentingHPOs: N/A

HPOsFreeText: N/A

FamilyNotHPOs: HP:0001270, HP:0007018, HP:0001252, HP:0000750, HP:0000252 (Motor delay, Attention deficit hyperactivity disorder, Muscular hypotonia, Delayed speech and language development, Microcephaly)

NotHPOsFreeText: N/A

FamilyGenotypingMethod: 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. Sanger sequencing confirmed the proband's siblings are homozygous for the variant and that each parent is a carrier.

FamilyPreviousTesting: Amino acid and organic acid testing showed elevated urinary 2-aminoadipic, 2-ketoadipic, and glutaric acids in the proband as well as in two elder brothers, ages 5 and 7.

FamilyAddInfo: Parents are consanguineous, All three affected siblings are clinically normal

Variant: NM_018706.6:c.915G>C (p.Gln305His)

CAID: CA5407680 http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA5407680

gnomAD: Other 0.0001630, https://gnomad.broadinstitute.org/variant/10-12131182-G-C

Affected: 3

Unaffected: 2

AddSegregationInfo: Candidate gene sequencing

CaseII-2: Female, Turkey

CasePresentingHPOs: HP:0001558, HP:0000337, HP:0045075, HP:0000463, HP:0000343, HP:0000278, HP:0000470, HP:0000767, HP:0002375, HP:0002033, HP:0006829 (Decreased fetal movement, Wide forehead, Sparse eyebrow, Anteverted nares, Long philtrum, Retrognathia, Short neck, Pectus excavatum, Decreased spontaneous movement, Poor suck, Severe muscular hypotonia)

HPOsFreeText: Birth weight was 2050 g and length was 43 cm, Absent Moro reflex, Required ventilator support from birth

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Born with fractures of the right humerus and left femur

CasePreviousTesting: N/A

CaseGenotypingMethod: Whole blood smaples were obtained from the parents and the probands and DNA was extracted. WES was performed on II-2, with the results being sequenced and filtered for pathogenicity. Once the LMOD3 variant was detected, PCR amplification and Sanger sequencing was used to amplify exon 1 and confirm the results.

Variant: NM_198271.4:c.138dup (p.Ser47Glnfs*13)

ClinVar: 162215 https://www.ncbi.nlm.nih.gov/clinvar/variation/162215/

gnomAD: European (Non-Finnish) 8.879e-6, Overall 8.049e-6 https://gnomad.broadinstitute.org/variant/3-69171399-T-TG

Biallelic

Case#: Patient 2, male, 5 years (report), Turkish

CaseHPOs: HP:0003235, HP:0002240 (hypermethioninemia, hepatomegaly)

CaseHPOsFreeText: Identified by newborn screening.

CaseNotHPOsFreeText: No signs of hepatic failure, no clinical symptoms.

CasePreviousTesting: On day of life 3, elevated methionine concentration in blood of 390 µmol/L (normal 20–40). Total homocysteine in plasma was 59 µmol/L (normal 5–15). At later age (not specified), plasma methionine ranged from 1000 to 2070 µmol/L, while total homocysteine in plasma ranged from 30 to 55 µmol/L.

CaseMethod1: PCR

CaseMethod2: Sequencing

CaseGenotypingMethod: All 9 exons and intron-exon junctions of MAT1A were amplified by PCR from genomic DNA. This was followed by SSCP analysis and sequence analysis of PCR products with abnormal mobility.

Variant: c.125T>C (p.Leu42Pro) (homozygous)

ClinVar: N/A

CAID: CA377363776

gnomAD: The highest population minor allele frequency in gnomAD is 0.000008791 (European non-Finnish); no homozygotes in any population.

VariantEvidence: When expressed in E. Coli, the variant resulted in reduction of methionine adenosyltransferase (MAT) and tripolyphosphatase (PPPase) activities to ~10 and ~20% of wild type respectively (Fernandez-Irigoyn et al, 2010, PMID 20675163; Fig 2).

