Case# PA7

Variant NM_000709.3(BCKDHA):c.905A>C (p.Asp302Ala)

ClinVar 93377

VariantEvidence none

Case# PA8

Variant NM_000709.3(BCKDHA):c.940C>T (p.Arg314Ter)

ClinVar 224072

gnomAD 0.000008958(European (Non-Finnish)), 0.000004064(Overall)

Case# PA9

Variant NM_000709.3:c.1270C>T

CAID CA406015510

Case# PA6

Variant NM_000709.3:c.896A>C

CAID CA406013345

VariantEvidence none

Case23: Female, Chinese, 3y (end of study)

HPOsFreeText: 1.8y onset, poor neurological outcome

NotHPOsFreeText: not responsive to vitamin B12

Case23 (Level 2)

CasePreviousTesting: same as group

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant1: NM_000255.3:c.729_730insTT (p.Asp244LeufsTer?)

ClinVar: 552079

CAID: CA3847064

gnomAD: 0.0002008 (East Asian)

Variant2: NM_000255.3:c.1295A>C (p.Glu432Ala)

ClinVar: n/a

CAID: CA364398703

gnomAD: n/a

CaseAddInfo: heterozygous mutations in trans

Case22: Female, Chinese, 1.3y (end of study)

HPOsFreeText: 11 mo onset, poor neurological outcome

NotHPOsFreeText: not responsive to vitamin B12

Case22 (Level 2)

CasePreviousTesting: same as group

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant1: NM_000255.3:c.729_730insTT (p.Asp244LeufsTer?)

ClinVar: 552079

CAID: CA3847064

gnomAD: 0.0002008 (East Asian)

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

ClinVar: 203846

CAID: CA312764

gnomAD: 0.0001504 (East Asian)

CaseAddInfo: heterozygous mutations in trans

Case21: Male, Chinese, 9 mo (age at death)

HPOsFreeText: neonatal onset

NotHPOsFreeText: not responsive to vitamin B12

Case21 (Level 2)

CasePreviousTesting: same as group

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant1: NM_000255.3:c.729_730insTT (p.Asp244LeufsTer?)

ClinVar: 552079

CAID: CA3847064

gnomAD: 0.0002008 (East Asian)

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

ClinVar: 203846

CAID: CA312764

gnomAD: 0.0001504 (East Asian)

CaseAddInfo: heterozygous mutations in trans, died at 9 mo

Case20: Female, Chinese, 1 y (end of study)

HPOsFreeText: neonatal onset, poor neruological outcome

NotHPOsFreeText: not responsive to vitamin B12

Case20 (Level 2)

CasePreviousTesting: same as group

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant1: NM_000255.3:c.729_730insTT (p.Asp244LeufsTer?)

ClinVar: 552079

CAID: CA3847064

gnomAD: 0.0002008 (East Asian)

Variant2: NM_000255.3:c.1105C>T (p.Arg369Cys)

ClinVar: 218989

CAID: CA347895

gnomAD: 0.00006536 (South Asian)

CaseAddInfo: heterozygous mutations in trans

Case19: Female, Chinese, 2.4 y (end of study)

HPOsFreeText: 1.8 y onset

NotHPOsFreeText: normal cognitive and motor development without further episodes of metabolic decompensation, not responsive to vitamin B12

Case19 (Level 2)

CasePreviousTesting: same as group

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant1: NM_000255.3:c.655A>G (p.Asn219Asp)

ClinVar: n/a

CAID: CA364404270

gnomAD: n/a

Variant2: NM_000255.3:c.729_730insTT (p.Asp244LeufsTer?)

ClinVar: 552079

CAID: CA3847064

gnomAD: 0.0002008 (East Asian)

CaseAddInfo: heterozygous mutations in trans

Case17: Male, Chinese, 6.5y (end of study)

HPOsFreeText: neonatal onset

NotHPOsFreeText: not responsive to vitamin B12, normal cognitive and motor development without further episodes of metabolic decompensation

Case17 (Level 2)

CasePreviousTesting: same as group

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant1: NM_000255.3:c.554C>T (p.Ser185Phe)

ClinVar: n/a

CAID: CA364404475

gnomAD: n/a

Variant2: NM_000255.3:c.2062G>T (p.Glu688Ter)

ClinVar: n/a

CAID: CA3846672

gnomAD: 0.00006152 (African)

CaseAddInfo: heterozygous mutations in trans.

Case16: Male, Chinese, 2.4 y (end of study)

HPOsFreeText: neonatal onset, poor neurological outcome

NotHPOsFreeText: not responsive to vitamin B12

Case16 (Level 2)

CasePreviousTesting: same as group

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant1: NM_000255.3:c.454C>T (p.Arg152Ter)

ClinVar: 426467

CAID: CA3847107

gnomAD: 0.00009813 (South Asian)

Variant2: NM_000255.3:c.1280G>A (p.Gly427Asp)

ClinVar: 218990

CAID: CA347873

gnomAD: 0.0004011 (East Asian)

CaseAddInfo: heterozygous mutations in trans.

Case18: Male, Chinese, 5 mo (age at death)

HPOsFreeText: neonatal onset

NotHPOsFreeText: not responsive to vitamin B12

Case18 (Level 2)

CasePreviousTesting: same as group

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant1: NM_000255.3:c.626dupC

ClinVar: n/a

CAID: CA825482501

gnomAD: n/a

Variant2: NM_000255.3:c.1531C>T (p.Arg511Ter)

ClinVar: 556124

CAID: CA3846855

gnomAD: 0.00003270 (South Asian)

CaseAddInfo: variant 1 is novel. Heterozygous mutations in trans.

Case15: Male, Chinese, 6 mo (age at death)

HPOsFreeText: neonatal onset

NotHPOsFreeText: not responsive to vitamin B12

Case15 (Level 2)

CasePreviousTesting: same as group

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant1: NM_000255.3:c.433G>A (p.Gly145Ser)

ClinVar: n/a

CAID: CA364404739

gnomAD: n/a

Variant2: NM_000255.3:c.1677-1G>A (p.=)

ClinVar: 550012

CAID: CA3846767

gnomAD: 0.0003511 (East Asian)

CaseAddInfo: heterozygous mutations in trans. Died at 6 mo.

Case14: Male, Chinese, 2.4 y (onset)

HPOsFreeText: 2.4 y onset

NotHPOsFreeText: not responsive to vitamin B12

Case14 (Level 2)

CasePreviousTesting: same as group

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant1: NM_000255.3:c.421G>A (p.Ala141Thr)

ClinVar: 557678

CAID: CA364404763

gnomAD: n/a

Variant2: NM_000255.3:c.810_811dupGG (p.?)

ClinVar: n/a

CAID: CA3847032

gnomAD: n/a

CaseAddInfo: lost to follow up. heterozygous mutations in trans. both novel mutations

Case12: Female, Chinese, 1.5 y (end of study)

HPOsFreeText: 11 mo onset, poor neruological outcome

NotHPOsFreeText: not responsive to vitamin B12

Case 12 (Level 2)

CasePreviousTesting: same as group

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant1: NM_000255.3:c.323G>A (p.Arg108His)

ClinVar: 100707

CAID: CA235522

gnomAD: 0.00006152 (African)

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

ClinVar: 218998

CAID: CA347913

gnomAD: 0.0001504 (East Asian)

CaseAddInfo: heterozygous mutations in trans

Case11: Female, Chinese, 2 y (age at death)

HPOsFreeText: 9 mo onset, responsive to vitamin B12

Case11 (Level 2)

CasePreviousTesting: same as group

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant1: NM_000255.3(MMUT):c.323G>A (p.Arg108His)

ClinVar: 100707

CAID: CA235522

gnomAD: 0.00006152 (African)

Variant2: NM_000255.3:c.1514T>C (p.!le505Thr)

ClinVar: n/a

CAID: CA364397174

gnomAD: n/a

CaseAddInfo: heterozygous mutations in trans, died at 2 y

Case10: Male, Chinese, 6.4 y (onset)

HPOsFreeText: 6.4 y onset

NotHPOsFreeText: not responsive to vitamin B12

Case10 (Level 2)

CasePreviousTesting: same as group

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant1: NM_000255.3(MMUT):c.323G>A (p.Arg108His)

ClinVar: 100707

CAID: CA235522

gnomAD: 0.00006152 (African)

Variant2: NM_000255.3:c.729_730insTT (p.Asp244LeufsTer?)

ClinVar: 552079

CAID: CA3847064

gnomAD: 0.0002008 (East Asian)

CaseAddInfo: lost to follow up, heterozygous mutations in trans

Case13: Female, Chinese, 7 mo (onset)

HPOsFreeText: 7 mo onset

NotHPOsFreeText: not responsive to vitamin B12

Case13 (Level 2):

CasePreviousTesting: same as group

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant1: NM_000255.3:c.419T>C (p.Leu140Pro)

ClinVar: n/a

CAID: CA364404765

gnomAD: n/a

Variant2: NM_000255.3:c.424A>G (p.Thr142Ala)

ClinVar: n/a

CAID: CA364404758

gnomAD: n/a

CaseAddInfo: Variant 1 is novel. Heterozygous mutations in trans. lost to follow up.