Points: 0.5

CaseSA6P: Male, 7 months onset, European

CasePresentingHPOs: HP:0001988, HP:0000825, HP:0001250, HP:0001987 (Recurrent hypoglycemia, Hyperinsulinemic hypoglycemia, Seizures, Hyperammonemia)

HPOsFreeText: N/A

CaseNotHPOs: N/A

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

Case# Lo, M, 10mo diagnosis---originally 1985

CasePresentingHPOs HP:0011968, HP:0001531, HP:0000737, HP:0007281, HP:0002013, HP:0001259, HP:0005979, HP:0001943

CaseMethod1 PCR

CaseMethod2 Sanger

CaseGenotypingMethod

Variant NM_000709.3:c.1265A>G(p.Lys422Arg), NM_000709.3:c.137dup(p.Ser47IlefsTer4)

CAID CA406015486(miss), CA658820686(dup)

VariantEvidence 3978850: in patient fibroblasts-rate of decarboxylation of alpha-ketoacids: alpha ketoisovalerate[0<0.31], Pyruvate[0.40>.34], alpha-ketoglutarate[0.14>0.10] (Table 1), BCKDH activity[0.0003<0.107] (Table 2), E1 subunit activity[0.6<5.9] (Table 3)--[ProbandvsNormal]

CaseAddInfo 3978850( a positive dinitrophenylhydrazine test and elevated urinary a-ketoisocaproate, a-methyl-P-OH-valerate and a-OH-isoval- erate,

CasePMIDs 3978850, 2914958

Case1: female, 2.3y (initial referral), Italian, Family 1

CasePresentingHPOs: HP:0001508, (failure to thrive)

HPOsFreeText: birthweight 3060 g, SGOT 11 mo 150 U/L, SGPT 11 mo 160 U/L, 1.8y elevated plasma lactic acid 2.9 mmol/L. 3.9 y alkaline phosphatase 928 U/L, 4.7 y serum tricglycerides 184 mg/dl, fasting plasma methionine 644 umol/L. increased urinary methionine excretion 40 umol/mmol. increased lactic acid excretiom 107 umol/mmol. mild centrilobular fibrosis in liver. some eosinophils present in liver.

CaseNotHPOs: HP:0001404, HP:0001397, HP:0012465, HP:0001396, (hepatocellular necrosis, hepatic steatosis, elevated hepatic iron concentration, cholestasis)

NotHPOsFreeText: hepatic dysfunction. normal tHcy.

CaseAddInfo: proband, second child, low methionine diet (300 mg/day) at 2.4 y decreased plasma methionine to normal.

Case (Level 2)

Variant1: NM_018960.5(GNMT):c.149T>C (p.Leu50Pro)

ClinVar: 4169

CAID: CA248600

gnomAD: 0.0002319 (East Asian)

Variant2: NM_018960.5(GNMT):c.529C>A (p.His177Asn)

ClinVar: 4170

CAID: CA248601

gnomAD: 0.0006993 (European non-Finnish)

CaseAddInfo: compound heterozygote, Leu50Pro maternal mutation, His177Asn paternal mutation. The amino acid shifts are one # different than those predicted in the paper (Leu49Pro and His176Asn).

CaseST-11: Male, 1 day onset, Turkey

CasePresentingHPOs: HP:0001319, HP:0000316, HP:0008070, HP:0001410, HP:0001987, HP:0004322, HP:0004482 (Neonatal hypotonia, Hypertelorism, Sparse hair, Liver dysfunction, Hyperammonemia, Short stature, Relative macrocephaly)

HPOsFreeText: N/A

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

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

CasePreviousTesting: See Group

CaseGenotypingMethod: See Group

Variant: NM_001123.3:c.905T>C (p.Phe302Ser)

CAID: CA377399030, http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA377399030

gnomAD: Not in gnomAD

FamilyST-VII: Iran

FamilyPresentingHPOs: HP:0002197, HP:0000316, HP:0001889 (Generalized-onset seizure, Hypertelorism, Megaloblastic anemia)

HPOsFreeText: N/A

FamilyNotHPOs: HP:0002904, HP:0001410, HP:0001943, HP:0001257, HP:0001347, HP:0001251, HP:0001276 (Hyperbilirubinemia, Decreased liver function, Hypoglycemia, Spasticity, Hyperreflexia, Ataxia, Hypertonia)

NotHPOsFreeText: N/A

FamilyGenotypingMethod: See Group

FamilyPreviousTesting: See Supp Info for methionine/transaminase values; Normal height, weight and head circumference at birth

FamilyAddInfo: Family is consanguineous, Family is homozygous for this variant

Variant: NM_006721.4(ADK):c.1022A>G (p.His341Arg)

ClinVar: 265772, https://www.ncbi.nlm.nih.gov/clinvar/variation/265772/

gnomAD: Not in gnomAD

Affected: 2

Unaffected: 0

Segregations: 1

AddSegregationInfo: Not enough info

Case#: PD, 44 years, female

CasePresentingHPOsFreeText: apo-B plasma levels <5% of normal; total cholesterol 2.57 (normal range 4-6.5 mmol/l), triglyceride 0.59 (normal range 0.3-1.8 mmol/l), apoB 17 (normal range 60-140 mg/dl).

CasePreviousTesting: On Western blot a truncated species of apo-B was found in the VLDL/chylomicron fraction (fig. 3), predicted based on size to be at the 5’ end of exon 26. The protein was absent from the HDL or infranate fractions.

CaseGenotypingMethod: Genomic DNA was digested with a panel of restriction enzymes and Southern blotted. Based on truncated apoB size (detected on Western blot), the variant was predicted to be at the end of exon 26. Approximately 1.7 kb of genomic DNA including exon 26 was amplified by the polymerase chain reaction (PCR), cloned into M13, and sequenced.

Variant: NM_000384.3(APOB):c.5463del (p.His1822fs), heterozygous

ClinVar: 17882

gnomAD: 0.000008797 (European non-Finnish)

Individual

Case#: 13940

Caseprevioustesting: SSCP Scan

Casemethod1: SSCP Scan

Casemethod2: PCR, Sanger Sequencing

Variant: NC_000022.11:g.29673267A>T (splice site acceptor, predicted/proven null)

CAID: CA16040592

gnomAD: not found

Individual

Case#: 16221

Caseprevioustesting: SSCP Scan

Casemethod1: PCR, Sanger

Variant: NM_000268.3:c.544G>T (nonsense)

CAID: CA021432

Caseaddinfo: de novo

gnomad: not found

Case#: 16773

Caseprevioustesting: SSCP Scan

Casemethod1: PCR, Sanger

Variant: NM_000268.3:c.1579G>T (nonsense)

CAID: CA021349

Caseaddinfo:

gnomad: no data

Variant: carrier status, considering that she was clinically asymptomatic and that she also had normal values for alpha-glucosidase activity on DBS analysis but she was heterozygous only for the c.2560C>T (rs121907943, p.Arg854Ter) pathogenic mutation

Variant: found reduced alpha-glucosidase activity on DBS assessment and the same pattern of mutations seen on the sister described above (II-6): (c.-32-13T>G (rs386834236, intronic) and c.2560C>T (rs121907943, p.Arg854Ter))

Case II-6: 56, Female

CasePresentingHPOs: hypertension, proximal weakness, with occasional cramping in both of her lower limbs, voice sounded particularly nasal, mild scapular winging, slight muscular atrophy on temporal, deltoids and thigh muscle extensors, mild lordosis

CasePresentingHPOsFreeText: Pain, temperature, vibration and proprioception were preserved as well as her coordination. myopathic pattern with reduced duration and amplitude of motor unit action potentials (MUAPs) specifically on thigh extensors

CaseNOTHPOS: dysphagia or dysphonia

CaseNOTHPOSFreeText: did not find paraspinal complex repetitive discharges, did not find any alteration on the tissue specimen analysis using hematoxylin and eosin

Variant: reduction in alpha-glucosidase activity (Neutral/acid: 61.0, reference: <30) and her GAA gene sequencing showed the two same pathologic heterozygous compound mutations found on our first subject (II-8) (c.-32-13T>G (rs386834236, intronic) and c.2560C>T (rs121907943, p.Arg854Ter))

*she did not have the third variant found on subject II-8 (c.1551+42G>A (rs115427918, intronic))

RescueModel Mus Musculus

GeneAlteration knock-in strain was generated by homologous recombination of the targeting vector with 129Sv/J embryonic stem cells genome. A 450-bp DNA segment encompassing 5′ UTR and exon 1 of the CASQ2 was used to screen RPCI-21 PAC mouse genomic library. Positive clones were sequenced to define the structure of an 8.5-Kb region encompassing promoter, exon 1, and part of intron 1 of mouse CASQ2. This region was cloned in 3 parts and assembled into the targeting vector pFlrt (supplemental Figure I). The linearized targeting vector was electroporated into 129Sv/J embryonic stem cells. The clone selected with G418 and gancyclovir was injected into C57BL/6NCrL blastocyts and transferred to pseudopregnant CD-1 females. Genotype was determined by sequencing of DNA extracted from tail biopsy specimens (DNasy Tissue Kit, Qiagen).

RescueVariant murine cardiac CASQ2 , WT?

RescuePhenotype HP:0004756

RescueMethod The AAV recombinant virus (AAV9-CASQ2) and the empty virus (AAV9-GFP) were produced by the AAV Vector Core of the Telethon Institute of Genetics and Medicine (Napoli, Italy) according to a previously published protocol.18 The viral titer was 2.6×1012 genome copies per milliliter for AAV9-CASQ2 and 4.3×1012 genome copies per milliliter for AAV9-GFP. The infection was performed by intraperitoneal injection in pups on postnatal day 3 (100 μL), which recovered until euthanasia was performed at 6, 9, or 12 months. Three-month-old mice were anesthetized with Avertin (0.025 mg/kg), and viral particles (200 μL) were injected into the tail vein and studied 2 months later. ECG recordings in mice of either sex were performed with implantable loop recorders (Data Sciences International).