Case1: Male, Chinese, 1.8 y (end of study)

HPOsFreeText: neonatal onset, poor neurological outcome

NotHPOsFreeText: not responsive to vitamin B12

Case9 (Level 2)

CasePreviousTesting: same as group

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant1: NM_000255.3:c.322C>T (p.Arg108Cys)

ClinVar: 1887

CAID: CA249732

gnomAD: 0.0008393 (Latino)

Variant2: NM_000255.3(MMUT):c.323G>A (p.Arg108His)

ClinVar: 100707

CAID: CA235522

gnomAD: 0.00006152 (African)

Variant3: NM_000255.3:c.2168G>A (p.Gly723Asp)

ClinVar: n/a

CAID: CA3846630

gnomAD: 0.00006533 (South Asian)

CaseAddInfo: Variant 1 in trans to Variants 2 and 3 (cis). Variant 3 is novel

Case8: Male, Chinese, 8 mo (onset)

HPOsFreeText 8 mo onset, responsive to vitamin B12

Case8 (Level 2)

CasePreviousTesting: same as group

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant1: NM_000255.3:c.278G>A (p.Arg93His)

ClinVar: 1880

CAID: CA249728

gnomAD: 0.0004777 (European-Finnish)

Variant2: NM_000255.3:c.567T>G (p.Asn189Lys)

ClinVar: 618734

CAID: CA364404447

gnomAD: n/a

CaseAddInfo: lost to follow up, heterozygous mutations in trans

Case7: Male, Chinese, 33 d (age at death)

HPOsFreeText: Neonatal onset

NotHPOsFreeText: Not responsive to vitamin B12

Case7 (Level 2)

CasePreviousTesting: Same as group

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant1: NM_000255.3:c.103C>T (p.Gln35Ter)

ClinVar: n/a

CAID: CA364405751

gnomAD: 0.00009950 (Ashkenazi Jewish)

Variant2: NM_000255.3:c.494A>G (p.Asp165Gly)

ClinVar: n/a

CAID: CA138799926

gnomAD: 0.00005443 (East Asian)

CaseAddInfo: died at 33d, heterozygous mutations in trans

Case6: Female, Chinese, 1 mo (onset)

HPOsFreeText: not responsive to vitamin B12

Case6 (Level 2)

CasePreviousTesting: same as group

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant1: NM_000255.3:c.68C>G (p.Ser23Ter)

ClinVar: n/a

CAID: CA364405860

gnomAD: n/a

Variant2: NM_000255.3:c.323G>A (p.Arg108His)

ClinVar: 100707

CAID: CA235522

gnomAD: 0.00006152 (African)

CaseAddInfo: lost to follow up, heterozygous mutations in trans

Case17 4yo diagnosis, Anglo-Celtic

CasePresentingHPOs HP:0001083

(Lens dislocation)

HPOsFreeText 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 was not responsive to the treatment, plasma homocysteine levels went unchanged. This patient was given additional oral betaine(3g twice per day) with a low protein diet.

CaseNotHPOs HP:0001249

(Intellectual disability)

NotHPOsFreeText no other sequelae

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 NM_000071.2(CBS):c.919G>A (p.Gly307Ser), homozygous

ClinVar 117

gnomAD MAF= 0.00041539(Other), no homozygotes

CaseAddInfo n/a

CasePMIDs n/a

Case29 Male, 1yo diagnosis (Family 25)

CasePresentingHPOs HP:0001256, HP:0001263

(Mild intellectual disability, Developmental delay)

HPOsFreeText Diagnosed was on the basis of developmental delay. 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 no other sequelae

CasePreviousTesting n/a

Variant 1. NM_000071.2(CBS):c.536_553del18 (p.Asp179_Leu184del), 2. NM_000071.2(CBS):c.325T>C (p.Cys109Arg), comp het

ClinVar 1. 198388, 2. 212878

gnomAD 1. MAF=0.00002683(European(non-Finnish), no homozygotes, 2. MAF= 0.0001631(Other), no homozygotes

CaseAddInfo son of patient 30

CasePMIDs n/a

Case25 9yo diagnosis, Australia, Anglo-Celtic

CasePresentingHPOs HP:0001083, HP:0002342, HP:0000939, HP:0001263, HP:0001369

(Lens dislocation, Moderate intellectual disability, Osteoporosis, Developmental delay, Arthritis)

HPOsFreeText Patient had severe osteoporosis, juvenile arthritis and was diagnosed on the basis of dislocated lenses and developmental delay. 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. (PMID:9156316): Total homocysteine was 252μmol/l before and 34μmol/l after treatment of betaine, folic acid, and low-dose aspirin.

CaseMethod1 PCR

CaseMethod2 Sanger

CaseGenotypingMethod PCR amplified then direct sequenced the entire coding region of CBS. (PMID: 9156316): Restriction analysis confirmed the presence of two variants. The variant c.430G>A destroys a TaqI site. Thus, TaqI digestion yielded uncleaved 155bp fragments in both the proband and his father, while the WT was cleaved into 107 and 48bp fragments. The c.1316G>A variant likewise destroys a HapII site. The proband and his father had uncleaved 120bp fragments while the WT had 87 and 33bp fragments on HapII digestion. As both variants were detected in the proband, father, and two paternal aunts, c.1316G>A and c.430G>A were proposed to be in cis.

Variant 1. NM_000071.2:c.892dup (p.Gln298ProfsTer?), 2. NM_000071.2(CBS):c.430G>A (p.Glu144Lys); 3. NM_000071.2(CBS):c.1316G>A (p.Arg439Gln), comp het, 2&3 in cis

ClinVar 1. n/a, 2. 122; 3. 189088

CAID 1. register

gnomAD 1. MAF=0.00001777(European(Non-finnish)), no homozygotes 2. MAF=0.00009799(South Asian), no homozygotes, 3. MAF=0.00005341(African), no homozygotes

CaseAddInfo n/a

CasePMIDs 9156316

Case15 3yo diagnosis, Anglo-Celtic

CasePresentingHPOs HP:0001083, HP:0001256

(Lens dislocation, Mild intellectual disability)

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

CaseNotHPOs n/a

NotHPOsFreeText no other sequelae

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 NM_000071.2(CBS):c.919G>A (p.Gly307Ser), homozygous

ClinVar 117

gnomAD MAF= 0.00041539(Other), no homozygotes

CaseAddInfo n/a

CasePMIDs n/a

CaseGM01781: Female, 18 years report, Caucasian, Europe

CasePresentingHPOs: HP:0003153 (Cystathioninuria)

HPOsFreeText: N/A

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Proband is a compound heterozygote for these variants, Variants were confirmed in trans by parental testing

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.

Variant1: 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

Variant2: NM_001902.5:c.718C>G (p.Gln240Glu)

ClinVar: 2940 https://www.ncbi.nlm.nih.gov/clinvar/variation/2940/

gnomAD: European (Non-Finnish) 1.759e-5, Overall 7.957e-6 https://gnomad.broadinstitute.org/variant/1-70896071-C-G

Patient11: 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_001354598.2:c.868G>A

CAID: CA391720951, http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA391720951

gnomAD: n/a

Patient10: 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_002225.4:c.436A>G

CAID: CA391716744, http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA391716744

**In ClinVar as: NM_002225.3(IVD):c.445A>G (p.Lys149Glu), https://www.ncbi.nlm.nih.gov/clinvar/variation/553403/

gnomAD: n/a

Case: Male, 5 days onset, Iran

CasePresentingHPOs: HP:0002572, HP:0004923 (Episodic vomiting, Hyperphenylalaninemia)

HPOsFreeText: Brown eyes

CaseNotHPOs: HP:0001249, HP:0032807 (Intellectual disability, Neonatal seizure)

NotHPOsFreeText: N/A

CaseAddInfo: Parents are consanguineous first cousins

CasePreviousTesting: Screened for HPAs by Guthrie test and on special diet

CaseGenotypingMethod: Genomic DNA was extracted from peripheral blood. PAH gene was screened but no mutations were found. BH4-metabolizing genes were screened via STR markers, with PCBD1 having a homozygous pattern. Direct sequencing revealed a homozygous termination codon extension.

Variant: NM_000281.4(PCBD1):c.313T>C (p.Ter105Gln)

ClinVar: 428616, https://www.ncbi.nlm.nih.gov/clinvar/variation/428616/

gnomAD: 0.00003301, https://gnomad.broadinstitute.org/variant/10-72643709-A-G

CaseAHN5.1: Australia, White

CasePresentingHPOs: HP:0008353 (Neutral hyperaminoaciduria)

HPOsFreeText: N/A

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Variant 1 is in-trans with Variants 2 and 3, which are in cis; Variant segregates with the disease

CasePMIDs: 17555458

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.196G>A (p.Gly66Arg)

CAID: CA359061114 http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA359061114

gnomAD: Not in gnomAD

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

Variant3: 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

CaseAHN9.2: 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(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

Variant2: NM_001003841.3:c.1550A>G (p.Asp517Gly)

CAID: CA3183226, http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA3183226

gnomAD: 0.00004675, https://gnomad.broadinstitute.org/variant/5-1221277-A-G

Case1: Newborn, Indian (consanguineous)

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 0.88 nM/ml (ref <0.8). 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.295C>T (p.Gln99Ter) (homozygous)

ClinVar: 281177

gnomAD: 0.00006537 South Asian

VariantEvidence: LOF

CaseAddInfo:

CasePMIDs:

Case4: Male, 1y 2m, Chinese

HPOsFreeText: (at 21 days) 0.77 C4 umol/L, 0.07 C4/C2, 0.86 C4/C3

NotHPOsFreeText: normal urine isobutyrylglycine

Case4 (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.444G>T (p.Pro148=)

ClinVar: n/a

CAID: CA6372946

gnomAD: 0.0002772 (Other)

CaseAddInfo: trans heterozygous, inherited one allele from each parent

Case1: Female, 1y 7m, Chinese

HPOsFreeText: (at 13 days) C4 1.31 umol/L, C4/C2 0.21, C4/C3 1.96

NotHPOsFreeText: normal urine isobutyrylglycine

Case1 (Level 2)

CasePreviousTesting: same as group

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant1: NM_014384.2:c.235C>G (p.Arg79Gly)

ClinVar: n/a

CAID: CA6372884

gnomAD: 0.0002718 (East Asian)

Variant2: NM_014384.2:c.1000C>T (p.Arg334Cys)

ClinVar: n/a

CAID: CA6373194

gnomAD: 0.0003007 (East Asian)

CaseAddInfo: trans heterozygous, inherited one allele from each parent

Case# Patient 11, Female, 5mo diagnosis

Variant NM_000320.2(QDPR):c.185+3delinsAC (p.W35fs)

CAID CA658795310

VariantEvidence frameshift

Case# Patient 17, Female, Turkish, 2mo diagnosis

CasePresentingHPOs HP:0004923

CasePreviousTesting screened for BH4 deficiency using dried blood and urine spots, Final di-agnosis was made by measurements of neurotransmitter and pterinmetabolites in cerebrospinal fluid (CSF) and urine, in addition toDHPR enzyme activity, which was assessed in whole red bloodcells

CaseMethod1 PCR

CaseMethod2 DGGE

Variant NM_000320.2:c.741A>T(p.R221Ter)

CAID CA658795309

VariantEvidence null

Case30 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 25(nv 2–10 mmol/mol creatinine) and 3-OH-glutarate 571(nv 2–14 mm/mol creatinine). In patient fibroblasts, %GCD activity was 1. 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 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

Case22 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 1028(nv 2–10 mmol/mol creatinine) and 3-OH-glutarate 291(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.

Variant NM_000159.3:c.541G>A(p.Glu181Lys), homozygous

ClinVar n/a

CAID CA404318015

gnomAD not in gnomAD

CaseAddInfo n/a

CasePMIDs see note on previous studies

Case36 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 24(nv 2–10 mmol/mol creatinine) and 3-OH-glutarate 221(nv 2–14 mm/mol creatinine). In patient fibroblasts, %GCD activity was 22. 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 V400M variant creates an NlaIII restriction site. Thus, while the patient's DNA fragments would cleave, control fragments would not be cleaved. Likewise, the R227P variant creates a NlaIV site. This would lead to cleaved patient DNA fragments and uncleaved control fragments.

Variant 1. NM_000159.3(GCDH):c.1198G>A (p.Val400Met), 2. NM_000159.3(GCDH):c.680G>C (p.Arg227Pro), compound heterozygous

ClinVar 1. 2088, 2. 2089

gnomAD 1. MAF= 0.0002769(Other), no homozygotes, 2. MAF= 0.0003566(European(non-Finnish)), no homozygotes

CaseAddInfo n/a

CasePMIDs n/a

Case12 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 8188(nv 2–10 mmol/mol creatinine) and 3-OH-glutarate 3573(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 n/a

CasePMIDs n/a

Case19 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 8955(nv 2–10 mmol/mol creatinine) and 3-OH-glutarate 2262(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 R372K variant destroys an MnlI restriction site. Thus, while the patient's DNA fragments would not cleave, control fragments would be cleaved.

Variant 1. NM_000159.3(GCDH):c.1115G>A (p.Arg372Lys), 2. NM_000159.3(GCDH):c.1173delG (p.Asn392Metfs), compound heterozygous

ClinVar 1. 557606, 2. 370106

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

CaseAddInfo n/a

CasePMIDs see note on previous studies

Case8 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 1098(nv 2–10 mmol/mol creatinine) and 3-OH-glutarate 148(nv 2–14 mm/mol creatinine). In patient fibroblasts, %GCD activity was 6.

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 1. NM_000159.3(GCDH):c.877G>A (p.Ala293Thr), 2. NM_000159.3(GCDH):c.268G>A (p.Glu90Lys), compound heterozygous

ClinVar 1. 2083, 2. 459949

gnomAD 1. MAF=0.0004013(African), no homozygotes, 2. MAF= 0.00008011(African), no homozygotes

CaseAddInfo n/a

CasePMIDs n/a

CaseF-15 female, 1yo onset, Ethnicity: North Indian, Hindu

CasePresentingHPOs HP:0006846, HP:0001254, HP:0011968, HP:0002098, HP:0002013, HP:0001942, HP:0001943, HP:0003571

(Acute encephalopathy, Lethargy, Poor feeding, Respiratory distress, Vomiting, Metabolic acidosis, Hypoglycemia, Propionicacidemia)

HPOsFreeText Ketones present, acycarnitines 6.2(normal <3.6nmol/mL), died after acute crisis(1yo)

CaseNotHPOs n/a

CasePreviousTesting Also screened for variants in PCCA

Variant NM_000532.4(PCCB):c.493C>T (p.Arg165Trp)

ClinVar 217894

gnomAD MAF= 0.00001456(European(non-Finnish), no homozygotes

CaseAddInfo nonconsanguineous, no abnormality in family history noted

CasePMIDs n/a

Case5: Male, 16 yrs report

CasePresentingHPOs: HP:0012072, HP:0010864, HP:0000734 (Aciduria, Severe intellectual disability, Disinhibition)

HPOsFreeText: N/A

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo:

CasePreviousTesting: Patient fibroblasts were analyzed for mutations in SLC25A21 and activities of KATII with no abnormalities; Metabolic work-up at 17 years of age revealed strongly elevated AAA (549 mmol/mol creatinine; ref <14) and KAA

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.2329T>C (p.Ser777Pro)

CAID: CA376014875 http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_canonicalid?canonicalid=CA376014875

gnomAD: 0.000008948, https://gnomad.broadinstitute.org/variant/10-12159681-T-C

Case2: male, 9.6 y (evaluation), Italian, Family 1

CasePresentingHPOs:

HPOsFreeText: birth weight 3850 g, 5y slightly elevated serum transaminases SGOT 60 U/L, SGPT 52 U/L. elevated alkaline phosphatase 1042 U/L). elevated triglycerides (342 mg/dl). fasting plasma methionine elevated to 426 umol/L. elevated tHcy 13 umol/L, elevated urinary methionine 34 umol/mmol. increased pyruvate excretion.

CaseNotHPOs:

NotHPOsFreeText: normal neurological examination and normal somatic growth. normal serum amylase and lipase. hepatic dysfunction.

CaseAddInfo: proband, first child

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).

Case3 Male, 5yo report, USA, Lumbee

CasePresentingHPOs HP:0003150

(Glutaricaciduria)

HPOsFreeText Patient had elevated glutaric acid and small 3-hydroxyglutaric acid in urine. Patient 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 HP:0002540, HP:0001344, HP:0011471

(Non-ambulatory, Nonverbal)

NotHPOsFreeText Patient did not present with acute metabolic decompensation.

CasePreviousTesting Patient's NBS detected glutarylcarnitine of 1.07μM(cutoff at >0.38). GCD enzyme activity was 0%.

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

Case1: Female, Anglo-Celtic, Birth onset

CasePresentingHPOs: HP:0001511, HP:0008872, HP:0025404, HP:0000737, HP:0010845, HP:0001263, HP:0001290, HP:0002059, HP:0012448, HP:0002079, HP:0002474, HP:0002421 (Intrauterine growth retardation, Feeding difficulties in infancy, Abnormal visual fixation, Irritability, EEG with genaralized slow activity, Developmental delay, Hypotonia, Cerebral atrophy, Delayed myelination, Thinning of the corpus callosum, Communication delay, Poor head control)

HPOsFreeText: Birth weight was 2160 g, Inability to follow with eyes at four months, EEG showed poorly modulated background, MRI showed markedly increased signals in the brain stem, upper pons, basal ganglia and thalami, Inability to sit unsupported

CaseNotHPOs: HP:0001250, HP:0001946 (Seizures, Ketosis)

NotHPOsFreeText: N/A

CaseAddInfo: Parents were not consanguineous, parental testing confirms in trans

CasePreviousTesting: Routine metabolic investigations at 4 months (plasma and CSF amino acids, urine amino and organic acids, transferrin isoforms, very long chain fatty acids) were uninformative, Increased excretion of SCPC, Increased levels of urine metabolites derived from methacrylate such as S-(2-carboxypropyl)cysteine (18,27 μmol/mmol of creatinine; reference <5.3), N-acetyl-S-(2-carboxypropyl)cysteine (30,42 μmol/mmol of creatinine; reference <4.2), S-(2-carboxypropyl)cysteine carnitine ester (5.8,5.3 μmol/mmol of creatinine; reference <1.0), and S-(2-carboxypropyl)cysteamine (13,16 μmol/mmol of creatinine; reference <10), Increased erythro-2,3-dihydroxy-2-methylbutyrate compared to controls, The coding region of the E1α gene was sequenced and was found normal

CaseGenotypingMethod: The HIBCH gene was sequenced after the metabolite testing. Other details were not described.

Variant1: NM_014362.4(HIBCH):c.1033G>A (p.Gly345Ser)

ClinVar: 208532 https://www.ncbi.nlm.nih.gov/clinvar/variation/208532/

gnomAD: European (Non-Finnish) 8.805e-6, Overall 3.982e-6 https://gnomad.broadinstitute.org/variant/2-191073618-C-T

Variant2: NM_014362.4(HIBCH):c.129dup (p.Gly44Argfs*20)

ClinVar: 208531 https://www.ncbi.nlm.nih.gov/clinvar/variation/208531/

gnomAD: South Asian 9.834e-5, Overall 1.597e-5 https://gnomad.broadinstitute.org/variant/2-191161628-C-CT

CaseST-7: Male, 1 day onset, Italy

CasePresentingHPOs: HP:0006579, HP:0001508, HP:0003215, HP:0000825, HP:0003256, HP:0006254, HP:0008947, HP:0006580, HP:0011968, HP:0002572, HP:0001251 (Prolonged neonatal jaundice, Failure to thrive, Dicarboxylic aciduria, Hyperinsulinemic hypoglycemia, Coagulopathy, Elevated alpha-fetoprotein, Infantile muscular hypotonia, Portal fibrosis, Feeding difficulties, Episodic vomiting, Ataxia)

HPOsFreeText: Small gallbladder stone

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.199C>T (p.Gln67Ter)

CAID: CA5563907, http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA5563907

gnomAD: 0.000008818, https://gnomad.broadinstitute.org/variant/10-76074480-C-T

Case# HCU-101-3, 10yo report, Iran, Sistani

CasePresentingHPOs HP:0001249, HP:0001083, HP:0002970, HP:0001634, HP:0003235, HP:0000924

(Intellectual disability, Ectopia lentis, Genu varum, Mitral valve prolapse, Increased methionine in blood, Skeletal abnormality)

HPOsFreeText skeletal abnormality/elongated long bones, elevated, elevated plasma homocysteine, elevated urinary cysteine, elevated plasma methionine

CaseNotHPOs HP:0001010, HP:0000501

(Hypopigmentation, Glaucoma)

NotHPOsFreeText n/a

CasePreviousTesting Plasma homocysteine 330μm/l(upper normal limit 15μm/l), plasma methionine 142μm/l(normal range 6-40μm/l)

CaseMethod1 Sanger

CaseMethod2 n/a

CaseGenotypingMethod Sanger sequenced exon 3 of the CBS gene

Variant NM_000071.2(CBS):c.346G>A (p.Gly116Arg), homozygous

ClinVar 188787

gnomAD MAF=0.00002892(Latino), no homozygotes

CaseAddInfo from the province of Sistan and Baluchestan. no known consanguinity although the patient is homozygous for a founder variant

CasePMIDs n/a

CaseIV-2: Female, 4 months onset, Lebanese

CasePresentingHPOs: HP:0002463, HP:0002376, HP:0001332, HP:0002510, HP:0002522, HP:0000648, HP:0011471, HP:0011968, HP:0001508, HP:0002453, HP:0007374, HP:0002518, HP:0007042, HP:0011931, HP:0012751, HP:0200147, HP:0000252, HP:0001141, HP:0000486, HP:0002300, HP:0002421, HP:0002540, HP:0008936, HP:0002509, HP:0040288, HP:0002650, HP:0002828, HP:0002827, HP:0010465, (Language impairment, Developmental regression, Dystonia, Spastic quadriplegia, Areflexia of lower limbs, Optic atrophy, Gastrostomy tube feeding in infancy, Poor feeding, Failure to thrive, Abnormality of the globus pallidus, Atrophy/Degeneration involving the caudate nucleus, Abnormality of the periventricular white matter, Focal white matter lesion, Abnormality of the cerebellar peduncle, Abnormal basal ganglia MRI signal intensity, Neuronal loss in basal ganglia, Microcephaly, Severe visual impairment, Strabismus, Mutism, Poor head control, Inability to walk, Truncal hypotonia, Limb hypertonia, Nasogastric tube feeding, Scoliosis, Multiple joint contractures, Hip dislocation, Precocious puberty in females,

HPOsFreeText: Normal early developmental milestones, Not able to bear weight, Unable to fix or follow, Intermittent roving eye movement,

CaseNotHPOs: HP:0000240, HP:0001999, HP:0025336, (Abnormality of head circumference, Dysmorphic facies, Delayed ability to sit,

NotHPOsFreeText:

CaseAddInfo: Parents are consanguinous (first cousins),

CasePreviousTesting: Elevated hydroxy-C4-carnitine at 0.88 μM for patient 1 (control < 0.3 μM).

CaseGenotypingMethod: Whole exome sequencing was performed on the proband's sibling, with all SNVs and variants detected called, investigated, and filtered for pathogenicity. Mitochondrial DNA mutation tests were negative, as well as a southern blot for deletions in the muscle. Sanger sequencing of SURF1, SCO1, SCO2, COX10, NDUFV1, NDUFS7, and PANK2 revealed no pathogenic mutation. The WES identified a homozygous variant in HIBCH that was orthogonally validated. PCR-based Sanger sequencing was performed on the proband and the parents to confirm the presence of the mutations and recessive inheritance.

Variant: NM_014362.3:c.196C>T (p.Arg66Trp)

ClinVar: 190268 https://www.ncbi.nlm.nih.gov/clinvar/variation/190268/

gnomAD: Latino 2.892e-5, Overall 1.193e-5 https://gnomad.broadinstitute.org/variant/2-191161562-G-A

Case#: Patient 3, M, 6 days (onset), Tunisia

CasePresentingHPOs: HP:0100022, HP:0011968, HP:0000737, HP:0001250, HP:0001257, HP:0001254, HP:0001993, HP:0012088

CasePreviousTesting: Clinical diagnosis of classic MSUD due to elevated levels of plasma BCAAs and presence of L-alloisoleucine.

CaseMethod1: PCR

CaseMethod2: Direct sequencing

CaseGenotypingMethod: Entire coding regions and flanking intronic regions of BCKDHA, BCKDHB and DBT amplified from genomic DNA and sequenced.

Variant: NM_001918.3:c.1333_1336delAATG (homozygous)

CAID: CA658820833

gnomAD: Not in gnomAD

VariantEvidence: NMD unlikely. The truncated protein, however, is likely unable to self-assemble (several copies of E2 form the core of the BCKD complex).

CaseAddInfo: Consanguineous parents.

Case#: OA-20, F, 10 months (report), Indian

CasePresentingHPOs: Classic form of MSUD.

CasePreviousTesting: Initially diagnosed with MSUD based on clinical features and biochemical tests (urine screening by DNPH and thin layer chromatography for amino acids). Confirmed diagnosis by blood amino acid quantification using HPLC.

CaseMethod1: PCR

CaseMethod2: Sequencing

CaseGenotypingMethod: Genomic DNA extracted from peripheral blood leukocytes. PCR of exonic and flanking intronic regions of BCKDHA, BCKDHB, and DBT.

Variant: 1. NM_001918.3:c.827T>C (p.Phe276Cys); 2. NM_001918.3:c.190G>A (p.Val64Ile).

ClinVar: 1. 11943

CAID: CA341345584

gnomAD: 1. European (Non-Finnish) 0.0002210, Overall 0.0001154; 2. Not in gnomAD

VariantEvidence: p.Phe276Cys results in an enzyme activity of the DBT protein similar to wild type (PMID: 2914958, 1847055).

CaseAddInfo: Unsure if the variants are in trans.

Patient10: Female, 11yo, sibling to patient 11

CasePresentingHPOs:

HPOsFreeText: intermittent isovaleric acidemia, C5 (DBS) = 15.7 μmol l−1, IVA incorp (%) = 7, IVG in urine mmol permol creatinine = 4376, pH = 7.35, Protein intake (g kg−1 per day) = 0.64, treated with carnitine and glycine supplements, PDI/IQ in follow-up = 85

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

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

NotHPOsFreeText: sweaty feet odor

CaseAddInfo: diagnosed via family evaluation at 2 days old

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

Patient6: Female, 4yo

CasePresentingHPOs:

HPOsFreeText: Asymptomatic, C5 (DBS) = 3.1 μmol l−1, IVG in urine mmol permol creatinine = 230, Ammonium (μmol l−1) = 56, pH = 7.37, Protein intake (g kg−1 per day) = 1.5, PDI/IQ in follow-up = 104

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 10 days old

CasePMIDs:

CasePreviousTesting: same as group

CaseGenotypingMethod: same as group

Variant1: NM_002225.3:c.158G>A listed in ClinVar as: NM_002225.4(IVD):c.149G>A (p.Arg50His)

CAID: CA7480513, http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA7480513

ClinVar: 370843, https://www.ncbi.nlm.nih.gov/clinvar/variation/370843/

gnomAD: 0.00002784, http://gnomad.broadinstitute.org/variant/15-40699841-G-A

Variant2: NM_002225.3(IVD):c.941C>T (p.Ala314Val)

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

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

Case1: male, German, 4 days (onset)

CasePresentingHPOs:

HPOFreeText: Neonatal screening suggestive of MBD deficiency. Treatment since 9 months with 90 mg L-carnitine per kg body weight. Normal at 6 years old.

CaseNotHPOs:

CasePreviousTesting: Day 4: elevated C5 acylcarnitine (0.57 umol/l, ref <0.31) confirmed by 3 of 4 subsequent tests of DBS. Urinary organic acids showed normal N-isovalerylglycine but increased N-methylbutyrylglycine.

CaseGenotypingMethod: Genomic DNA extracted from DBS or peripheral blood. All exons and intron-exon boundaries of ACADSB sequenced.

Variant: NM_001609.3(ACADSB):c.38G>A (p.Arg13Lys) (homozygous)

ClinVar: 299068

gnomAD: European (Finnish): 0.4494; 1546 homozygotes

VariantEvidence: High frequency, listed as benign in ClinVar.

CaseAddInfo:

CasePMIDs:

Case#: Patient 13, 3 days (onset), China

CasePresentingHPOs: HP:0012758, HP:0008344

HPOsFreeText: Thiamine-responsive form of MSUD.

CasePreviousTesting: Diagnosed based on elevated blood levels of leucine, valine and the ratio of leucine to phenylalanine (tandem mass spec), as well as abnormal urine BCKAs (gas phase chromatography-mass spec). . CaseMethod1: PCR

CaseMethod2: Sequencing

CaseGenotypingMethod: Genomic DNA from peripheral blood leukocytes from patients and parents extracted. PCR amplification of exons and intron/exon boundaries for BCKDHA, BCKDHB, DBT.

Variant: 1. NM_001918.3(DBT):c.51+5G>C

ClinVar: 1. 94004

CAID: 2. CA658820834

gnomAD: 1. East Asian 6.165e-4, overall 3.229e-5 ; 2. Not in gnomAD

VariantEvidence: Predicted LOF

CaseAddInfo: Parents tested.

Case35 Male, 7.1yo report, Ethnicity: Syrian

CasePresentingHPOs HP:0003571, HP:0002789

(propionicacidemia, tachypnea)

HPOsFreeText tachypnea(6do). 55 IQ on Culture Free Test

CaseNotHPOs n/a

CasePreviousTesting Diagnosis by NBS. Patient fibroblasts had 1.5% PCC activity of control.

Variant NM_000532.4:c.1313C>T (p.Ala438Val), homozygous

ClinVar n/a

CAID CA354649295

gnomAD not in gnomAD

CaseAddInfo no parental consanguinity

CasePMIDs n/a

Case34 female, 5.5yo report, Ethnicity: German

CasePresentingHPOs HP:0003571, HP:0012523, HP:0001531, HP:0002791, HP:0001944

(propionicacidemia, oral aversion, failure to thrive in infancy, bradypnea, dehydration)

HPOsFreeText feeding refusal, failure to thrive, bradypnea, dehydration(2do). 55 IQ on Culture Free Test

CaseNotHPOs n/a

CasePreviousTesting Diagnosed on newborn screen. PCC activity in patient fibroblasts was 0.9% of control

Variant NM_000532.4:c.483dup (p.Gly162TrpfsTer?), homozygous

ClinVar n/a

CAID to register

gnomAD not in gnomAD

CaseAddInfo no parental consanguinity

CasePMIDs n/a

CaseEG1291: Female, 5 days onset

CasePresentingHPOs: HP:0200134, HP:0012469, HP:0032794, HP:0032667, HP:0002187, HP:0032792, HP:0002069, HP:0007359, HP:0010841, HP:0002188, HP:0006913, HP:0002079, HP:0001290 (Epileptic encephalopathy, Infantile spasms, Myoclonic seizure, Myoclonic status epilepticus, Profound intellectual disability, Tonic seizure, Bilateral tonic-clonic seizure, Focal-onset seizure, Multifocal epileptiform discharges, Delayed CNS myelination, Frontal cortical atrophy, Hypoplasia of the corpus callosum, Generalized hypotonia)

HPOsFreeText: N/A

CaseNotHPOs: HP:0002521 (Hypsarrhythmia)

NotHPOsFreeText: N/A

CaseAddInfo: Proband confirmed de novo heterozygous by parental sequencing; Father had two seizures as an adult, no further seizures while on LTG

CasePreviousTesting: Currently on levitiracetam and valproate; previously on pyridoxine, prednisolone, vigabatrin, zonisamide and ketogenic diet

CaseGenotypingMethod: About 10% of the original 290 genes where a single de novo variant had been identified was chosen for targeted sequencing in a new cohort. Most of these individuals had been tested for mutations of known and novel candidate genes as well as pathogenic copy-number variants. These genes were sequenced in the parents and proband to look for de novo variants.

Variant: NM_004171.4(SLC1A2):c.244G>C (p.Gly82Arg)

ClinVar: 254265, https://www.ncbi.nlm.nih.gov/clinvar/variation/254265/

gnomAD: Not in gnomAD

Case8: Female, South America

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

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.

Variant1: NM_198271.4:c.723_733del (p.Asp242Glufs*4)

CAID: CA2488932 http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/allele?hgvs=NM_198271.4%3Ac.723_733del

gnomAD: Latino 2.896e-4, Overall 4.416e-5 https://gnomad.broadinstitute.org/variant/3-69168772-AAGCTCCCATCC-A

Variant2: NM_198271.4:c.976G>C (p.Gly326Arg)

CAID: CA353473660 http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/allele?hgvs=NM_198271.4%3Ac.976G%3EC

gnomAD: Not in gnomAD

Case9: Female, Italy

CasePresentingHPOs: HP:0001290, HP:0003324, HP:0002093, HP:0011968, HP:0001283, HP:0003798, HP:0001561, HP:0001558, HP:0000602, HP:0001622, HP:0001371 (Generalized hypotonia, Generalized muscle weakness, Respiratory insufficiency, Feeding difficulties, Bulbar weakness, Nemaline bodies, Polyhydramnios, Decreased fetal movements, Ophthalmoplegia, Preterm delivery, Contractures)

HPOsFreeText: N/A

CaseNotHPOs: HP:0001627 (Cardiac abnormality)

NotHPOsFreeText: N/A

CaseAddInfo: Severe congenital myopathy, Deceased in 4 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.860delT (p.Phe287Serfs*3)

CAID: CA913203455 http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA913203455

gnomAD: Not in gnomAD

CaseOS-152_II-1: Female, 1 yr report, United Kingdom

CasePresentingHPOs: HP:0001992, HP:0002013, HP:0002013, HP:0040293, HP:0010628, HP:0007359 (Organic aciduria, Vomiting, Bilateral tonic-clonic seizure, Right hemiplegia, Facial palsy, Focal-onset seizure)

HPOsFreeText: N/A

CaseNotHPOs: HP:0001263 (Global developmental delay)

NotHPOsFreeText: N/A

CaseAddInfo: Parents are non-consanguineous, Proband is confirmed compound heterozygous for these variants via parental testing, R353C variant inherited from father but R393H supposedly de novo or mosaic

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

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

Variant1: 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

Variant2: NM_000666.3(ACY1):c.1178G>A (p.Arg393His)

ClinVar: 18114 https://www.ncbi.nlm.nih.gov/clinvar/variation/18114/

gnomAD: 0.006571 (3 homozygotes), https://gnomad.broadinstitute.org/variant/3-52023042-G-A

Case 8 (Level 2)

CasePreviousTesting: same as group

CaseMethod1: PCR

CaseGenotypingMethod: same as group

__

Variant1: NM_015506.2(MMACHC):c.394C > T (p.Arg132Ter)

ClinVar: 1423

CAID: CA251787

gnomAD: 0.0007190 (South Asian)

__

Variant2: NM_015506.2(MMACHC):c.609G > A (p.Trp203Ter)

ClinVar: 30800

CAID: CA259906

gnomAD: 0.0005562 (East Asian)

CaseAddInfo: proband genotype, unknown cis or trans, no phenotypic info, sibling (fetus) carrier of p.R132X maternal variant

Case 9 (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.637G>T (p.Glu213Ter)

ClinVar: n/a

CAID: CA340133779

gnomAD: n/a

CaseAddInfo: proband genotype, unknown cis or trans, no phenotypic info, sibling (fetus) carrier of p.W203X maternal variant

Case1: Female, 9 days at follow up, North African ancestry

CasePresentingHPOs: HP:0002013, HP:0001944,HP:0031123, HP:0000388 (emesis, dehydration, recurrent gastroenteritis, otitis media)

HPOsFreeText: NBS C4-acylcarnitine 2.9 umol/L, plasma C4 acylcarnitine 1.35 umol/L, isoC4-acylcarnitine 0.090 umol/g protein,

CaseNotHPOs:

NotHPOsFreeText: normal urine isobutyrylglycine

CaseAddInfo: repeatedly hospitalized during first 2 years of life.

CasePMIDs:

Case (Level 2)

CasePreviousTesting: same as group

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant: NM_014384.2(ACAD8):c.988C>T (p.Arg330Trp)

ClinVar: 5357

CAID: CA117438

gnomAD: 0.00006155 (African)

CaseAddInfo: homozygous mutations

FamilyHA: White, Great Britian

FamilyPresentingHPOs: HP:0000988, HP:0002344, HP:0002359, HP:0002355, HP:0007210, HP:0000712, HP:0002066, HP:0000639, HP:0001347, HP:0008353 (Skin rash, Progressive neurologic deterioration, Frequent falls, Difficulty walking, Lower limb amyotrophy, Emotional lability, Gait ataxia, Nystagmus, Hyperreflexia, Neutral hyperaminoaciduria)

HPOsFreeText: N/A

FamilyNotHPOs: N/A

NotHPOsFreeText: N/A

FamilyGenotypingMethod: Homozygosity mapping and fine mapping in ten members of two consanguineous families (the siblings in whom Hartnup disorder was originally discovered; family A) and in siblings from the US revealed linkage of Hartnup disorder to 5p15 only in family A, with a maximum combined multipoint lod score of 2.31 at 11.24 cM. This confirmed a previous study showing linkage to the same region. 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 splice site mutation that perfectly segregated with the phenotype.

FamilyPreviousTesting: Consistent testing showed the consistent phenotypes were the hyperaminoaciduria among all family members; Linkage analysis for SLC3A2, SLC7A8, SLC1A5, SLC6A18, and SLC6A20 were all negative for amy correlation

FamilyAddInfo:

FamilyProband: Case EHAA

FamilyPMIDs: 13358233

Variant: NM_001003841.3(SLC6A19):c.1173+2T>G

ClinVar: 449553 https://www.ncbi.nlm.nih.gov/clinvar/variation/449553/

gnomAD: 0.00008019, https://gnomad.broadinstitute.org/variant/5-1217062-T-C?dataset=gnomad_r2_1

Affected: 4

Unaffected: 5

pLOD: 2.31 at 11.24 cM

AddSegregationInfo: Family is consanguineous (first cousins), Candidate gene sequencing, Pedigree in Figure 1aA

Case5 Female, 6yo diagnosis, Korean

CasePresentingHPOs HP:0001083, HP:0003235

(Ectopia lentis, Increased blood methionine)

HPOsFreeText pretreatment homocysteine was normal, increased post-treatment total homocysteine, patient treated with low-methionine diet, vitamin B6, folate, vitamin B12, and betaine after diagnosis

CaseNotHPOs HP:0001249

(Intellectual disability)

NotHPOsFreeText Marfan syndrome, vascular occlusion

CasePreviousTesting pretreatment/posttreatment methionine: 697/648 μM(nv 7-47), pretreatment homocysteine: 3 μM(nv 0-5), posttreatment total homocysteine 121 μM(nv 5-15)

CaseMethod1 PCR

CaseMethod2 Sanger

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

Variant 1. NM_001178008.1:c.464C>T(p.Ala155Val), 2. NM_000071.2(CBS):c.1039G>A (p.Gly347Ser), compound heterozygous

ClinVar 1. n/a, 2. 188801

CAID 1. CA410601498

gnomAD 1. not in gnomAD, 2. MAF=0.00006497(European(non-Finnish)), no homozygotes

CaseAddInfo A155V inherited from father, G347S from mother

CasePMIDs n/a

CaseHE9: 6 month onset

CasePresentingHPOs: HP:0001250, HP:0001943, HP:0001942, HP:0012120 (Seizure, Hypoglycemia, Metabolic acidosis, Methylmalonic aciduria)

HPOsFreeText: Elevated proionyl-carnitine and methyl-citrate in urine

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Proband is homozygous for this variant,

CasePreviousTesting: Fibroblast cell lines taken from 150 patients with mild but clear MMAuria were studied; Urinary MMA at 143–184 mmol/mol creat. (ref 0.3-1.1); Low propionate incorporation in fibroblasts both +/- OHCbl of 3.0 nmol/16 h/mg (ref 3.5–24.4 −OHCbl, 4.3–28.9 +OHCbl); Not responsive to Vitamin B12 treatment

CaseGenotypingMethod: Genomic DNA and RNA extraction was performed and the MCEE gene was sequenced to identify any likely pathogenic variants.

Variant: NM_032601.3:c.158T>G (p.Ile53Arg)

CAID: CA347189175, http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA347189175

gnomAD: Not in gnomAD

Case# 16392, Spanish, 4yo report

Variant NM_000709.3(BCKDHA):c.659C>T (p.Ala220Val), NM_000709.3(BCKDHA):c.853G>C (p.Ala285Pro)

ClinVar 93367(c.659), 93375(c.853)

gnomAD c.659=0.00005805(East Asian), 0.000008156(Overall), c.853=0.00007896(European (Non-Finnish)), 0.00003970(Overall)

VariantEvidence 4% of normal BCKD activity in patient fibroblasts

CaseAddInfo in trans

CaseCI-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.194T>C (p.Ile65Thr)

ClinVar: 636 https://www.ncbi.nlm.nih.gov/clinvar/variation/636/

gnomAD: 0.0005574, https://gnomad.broadinstitute.org/variant/12-102894893-A-G?dataset=gnomad_r3

Variant2: NM_000277.3(PAH):c.261C>A (p.Ser87Arg)

ClinVar: 583 https://www.ncbi.nlm.nih.gov/clinvar/variation/583/

gnomAD: 0.00009294, https://gnomad.broadinstitute.org/variant/12-102894826-G-T?dataset=gnomad_r3

CaseDII-3: N/A

CasePresentingHPOs: N/A

HPOsFreeText: Pretreatment phenylalanine level at 725 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.782G>C (p.Arg261Pro)

ClinVar: 102832 https://www.ncbi.nlm.nih.gov/clinvar/variation/102832/

gnomAD: Not in gnomAD

Patient1: Male, 14 yo, Thai

CasePresentingHPOs: HP:0011968, HP:0001254, HP:0001250, HP:0001876, HP:0002919, HP:0001942, HP:0001987, HP:0002187, HP:0002300, HP:0002540, HP:0006846, HP:0002901, HP:0001510 (poor feeding, lethargy, seizures, pancytopenia, ketonuria, metabolic acidosis, hyperammonemia, profound mental retardation, inability to speak, inability to walk, acute encephalopathy, hypocalcemia, growth delay)

HPOsFreeText: acute neonatal IVA, hemoglobin = 10g/dL, WBC = 3370/mm^3, platelets = 27,000/mm^3, bicarbonate = 10mmol/L, anion gap = 36mmol/L, low serum calcium, ammonia = 420µmol/L, treated with total blood exchange, treated with low-protein diet and leucine-free formula (150–250mg/kg per day of l‐glycine, and 100mg/kg per day of l‐carnitine), urine isovalerylglycine = 515

CaseNotHPOs:

NotHPOsFreeText:

CaseAddInfo: non-consanguineous family, symptom onset at 7 days old, diagnosed at 2wks old

CasePMIDs:

CasePreviousTesting: urine organic acid analysis with GC/MS (PMID: 27082169)

CaseMethod1: PCR

CaseMethod2: Sequencing

CaseGenotypingMethod: Genomic DNA extracted from peripheral blood, IVD coding exons and flanking introns amplified with PCR (primer in table 1), PCR products purified and sequenced with Big Dye Primer Cycle Sequencing Kit and ABI 210 Genetic Analyzer

Variant1: c.457–3_2CA>GG / no ClinVar or CAID

Variant2: NM_002225.3:c.1025A>G (p.Tyr342Cys) listed in paper as (p.Tyr371Cys)

CAID: CA7480839, http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/allele?hgvs=NM_002225.3%3Ac.1025A%3EG

gnomAD: 0.000003977, https://gnomad.broadinstitute.org/variant/15-40708332-A-G

CaseLE19: Male, 16 yrs report

CasePresentingHPOs: HP:0000964, HP:0002650, HP:0000501, HP:0000518, HP:0000750, HP:0010862, HP:0002572 (Eczema, Scoliosis, Glaucoma, Cataract, Delayed speech and language development, Delayed fine motor development, Episodic vomiting)

HPOsFreeText: N/A

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Variants not confirmed in-trans due to lack of parental testing

CasePreviousTesting: Specific plasma MMA levels, urine MMA levels, urine MA levels, and MMA/MA ratio available in Table 2; Congenital unilateral persistent hyperplastic primary vitreous (PHPV), causing glaucoma and cataract

CaseGenotypingMethod: See Group

Variant1: NM_001243279.3(ACSF3):c.1075G>A (p.Glu359Lys)

ClinVar: 31136, https://www.ncbi.nlm.nih.gov/clinvar/variation/31136/

gnomAD: 0.001054, https://gnomad.broadinstitute.org/variant/16-89180844-G-A

Variant2: NM_001243279.3:c.1239+2T>G

CAID: CA397142431, http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA397142431

gnomAD: Not in gnomAD

Case# Case 1, F, Chinese, 4day old Age of Onset

CasePresentingHPOs HP:0011968, HP:0001276, HP:0002373, HP:0001954, 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_000056.4(BCKDHB):c.508C>T (p.Arg170Cys), NM_000056.3:c.659del

ClinVar 96590 (missense)

CAID CA658820635 (c.659del)

gnomAD 0.00001791 (European(Non-Finnish)), 0.000008123 (Overall)

CasePMIDs missense mentioned in 25381949

CaseSE7: Female, 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

CaseWG2797: N/A

CasePresentingHPOs: HP:0012120 (Methylmalonic aciduria)

HPOsFreeText: N/A

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Proband is asymptomatic and has an affected sibling with similar aciduria

CasePreviousTesting: Propionate Incorporation w/o hydroxycobalamin at 3.9 nmols/mg protein/18hr; Propionate Incorporation with hydroxycobalamin at 4.0 nmols/mg protein/18hr; Adenosylcobalamin synthesis percentage at 12.4%; Elevated methylmalonic values of 1400 µmol/mmol creatinine (reference 0.8-5.2 µmol/mmol creatinine)

CaseGenotypingMethod: Genomic DNA was extracted from patient fibroblasts, sequenced, and the three MCEE/thirteen MUT exons were analyzed and amplified. Any reported mutations were resequenced from fresh PCR product.

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

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

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

CaseII-3: Female, Turkey

CasePresentingHPOs: HP:0001561, HP:0001789, HP:0000494, HP:0000463, HP:0005280, HP:0011829, HP:0000319, HP:0002705, HP:0002033, HP:0006829, (Polyhydramnios, Hydrops fetalis, Downslanted palpebral fissures, Anteverted nares, Depressed nasal bridge, Thin philtrum, Smooth philtrum, High narrow palate, Poor suck, Severe muscular hypotonia,

HPOsFreeText: Birth weight was 1890 g and length was 42 cm, Absent Moro reflex, Required ventilator support from birth

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Born with fractures of the left humerus and pes equinovarus

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

Case2: 8 days onset

CasePresentingHPOs:

HPOFreeText: Identified by after metabolic studies performed for evaluation of clinical disease.

CaseNotHPOs:

CasePreviousTesting: DBS C5 carnitine 0.97 umol/L (ref <0.46); plasma alloisoleucine 1.5 umol/L (ref <2.1); urine C5 carnitine 35.8, 80.6 umol/mmol creatine (ref <3.0); urine 2-EHA 116, 132 U (ref <5); urine 2-MBG 20.2, 36.7 (ref <2.3); urine 2-methylbutyric acid conjugates as R-isomer 45%, 43% (ref 43-56%).

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

Variant: NM_001609.3:c.908G>C (p.Gly303Ala) (homozygous)

CAID: CA378620840

gnomAD: European (non-Finnish) 0.00002323

VariantEvidence:

CaseAddInfo:

CasePMIDs:

Case3: 11 months onset

CasePresentingHPOs:

HPOFreeText: Identified by after metabolic studies performed for evaluation of clinical disease.

CaseNotHPOs:

CasePreviousTesting: DBS C5 carnitine 1.75 umol/L (ref <0.46); urine C5 carnitine 2.7 umol/mmol creatine (ref <3.0); urine 2-EHA 32, 8, 15 U (ref <5); urine 2-MBG 14.5 umol/mmol creatine (ref <2.3); urine 2-methylbutyric acid conjugates as R-isomer 46%, (ref 43-56%).

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

Variant: NM_001609.3:c.443C>T (p.Thr148Ile) (homozygous)

ClinVar: 9202

gnomAD: South Asian 0.002678

VariantEvidence: Decreases ACADSB enzymatic activity to 2.8% of wild type in vitro (PMID: 20547083).

CaseAddInfo:

CasePMIDs:

Case4: 6 years onset

CasePresentingHPOs:

HPOFreeText: Identified by after metabolic studies performed for evaluation of clinical disease.

CaseNotHPOs:

CasePreviousTesting: DBS C5 carnitine 0.5, 0.55 umol/L (ref <0.46); urine 2-EHA 152, 19, 43 U (ref <5).

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

Variant: NM_001609.3:c.1102T>C (p.Ser368Pro) (homozygous)

CAID: CA5730911

gnomAD: Ashkenazi Jewish 0.002200; Overall 0.0002488

VariantEvidence:

CaseAddInfo:

CasePMIDs:

Case1752 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

Case16 Female, Finland

CasePresentingHPOs HP:0003826, HP:0000202, HP:0000347, HP:0001162, HP:0001829, HP:0010066, HP:0000157, HP:0002101, HP:0000238, HP:0007370, HP:0010664, HP:0007260

(Stillbirth, Oral cleft, Micrognathia, Postaxial hand polydactyly, Foot polydactly, Duplicated hallux, Tongue abnormality, Defective lung lobation, Hydrocephalus, Callosal agenesis, Fused thalami, Type II lissencephaly)

HPOsFreeText Crown-heel body length 47cm, body weight 2520g, abnormal larynx/trachea, uterus duplex, hydrocephalus/open-book, occipitotemporal hypoplasia, infratentorial hypoplasia, absent developed and identifiable mamillary bodies, absent anterior commissure, difficult to identify nucleus dentatus

CaseNotHPOs HP:0001627

(Congenital heart defect)

NotHPOsFreeText Hydranencephaly/anencephaly, true alobar or semilobar holoprosencephaly

CasePreviousTesting n/a

Variant NM_145014.2(HYLS1):c.632A>G (p.Asp211Gly), homozygous

ClinVar 1143

gnomAD MAF= 0.009594(European(Finnish)), no homozygotes

CaseAddInfo stillbirth, 34+0wk gestational age

CasePMIDs n/a

Case19 Male, Finland

CasePresentingHPOs HP:0000347, HP:0002101, HP:0000238, HP:0001341, HP:0007370, HP:0010664, HP:0002126, HP:0007260

(Micrognathia, Defective lung lobation, Hydrocephalus, Olfactory lobe agenesis, Callosal agenesis, Fused thalami, Polymicrogyria, Type II lissencephaly)

HPOsFreeText Crown-heel body length 49cm, body weight 3864g, brain weight 286g, abnormal larynx/trachea, hydrocephalus/open-book, occipitotemporal hypoplasia, infratentorial hypoplasia, occipitoschisis/keyhole, leptomeningeal heterotopia, neuroepithelial rosettes, absent developed and identifiable mamillary bodies, absent anterior commissure, difficult to identify nucleus dentatus, (Fig6:) markedly narrowed basis pontis with minute corticospinal tract and a larger tg above, dysplastic dentate nucleus with irregular neuronal strands and intermingled matrix-like small cells is seen in the cerebellar hilus

CaseNotHPOs HP:0001161, HP:0001829, HP:0001627, HP:0000157, HP:0007165

(Hand polydactyly, Foot polydactyly, Congenital heart defect, Tongue abnormality, Periventricular neuronal heterotopia)

NotHPOsFreeText Uterus duplex, hydranencephaly/anencephaly, true alobar or semilobar holoprosencephaly

CasePreviousTesting n/a

Variant NM_145014.2(HYLS1):c.632A>G (p.Asp211Gly), homozygous

ClinVar 1143

gnomAD MAF= 0.009594(European(Finnish)), no homozygotes

CaseAddInfo died 1 minute after birth, 40+6wk gestational age

CasePMIDs n/a

Case5: Female, 50 yrs report

CasePresentingHPOs: HP:0002355, HP:0100753, HP:0003089, HP:0003798, HP:0031237, HP:0031318 (Difficulty walking, Schizophrenia, Hamstring contracture, Nemaline bodies, Internally nucleated skeletal muscle fibers, Myofiber disarray)

HPOsFreeText: Jaw contractures, Pectoral contractures, Ankle dorsiflexor contractures, Quad contractures, Increased oxidative staining

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Daughter of Case 4, Sibling of Case 6

CasePreviousTesting: N/A

CaseGenotypingMethod: The TPM2 gene was sequenced and variants identified. No other information is provided.

Variant: NM_003289.3:c.14_16delAGA (p.Lys7del)

ClinVar: 140486 https://www.ncbi.nlm.nih.gov/clinvar/variation/140486/

gnomAD: Not in gnomAD

Case8: Female, 65 yrs report

CasePresentingHPOs: HP:0001263, HP:0030051, HP:0003326, HP:0003731, HP:0002540, HP:0003236, HP:0006466, HP:0005830, HP:0012785, HP:0009600, HP:0003798, HP:0031237, HP:0003555, HP:0001761 (Delayed milestones, Tip-toe gait, Myalgia, Quadriceps weakness, Non-ambulatory, Elevated serum creatine phosphokinase, Ankle contracture, Toe contractures, Flexion contracture of finger, Contracture of thumb, Nemaline bodies, Internally nucleated skeletal muscle fibers, Muscle fiber splitting, Pes cavus)

HPOsFreeText: Tendo-achilles release performed, NCPAP from 54 yrs, Jaw contractures, Nuclei clumps, Fiber type grouping, Required non-invasive nocturnal ventilatory support at age 54 years

CaseNotHPOs: HP:0001627 (Cardiac abnormality)

NotHPOsFreeText: N/A

CaseAddInfo: Parental testing not performed

CasePreviousTesting: ACTA1 was sequenced and returned negative.

CaseGenotypingMethod: The TPM2 gene was sequenced and variants identified. No other information is provided.

Variant: NM_003289.3:c.14_16delAGA (p.Lys7del)

ClinVar: 140486 https://www.ncbi.nlm.nih.gov/clinvar/variation/140486/

gnomAD: Not in gnomAD

Case1: Female, 45 yrs report

CasePresentingHPOs: HP:0008994, HP:0002015, HP:0003236, HP:0003273, HP:0031237, HP:0003555, HP:0100295, HP:0012548 (Proximal muscle weakness in lower limbs, Dysphagia, Elevated serum creatine phosphokinase, Hip contracture, Internally nucleated skeletal muscle fibers, Fiber splitting, Muscle fiber atrophy, Skeletal muscle fatty infiltration)

HPOsFreeText: N/A

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Proband of Family 1, Mother of Case 2, Variant is de novo

CasePreviousTesting: N/A

CaseGenotypingMethod: The TPM2 gene was sequenced and variants identified. No other information is provided.

Variant: NM_003289.3:c.14_16delAGA (p.Lys7del)

ClinVar: 140486 https://www.ncbi.nlm.nih.gov/clinvar/variation/140486/

gnomAD: Not in gnomAD

Case: Family 1 Moynihan Proband VII:7, female, Pakistani

CasePresentingHPOs: HP:0000407, HP:0004322, HP:0000135, HP:0100540, HP:0012385, HP:0002716, HP:0034920, HP:0034677, HP:0034391, HP:0030075, HP:0002862

CasePresentingHPOsFreeText: Eyelid swellings due to histiocytic deposits, diverse cytoplasmic inclusions in phagocytes and reticulum cells of bone marrow, Lymph node and eyelid show reactive features with small reactive lymphoid follicles and histiocytes within dilated sinuses resembling RDD. invasive ductal carcinoma grade 3 ER negative breast aged 43 years and carcinoma bladder (grade 3 papillary TCC) age 46 years

CaseNOTHPOs:

CaseNOTHPOSFreeText:

CasePreviousTesting: direct sequencing of PRF1 and UNC5B

CaseGenotypingMethod: autozygosity mapping, genotyping (genome-wide linkage scan was performed using Affymetrix 250k SNP arrays), direct sequencing, PCR

Variant: c.300+1G>A homozygous

ClinVarID: or CAID: 130339/CA155238

gnomAD: This splice site variant in intron 2 is present in gnomAD v4.0.0 at a MAF=0.0002197 (20/91048 alleles) in the South Asian population with 0 homozygotes.

AddCaseInfo: brother is affected as well

PreviouslyPublished: PMID: 9545394

VariantEvidence: n/a

Case: Family 2 Kismet Proband, Turkish, male

CasePresentingHPOs: HP:0000407, HP:0002240, HP:0004322, HP:0025289, HP:0033176, HP:0100727

CasePresentingHPOsFreeText: Rapidly growing orbital mass with SHML histopathology. Retropharyngeal lymphadenopathy. Filling of lymph node sinuses with histiocytes, plasma cells and lymphocytes. Histiocytes had a benign appearance, were CD-1a negative but positive for CD68 and S-100.

CaseNOTHPOs:

CaseNOTHPOSFreeText:

CasePreviousTesting: direct sequencing of PRF1 and UNC5B

CaseGenotypingMethod: genotyping (genome-wide linkage scan was performed using Affymetrix 250k SNP arrays), direct sequencing, PCR

Variant: p.Gly437Arg; p.Phe103X

ClinVarID: or CAID: 565; 30947

gnomAD: The missense variant G437R is present in gnomAD v4.0.0 with a MAF=0.00006666 (4/60002 alleles) in the Admixed American population and 0 homozygotes.

The nonsense variant, p.Phe103Ter found in exon 2/6, is present in gnomAD v4.0.0 with a MAF=0.0005201 (3/5768 alleles) in the Middle Eastern population and 0 homozygotes.

AddCaseInfo: 2 affected brothers

PreviouslyPublished: 16118898

VariantEvidence: Functional characterization of G437R in Xenopus oocytes showed that transport activity was approximately half of WT SLC29A3. Studies in NIH 3T3 fibroblasts demonstrated accelerated turnover of the G437R mutant compared to WT, suggesting decreased stability (Kang et al. 2010; PMID: 20595384)

CaseP4 male, 45yo report, Tunisia

CasePresentingHPOs HP:0012207, HP:0012865, HP:0012868, HP:0003251

(reduced sperm motility, sperm head anomaly, sperm tail anomaly, male infertility)

HPOsFreeText Patient's sperm volume: 2.2mL(lower reference limit: 1.5), sperm concentration: 56million/mL(reference: 15), total motility after 1hr: 0%(reference: 40), vitality: 79%(reference:53%), normal spermatozoa: 0%(reference: 4), abnormal acrosome: 78%, abnormal head morphology: 58%, abnormal base: 14%, absent flagella: 50%, short flagella: 32%, coiled flagella: 34%, flagella of irregular caliber: 50%, multiple anomalies index: 3.4%.

CaseNotHPOs n/a

CasePreviousTesting Previously screened all patients for DNAH1, CFAP43, and CFAP44 variants. Somatic karyotypes were normal.

Variant NM_144668.5:c.3007_3337del

ClinVar 586974

gnomAD not in gnomaD

CaseAddInfo consanguinity

CasePMIDs 29449551

CaseP3 male, 51yo report, Tunisia

CasePresentingHPOs HP:0012207, HP:0012865, HP:0012868, HP:0003251

(reduced sperm motility, sperm head anomaly, sperm tail anomaly, male infertility)

HPOsFreeText Patient's sperm volume: 1.7mL(lower reference limit: 1.5), sperm concentration: 56million/mL(reference: 15), total motility after 1hr: 5%(reference: 40), vitality: 73%(reference:53%), normal spermatozoa: 0%(reference: 4), abnormal acrosome: 56%, abnormal head morphology: 20%, abnormal base: 32%, absent flagella: 22%, short flagella: 59%, coiled flagella: 27%, flagella of irregular caliber: 37%, multiple anomalies index: 2.7%.

CaseNotHPOs n/a

CasePreviousTesting Previously screened all patients for DNAH1, CFAP43, and CFAP44 variants. Somatic karyotypes were normal.

Variant NM_144668.5:c.3007_3337del

ClinVar 586974

gnomAD not in gnomAD

CaseAddInfo consanguinity

CasePMIDs 29449551

CaseP2 male, 54yo report, Tunisia

CasePresentingHPOs HP:0012207, HP:0012865, HP:0012868, HP:0003251

(reduced sperm motility, sperm head anomaly, sperm tail anomaly, male infertility)

HPOsFreeText Patient's sperm volume: 1.8mL(lower reference limit: 1.5), sperm concentration: 6million/mL(reference: 15), total motility after 1hr: 1%(reference: 40), vitality: 80%(reference:53%), normal spermatozoa: 0%(reference: 4), abnormal acrosome: 56%, abnormal head morphology: 10%, abnormal base: 2%, absent flagella: 6%, short flagella: 32%, coiled flagella: 17%, flagella of irregular caliber: 68%, multiple anomalies index: 2.3%.

CaseNotHPOs n/a

CasePreviousTesting Previously screened all patients for DNAH1, CFAP43, and CFAP44 variants. Somatic karyotypes were normal.

Variant NM_144668.5:c.3007_3337del

ClinVar 586974

gnomAD n/a

CaseAddInfo consanguinity

CasePMIDs 29449551

Case#: II-9, male, Asian, 38 yr, proband

DiseaseAssertion: HCM, LQT1

FamilyInfo: HCM passed on in family (figure 1)

CasePresentingHPOs: HP:0001279, HP:0001712, HP:0001639 (Syncope, Left ventricular hypertrophy, Hypertrophic cardiomyopathy)

CaseHPOFreeText: physical examination; 12-lead ECG (SR, HR 57 bpm, P 80 ms, PR 164 ms, QRS 102 ms, QTc 423 ms); 2D echocardiographic study with S5-1 probe (MLVWT 14 mm, EF 62%, LVMI 94.27 g/m^2);

CaseNotHPOs: HP:0030148 (Heart murmurs)

CaseNotHPOFreeText: No inverted T wave: Normal QTc 440 ms

CasePreviousTesting: Additional Sanger sequencing for KCNQ1, MKLY2, TMEM70

CaseGenotypingMethod: Illumina-SolexaHiSeq 2000 sequencing from peripheral blood leococytes; bi-directional Sanger sequencing;

Variant: absent

ClinVar: absent

CAID: CA389036862 http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA389036862

gnomAD: absent

MultipleGeneVariants:

(1) KCNQ1 Variant:

Variant: NM_181798.1(KCNQ1):c.187C>T (p.Arg63Trp)

ClinVar: 53070 https://www.ncbi.nlm.nih.gov/clinvar/variation/53070/

CAID: n/a

gnomAD: absent

(2) MKLY2 Variant:

Variant: absent

ClinVar: absent

CAID: CA408526793 http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA408526793

gnomAD: African 0.00006453 https://gnomad.broadinstitute.org/variant/20-30412153-A-G

(3) TMEM70 Variant:

Variant: NM_017866.6(TMEM70):c.440T>C (p.Ile147Thr)

ClinVar: 571761 https://www.ncbi.nlm.nih.gov/clinvar/variation/571761/

CAID: n/a

gnomAD: East Asian 0.0004893 https://gnomad.broadinstitute.org/variant/8-74893513-T-C

Case#: Carelli proband, 20yo, Italian, male, onset in first year of life

DiseaseAssertion: Behr syndrome

FamilyInfo: Parents are unrelated. c.1705+1G>T was also identified in the affected mother and in nine maternal relatives, three of which (III-10, III-14 and III-18) are clinically unaffected (pedigree in Fig. 1A). The 43-year-old proband’s mother (Fig. 1A, III-4 in the pedigree) had isolated optic atrophy, most evident on the temporal side (Fig. 1C), which segregated as a dominant trait in her family (Fig. 1A). The other affected individuals in this family all suffered a non-syndromic optic atrophy, which started in childhood and progressively worsened, but remained as an isolated symptom. c.1146A>G (p.I382M) is present in the clinically unaffected father and sister. There were no MRI structural or metabolic abnormalities in the father (III-3) and the mother (III-4) of the proband, except for a few focal hyperintensities in the cerebral white matter (Fig. 1K–L). Neither signs of optic neuropathy nor abnormal nerve conduction velocities were recorded upon neuro-ophthalmological exam of the proband’s father and the elder sister (III-3 and IV-3).

CasePresentingHPOs: HP:0006934, HP:0000648, HP:0002066, HP:0002359, HP:0007663, HP:0002064, HP:0002403, HP:0001776, HP:0002395, HP:0003487, HP:0007078, HP:0012898, HP:0012897, HP:0003535, HP:0003387, HP:0001272, HP:0000543, HP:0010871, HP:0002313, HP:0001310, HP:0002075, HP:0000639

CaseHPOFreeText: Bilateral optic atrophy. Motor difficulties and visual acuity worsened during childhood. Nerve conduction studies showed delayed sensory-motor velocities with reduced amplitudes of motor potentials. Somatosensory evoked potentials could not be recorded and motor evoked potentials showed absent potentials at lower limbs and delayed latency at upper limbs. Brainstem evoked potentials were delayed bilaterally. Repeated urine organic acid gas chromatography/mass spectrometry analysis revealed mild elevation of 3-methylglutaconic acid (8–20 μmol/mmol creatinine). Sural nerve biopsy demonstrated a marked reduction of the large-diameter myelinated axons (Fig. 1E–G). Teased fibre analysis disclosed regions of demyelination confined to the paranodal region, clustered in some axons, suggesting secondary demyelination. Ultrastructural examination confirmed the reduction of myelinated axons (not shown). Elevated serum lactic acid after standardized exercise. mild cerebellar atrophy and pathological accumulation of lactate in the CSF of lateral ventriculi. OCT examination of retinal nerve fibre layer (RNFL) thickness shows a diffuse reduction of thickness compatible with the severe optic atrophy. Positive Holmes rebound phenomenon. Vision was light perception with severe nystagmus in any direction of gaze. Compared to controls, mitochondrial ATP synthesis driven by complex I substrates (pyruvate and malate) was incrementally reduced in the mother’s fibroblasts bearing the c.1705+1G>T (III:4) mutation and in the proband’s fibroblasts (IV:4) carrying both mutations.

CaseNotHPOs:

CaseNotHPOFreeText: cognitive delay

GenotypingMethod: Direct sequencing of the entire coding regions of the OPA1 gene and the flanking intronic boundaries. Sequence analysis of the OPA3 gene excluded the presence of pathogenic mutations.

PreviouslyPublished:

Variant: c.1705+1G>T; c.1146A>G (p.I382M)

CAID: CA10576007

SupplementalData:

CaseVII-3: Male, 4 years report, Turkey

CasePresentingHPOs: HP:0004912, HP:0002540, HP:0002970 (Hypophosphatemic rickets, Inability to walk, Genu varum)

HPOsFreeText: -3.44 standard deviation score

CaseNotHPOs: HP:0000083, HP:0002024, HP:0001947 (Renal failure, Malabsorption, Renal tubular acidosis)

NotHPOsFreeText: Other tubulopathies that cause secondary hypophosphatemia, Vitamin D-dependent rickets type 1 and 2

CaseAddInfo: Variant is asserted de novo and confirmed by parental testing, Proband is hemizygous for this mutation, Mutation is at splice donor site at Intron 2

CasePreviousTesting: Calcium levels (9.0 mg/dL), Decreased serum phosphate levels (3.1 mg/dL; reference 3.8-6.5), Elevated serum ALP (393 IU/L; reference 90-325), 25OHD3 levels (4.9 ng/ml), 1,25(OH)2D3 levels (N/A pg/mL), PTH levels (154.7 ng/L), TPR levels (58.7), Decreased TmP/GFR levels (1.82; reference 2.9-6.1)

CaseGenotypingMethod: Genomic DNA was isolated from peripheral blood leukocytes, amplified, and directly sequenced in all the coding exons and intron-exon boundaries of PHEX. If no mutations were found, FGF23 followed by consecutive analysis of DMP1, ENPP1, CLCN5, SLC34A3 and SLC34A1 was performed. Significance of found mutations was predicted by computational programs and sorted.

Variant: NM_000444.5(PHEX):c.187+1G>T

CinVar: 438537 https://www.ncbi.nlm.nih.gov/clinvar/variation/438537/

gnomAD: Not in gnomAD

CaseII:II-2: Female, 37 years, Turkey