RescueEvidence (Fig.5&8) Epinephrine/Isoprenol administration had lead to diminished incidence of VT in CASQ infected mice vs R33Q mice.

ModelVariant NM_001232.3(CASQ2):c.97C>T (p.Arg33Ter), ClinVar: 41043

Case#: Consequence 29 Table 1 in Data Supplement (1 cases)

DiseaseAssertion: HCM

FamilyInfo: N/A

CasePresentingHPOs: N/A

CaseHPOFreeText: Cardiac magnetic resonance imaging, BSA, NYHA functional class, obstructive HCM, atrial fibrillation, coronary artery disease, NSVT, Syncope, SCD family hitory, wall thickness

CaseNotHPOs: N/A

CaseNotHPOFreeText: N/A

CasePreviousTesting: Additional oligonucleotide hybridization–based DNA sequencing and dideoxy-based DNA sequencing for MYBPC3, TMP1, TNNC1, TNNI2, TNNI3, TNNT2, ACTC1, MYL3

CaseGenotypingMethod: oligonucleotide hybridization–based DNA sequencing and dideoxy-based DNA sequencing

Variant: NM_000257.4(MYH7):c.2782G>A (p.Asp928Asn)

ClinVar: 164313 https://www.ncbi.nlm.nih.gov/clinvar/variation/164313/

CAID: n/a

gnomAD: absent

Case#: Consequence 14 Table 1 in Data Supplement (1 cases)

DiseaseAssertion: HCM

FamilyInfo: N/A

CasePresentingHPOs: N/A

CaseHPOFreeText: Cardiac magnetic resonance imaging, BSA, NYHA functional class, obstructive HCM, atrial fibrillation, coronary artery disease, NSVT, Syncope, SCD family hitory, wall thickness

CaseNotHPOs: N/A

CaseNotHPOFreeText: N/A

CasePreviousTesting: Additional oligonucleotide hybridization–based DNA sequencing and dideoxy-based DNA sequencing for MYBPC3, TMP1, TNNC1, TNNI2, TNNI3, TNNT2, ACTC1, MYL3

CaseGenotypingMethod: oligonucleotide hybridization–based DNA sequencing and dideoxy-based DNA sequencing

Variant: NM_000257.4(MYH7):c.1208G>A (p.Arg403Gln)

ClinVar: 14087 https://www.ncbi.nlm.nih.gov/clinvar/variation/14087/

CAID: n/a

gnomAD: absent

Patient: P100

Variant: c.120_121delAG p.Gly41Argfs*16

CAID: CA2580612030

Functional effect: Decreased expression of full-length mutant allele compared to wt allele. Observed in blood.

Giannandrea_Family_MRX72

Family#PresentingHPOs: HP:0002342, HP:0010864, HP:0001250, HP:0000729

FamilyNotHPOs:

CountryOrigin: Italy

Ethnicity:

Race:

Family#PreviousTesting: X-linked intellectual disability was mapped to Xq28

Family#Method1: PCR

Family#Method2: Sanger

Family#GenotypingMethod: Both exons of the RAB39B gene were amplified by PCR and directly sequenced

Affected: 5

Unaffected:

Segregations: 7

PublishedLOD:

eLOD: 2.1

AddSegregationInfo:

ProbandLabel: Giannandrea_Family_MRX72_Proband III-4

Variant: NM_171998.4(RAB39B):c.215+1G>A

ClinVar: 10542

CAID:

gnomAD:

FamilyAddInfo:

FamilyPMIDs: 11050621

TissueExpression/Uberon ID: UBERON:0000955

ExpressionEvidence: qRT-PCR on several human and mouse tissues revealed that RAB39B was enriched in the brain. In theh developing mouse brain, Rab39b expression was constant between P1-P10 and P20-P180. Between P10-P20, Rab39b was upregulated 2-fold. Rab39a transcript levels were 10x lesss that Rab39b. The authors established that Rab39b was a neuron-specific gene, with highest expression in the hippocampus, and specifically in neuronal precursors and neurons only.

Where in paper: Figure 2

Giannandrea_Family X (D-23)

Family#PresentingHPOs: HP:0001256, HP:0002342, HP:0010864, HP:0000256, HP:0004322,

FamilyNotHPOs: HP:0001999

other malformations

CountryOrigin: