CaseT23159: Female, 2 days onset

CasePresentingHPOs: HP:0200134, HP:0011167, HP:0010818, HP:0032667, HP:0031475, HP:0032816, HP:0010841, HP:0010845, HP:0006808, HP:0002079, HP:0012753, HP:0002059, HP:0007266, HP:0002273, HP:0011471, HP:0000121, HP:0000141, HP:0000696, HP:0001662, HP:0002045, HP:0000253, HP:0000341, HP:0001525, HP:0001007, HP:0002751, HP:0001371, HP:0001182 (Epileptic encephalopathy, Focal tonic seizure, Generalized tonic seizure, Myoclonic status epilepticus, Nonconvulsive status epilepticus, Neonatal multifocal myoclonic seizure, Multifocal epileptiform discharges, EEG with generalized slow activity, Cerebral hypomyelination, Hypoplasia of the corpus callosum, T2 hypointense basal ganglia, Cerebral atrophy, White matter dysmyelination/demyelination, Tetraparesis, Gastrostomy tube feeding in infancy, Nephrocalcinosis, Amenorrhea, Delayed eruption of permanent teeth, Bradycardia, Hypothermia, Progressive microcephaly, Narrow forehead, Severe failure to thrive, Hirsutism, Kyphoscoliosis, Flexion contracture, Tapered finger)

HPOsFreeText: Thinning of cortical gray matter

CaseNotHPOs: HP:0001272 (Cerebellar atrophy)

NotHPOsFreeText: Hypomyelination of cerebellum and brain stem

CaseAddInfo: Proband confirmed de novo heterozygous by parental sequencing

CasePreviousTesting: Currently on carbamazepime, lamotrigine and lacosamide; previously on pyridoxine, clonazepam, acetazolamide, valproate, phenobarbitone, phenytoin, prednisolone, vigabatrin, topiramate 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.254T>C (p.Leu85Pro)

ClinVar: 254266, https://www.ncbi.nlm.nih.gov/clinvar/variation/254266/

gnomAD: Not in gnomAD

Case43: 20 y (at biopsy), Lebanese

Case 43 (Level 2)

CasePreviousTesting: same as group. found 1.4 nmol/mg protein/18 h propionate incorporation, 1.1 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.1741C>T (p.Arg581Ter)

ClinVar: 554675

CAID: CA364395893

gnomAD: n/a

CaseAddInfo: mut0 type, homozygous mutations.

Case45: no age or ethnicity recorded

Case 45 (Level 2)

CasePreviousTesting: same as group. found 1.5 nmol/mg protein/18 h propionate incorporation, 1.6 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.1891delG (p.Ala631GlinfsTer17)

ClinVar: n/a

CAID: CA351288

gnomAD: 0.00002896 (Latino)

CaseAddInfo: mut0 type, homozygous mutations.

Case42: 1 y 7 mo (at biopsy), Hmong

Case 42 (Level 2)

CasePreviousTesting: same as group. found 4.5 nmol/mg protein/18 h propionate incorporation, 9.0 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.1663G>A (p.Ala555Thr)

ClinVar: 203850

CAID: CA312769

gnomAD: 0.0003262 (East Asian)

CaseAddInfo: mut- type, homozygous mutations. Additional heterozygous mutations in CUBN gene (Variant 1: NM_001081.3:c.3500G>T, CAID: CA376148913 , gnomAD: 0.00005014, East Asian ) (Variant 2: NM_001081.3:c.6211A>G, CAID: CA5423765 , gnomAD: 0.001003, East Asian )

Case41: 6 mo (at biopsy), Pakistani

Case 41 (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.1560G>C (p.Lys520Asn)

ClinVar: n/a

CAID: CA364397073

gnomAD: n/a

CaseAddInfo: mut0 type, homozygous mutations.

Case40: Middle Eastern

Case 40 (Level 2)

CasePreviousTesting: same as group. found 1.8 nmol/mg protein/18 h propionate incorporation, 1.6 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.1420C>T (p.Arg474Ter)

ClinVar: 554873

CAID: CA138795916

gnomAD: 0.00003267 (South Asian)

CaseAddInfo: mut0 type, homozygous mutations. documented consanguinity.

Case39: 1 y (at biopsy), Asian

Case 39 (Level 2)

CasePreviousTesting: same as group. found 1.4 nmol/mg protein/18 h propionate incorporation, 1.5 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)

CaseAddInfo: mut0 type, homozygous mutations. documented non-consanguinity.

Case38: 9 y (at biopsy), German/Polish

Case 38 (Level 2)

CasePreviousTesting: same as group. found 0.5 nmol/mg protein/18 h propionate incorporation, 0.5 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.1106G>A (p.Arg369His)

ClinVar: 203846

CAID: CA312764

gnomAD: 0.0001504 (East Asian)

CaseAddInfo: mut0 type, homozygous mutations.

Case37: 18 mo (at biopsy), Moroccan

Case 37 (Level 2)

CasePreviousTesting: same as group. found 0.5 nmol/mg protein/18 h propionate incorporation, 0.5 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.683G>A (p.Arg228Gln)

ClinVar: n/a

CAID: CA3847070

gnomAD: 0.00002905 (Latino)

CaseAddInfo: mut0 type, homozygous mutations.

Case36: 14 mo (at biopsy), Hispanic

Case 36 (Level 2)

CasePreviousTesting: same as group. found 0.7 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.406G>T (p.Val136Phe)

ClinVar: n/a

CAID: CA364404792

gnomAD: n/a

CaseAddInfo: mut0 type, homozygous mutations. documented consanguinity.

Case35: 5 y (at biopsy), Hispanic

Case 35 (Level 2)

CasePreviousTesting: same as group. found 0.5 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.322C>T (p.Arg108Cys)

ClinVar: 1887

CAID: CA249732

gnomAD: 0.0008393 (Latino)

CaseAddInfo: mut0 type, homozygous mutations.

Case33: 9 mo (at biopsy), Hispanic

Case 33 (Level 2)

CasePreviousTesting: same as group. found 1.0 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.322C>T (p.Arg108Cys)

ClinVar: 1887

CAID: CA249732

gnomAD: 0.0008393 (Latino)

CaseAddInfo: mut0 type, homozygous mutations.

Case32: 10 y (at biopsy), Arab

Case 32 (Level 2)

CasePreviousTesting: same as group. found 1.0 nmol/mg protein/18 h propionate incorporation, 1.0 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.278G>A (p.Arg93His)

ClinVar: 1880

CAID: CA249728

gnomAD: 0.0004777 (European- Finnish)

CaseAddInfo: mut0 type, homozygous mutations. documented consanguinity.

Case34: 3.5 wks (at biopsy), White/Hispanic

Case 34 (Level 2)

CasePreviousTesting: same as group. found 1.6 nmol/mg protein/18 h propionate incorporation, 1.8 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.322C>T (p.Arg108Cys)

ClinVar: 1887

CAID: CA249732

gnomAD: 0.0008393 (Latino)

CaseAddInfo: mut0 type, homozygous mutations.

Case31 sex unknown, Spain

CasePresentingHPOs HP:0001332, HP:0006846

(Dystonia, Acute encephalopathy)

HPOsFreeText Phenotypes were "mild," owing to static or improving dystonia, with maintained ability to walk without assistance. Patient may have gone undetected by routine screens as glutarate excretion was normal and 3-hydroxyglutarate was only slightly increased.

CaseNotHPOs HP:0002540

(Non-ambulatory)

NotHPOsFreeText n/a

CasePreviousTesting GC-MS measured glutarate of 12(nv 2–10 mmol/mol creatinine) and 3-OH-glutarate 175(nv 2–14 mm/mol creatinine). In patient fibroblasts, %GCD activity was 2.

CaseMethod1 SSCP

CaseMethod2 Sanger

CaseGenotypingMethod PCR amplified and SSCP screened 11 exons and intron boundaries of GCDH gene. Confirmed variants with Sanger sequencing and restriction digest. The 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

Case34 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. Patient may have gone undetected by routine screens as glutarate excretion was normal and 3-hydroxyglutarate was only slightly increased.

CaseNotHPOs HP:0002540

(Non-ambulatory)

NotHPOsFreeText n/a

CasePreviousTesting GC-MS measured glutarate of 7(nv 2–10 mmol/mol creatinine) and 3-OH-glutarate 99(nv 2–14 mm/mol creatinine). In patient fibroblasts, %GCD activity was 30. 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 Sibling (Case 35) presented with same enzyme activity, clinical onset and phenotype. Gluterate was 6mmol/mol creatinine and 3-OH-glutarate was 36mmol/mol creatinine.

CasePMIDs see note on previous studies

Case28 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 2(nv 2–10 mmol/mol creatinine) and 3-OH-glutarate 185(nv 2–14 mm/mol creatinine). In patient fibroblasts, %GCD activity was 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 V400M variant creates an NlaIII site. This would lead to cleaved patient DNA fragments and uncleaved control fragments.

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

ClinVar 2088

gnomAD MAF= 0.0002769(Other), no homozygotes

CaseAddInfo n/a

CasePMIDs see note on previous studies

Cas23 sex unknown, Spain

CasePresentingHPOs n/a

HPOsFreeText Clinical onset was "insidious," involving slowly progressive psychomotor delay, dystonic postures, lethargy, or macrocephaly. Patient is otherwise phenotypically asymptomatic. 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 7932(nv 2–10 mmol/mol creatinine) and 3-OH-glutarate 1074(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 R383C variant destroys an AciI restriction site. Thus, while the patient's DNA fragments would not cleave, control fragments would be cleaved. Likewise, the 387_388del variant destroys an AluI site. This would lead to uncleaved patient DNA fragments and cleaved control fragments.

Variant 1. NM_000159.3:c.387_388del(p.Glu129AspfsTer?), 2. NM_000159.3(GCDH):c.1147C>T (p.Arg383Cys), compound heterozygous

ClinVar 1. n/a, 2. 188852

CAID 1. register link

gnomAD 1. not in gnomAD, 2. MAF= 0.00008017, no homozygotes

CaseAddInfo n/a

CasePMIDs see note on previous studies

FamilyLabel Family 1

Family1PresentingHPOs HP:0003235

(Increased blood methionine)

HPOsFreeText increased pre-treatment blood homocysteine and post-treatment total homocysteine, affected individuals were treated with low-methionine diet, vitamin B6, folate, vitamin B12, and betaine after diagnosis

FamilyNotHPOs n/a

NotHPOsFreeText Marfan syndrome, vascular occlusion

CountryOrigin Korea

Ethnicity n/a

Race n/a

Family1PreviousTesting tested plasma homocysteine and methionine in both siblings, both metabolites were elevated

FamilyMethod1 PCR

FamilyMethod2 Sanger

FamilyGenotypingMethod PCR amplified all 16 coding exons of CBS gene, then direct sequenced. Deletion variant sequenced after cloning into pGEM T easy vector.

ProbandLabel Case 2

Variant 1. NM_001178008.1:c.700_702del(p.Asp234del), NM_000071.2(CBS):c.770C>T (p.Thr257Met), compound heterozygous

ClinVar 1. n/a, 2. 188927

CAID 1. register link

gnomAD 1. not in gnomAD, 2. MAF=0.0001396(Other), no homozygotes

FamilyAddInfo D234del inherited from father, T257M inherited from mother

FamilyPMIDs n/a

CaseD: Male, French, Quebec

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

HPOsFreeText: Elevated blood Sarcosine (229 μmol/l; reference 0-9), Elevated urine Sarcosine (1213 μmol/mmol; reference 0-36)

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: N/A

CasePreviousTesting: N/A

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

Variant: NM_007101.3:c.860C>T (p.Pro287Leu)

ClinVar: 39449 https://www.ncbi.nlm.nih.gov/clinvar/variation/39449/

gnomAD: European (Finnish) 1.692e-4, Overall 1.032e-4 http://gnomad.broadinstitute.org/variant/9-136594942-G-A

Case# Patient 1, Male, 40do diagnosis

Variant NM_000320.2:c.525A>G (p.G149R)

CAID CA438712209

Case# Patient 16, Female, 2mo diagnosis

Variant NM_000320.2:c.277G>A (p.Gly93Ser)

CAID CA356450231

CaseGSHSP44: Female, 44 yrs onset, Australia/UK

CasePresentingHPOs: HP:0040083, HP:0002317, HP:0002064, HP:0002460, HP:0002395, HP:0007350, HP:0003487, HP:0000012, HP:0012104, HP:0006913, HP:0001761, HP:0031520, HP:0006827 (Toe walking, Unsteady gait, Spastic gait, Distal muscle weakness, Lower limb hyperreflexia, Hyperreflexia in upper limbs, Babinski sign, Urinary urgency, Parietal cortical atrophy, Frontal cortical atrophy, Pes cavus, Groin pain, Atrophy of the spinal cord)

HPOsFreeText: 4/7 disability score, Hip replacement

CaseNotHPOs: HP:0001317, HP:0002464, HP:0100543, HP:0000973, HP:0006938 (Abnormal cerebellum morphology, Spastic dysarthria, Cognitive impairment, Cutis laxa, Impaired vibration sensation at ankles)

NotHPOsFreeText: N/A

CaseGenotypingMethod: Exome sequencing data of 160 index patients with autosomal dominant spastic paraplegia and 275 index patients with autosomal recessive or sporadic spastic paraplegia was examined. 95 patients were screened through panel sequencing. Any determined ALDH18A1 mutations were confirmed by Sanger sequencing.

CasePreviousTesting: N/A

CaseAddInfo: Case is sporadic

Variant reported as c.1949G>T which does not exist in current transcripts

Variant: NM_002860.3:c.1955C>T (p.Ser652Phe)

CAID: CA377700120 http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA377700120

gnomAD: Not in gnomAD

GroupPresentingHPOs: HP:0001987, HP:0001942, HP:0003128, HP:0002912 (hyperammonemia, metabolic acidosis, lactic acidosis, methylmalonic acidemia)

HPOsFreeText: elevated blood levels of propionylcarnitine (C3) (median 13.56 umol/L), and elevated C3 to acetylcarnitine (C2) ratio (median 0.85). elevated urine methylmalonate levels (median 598.96 mmol/mol creatinine). 28/73.7% displayed recurrent vomiting (HP:0002572). 28/73.7% displayed disturbances of consciousness (HP:0004372). 23/60.5% displayed feeding difficulties in infancy (HP:0008872), 20/52.6% displayed respiratory distress (HP:0002098), 20/52.6% displayed failure to thrive (HP:0001508), 20/52.6% displayed dystonia (HP:0001332), 14/36.8% displayed mental retardation, 12/31.6% displayed convulsions.

GroupNotHPOs: normal plasma total homocysteine

NotHPOsFreeText:

NumberMales: 27

NumberFemales: 16

CountryOrigin: China

Ethnicity: Chinese

AgeRange: 33d- 10.3 y at last follow up

TotalNumber 43

FamilyInfo: 43

NoFamily: 0

GroupAddInfo: 9 identified by NBS, 34 recruited after being suspected of inherited metabolism disorders. 42 families from 14 provinces of the Mainland of China. 8 died during acute metabolic crisis, 7 lost to follow up, 28 alive at end of study.

Group (Level 2)

VariantCount: 43 (35 had two mutant alleles)

NonVariant: 0

OtherVariant: 0

GroupPreviousTesting: tandem mass spectrometry for blood levels, gas chromatography-mass spectrometry for urine,

GroupMethod1: PCR

GroupGenotypingMethod: genomic DNA extracted from peripheral blood leukocytes from patients and their parents. Exonic and flanking intronic regions of the MUT gene were amplified through PCR. PCR products were sequenced directly using forward and reverse primers on ABI 3700 sequencer. Mutation analysis using MUT reference NM_000255.3. Nucleotide numbering reflects cDNA numbering (initiation codon= codon1). All detected mutations were confirmed in the patients' parents. Novel variants were screened in 100 alleles from 50 control children. Novel variants were considered pathogenic when predicted to be deleterious by two algorithms.

Case1: Male, Chinese, 11 mo (at end of study)

Case1HPOs: HP:0012736 (profound psychomotor retardation)

HPOsFreeText: 10 mo at onset, poor neurological outcome

NotHPOsFreeText: not responsive to vitamin B12

Case1 (Level 2)

CasePreviousTesting: same as group

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

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

ClinVar: 100707

CAID: CA235522

gnomAD: 0.00006152 (African)

CaseAddInfo: homozygous mutations

Case4: Male, Chinese, 2.8 y (end of study)

HPOsFreeText: poor neurologic outcome, neonatal onset

NotHPOsFreeText: not responsive to vitamin B12

Case4 (Level 2):

CasePreviousTesting: NBS

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

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

ClinVar: 552079

CAID: CA3847064

gnomAD: 0.0002008 (East Asian)

CaseAddInfo: homozygous mutations

Case5: Female, Chinese, 1.6 y (onset)

HPOsFreeText: 1.6 y onset

NotHPOsFreeText: Not responsive to vitamin B12

Case5 (Level 2):

CasePreviousTesting: same as group

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

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

ClinVar: 203846

CAID: CA312764

gnomAD: 0.0001504 (East Asian)

CaseAddInfo: homozygous mutations, lost to follow up

Case3: Female, Chinese, 4 mo (end of study)

HPOsFreeText: Responsive to vitamin B12

Case3 (Level 2):

CasePreviousTesting: NBS

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant: NM_000255.3:c.599T>C (p.Ille200Thr)

ClinVar: n/a

CAID: CA364404381

gnomAD: n/a

CaseAddInfo: homozygous mutations, asymptomatic

Case2: Male, Chinese, 3 mo (age at death)

HPOsFreeText: Neonatal onset

NotHPOsFreeText: Not responsive to vitamin B12

Case2 (Level 2):

CasePreviousTesting: NBS (newborn screening)

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant: NM_000255.3:c.482G>T (p.Gly161Val)

ClinVar: n/a

CAID: CA364404636

gnomAD: n/a

CaseAddInfo: homozygous mutations, novel mutation

Case3: Female, 5 yrs report

CasePresentingHPOs: HP:0012072 (Aciduria)

HPOsFreeText: N/A

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo:

CasePreviousTesting: Partial biotinidase deficiency (19%) was confirmed; Urine AAA (552 mmol/mol creatinine; ref <91) and KAA (251 mmol/mol creatinine) were clearly increased. At 5 years of age, urine AAA and KAA as well as plasma AAA were persistently elevated.

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

Variant1: 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

Variant2: NM_018706.7(DHTKD1):c.1309G>T (p.Glu437Ter)

ClinVar: 574714 https://www.ncbi.nlm.nih.gov/clinvar/variation/574714/

gnomAD: 0.0001316, https://gnomad.broadinstitute.org/variant/10-12136221-G-T

Variant1: NM_001902.5:c.200C>T (p.Thr67Ile)

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

VariantEvidence: Mutant proteins were expressed in the Escherichia coli by site-directed mutagenesis, with the codon change confirmed by DNA sequencing. Recombinant CTH was expressed and the proteins were purified and the levels detected. The activity of the T67I mutant was 0.6 μmol min−1 mg−1 protein, 3.5-fold lower than the wild-type, with H2S generation from homocysteine and cysteine showing similar decreases of 4-fold and 2-fold respectively. The Q240E mutant was significantly lower, with only 0.03 μmol min−1 mg−1 protein and no detectable H2S generation. See Purification and Kinetic Properties of the Patient Mutations T67I and Q240E for details.

Furthermore, the variant was tested for PLP responsiveness. While the wild-type alleles exhibited ~80% content, the T67I and Q240E variants only displayed 19% and 0.3-1% saturation respectively. See Figure 3 for details.

CaseF6P: 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, Predicted to undergo NMD

CasePreviousTesting: Blood phenylalanine before treatment was 1260 μmol/l, Blood tyrosine before treatment was N/A 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(PAH):c.526C>T (p.Arg176Ter)

ClinVar: 102723 https://www.ncbi.nlm.nih.gov/clinvar/variation/102723/

gnomAD: 0.00008012, http://gnomad.broadinstitute.org/variant/12-103249094-G-A

CaseAL1: Male, 14 yrs report

CasePresentingHPOs: HP:0012120 (Methylmalonic aciduria)

HPOsFreeText: Malonic acidemia/uria

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Born to non-consanguineous Ashkenazi Jewish parents; Proband is confirmed homozygous via parental testing

CasePreviousTesting: Newborn screening identified these probands, with subsequent urine organic acid analysis by gas chromatography mass spectroscopy confirming CMAMMA; Initial urine MMA level was 891 μmol/mmol creatinine; Confirmatory testing showed a urine MMA of 745 μmol/mmol creatinine (normal <25) and MA of 60 μmol/mmol creatinine (normal <6); Plasma MMA level was 18 μmol/l (normal <0.5); No other organic acid abnormality was detected. Carnitine levels, acylcarnitine profiles, plasma amino acids, ammonia, blood gas values, total homocysteine, folate and vitamin B12 levels were all within reference ranges, and the mothers were not vitamin B12 deficient; MCD activity in fibroblasts were within the reference range

CaseGenotypingMethod: Genomic DNA was isolated from fibroblasts and all exons and flanking introns of MLYCD, along with ACSF3, were subject to Sanger sequencing.

Variant: NM_001243279.3:c.1411C>T (p.Arg471Trp)

ClinVar: 31137, https://www.ncbi.nlm.nih.gov/clinvar/variation/31137

gnomAD: 0.007046, https://gnomad.broadinstitute.org/variant/16-89211719-C-T

Case14 29yo diagnosis, Australia, Anglo-Celtic

CasePresentingHPOs HP:0001083, HP:0002751, HP:0000939

(Lens dislocation, Kyphoscoliosis, Osteoporosis)

HPOsFreeText Diagnosis was based on dislocated lens and skeletal deformity. Patient has severe kyphoscoliosis. Treatment began with 100-200mg/day for two weeks followed by an addition of 5mg/day folic acid. This patient had a partial response to the treatment. Homocystine levels were sharply reduced on pyridoxine and folate treatment, but levels did not fall below 20μmol (nv <60μmol) on this treatment alone. (PMID:9156316:): In this study, patient was found to have mild intellectual disability and osteoporosis at 50yo.

CaseNotHPOs HP:0001249

(Intellectual disability)

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): Serum homocysteine was 100μmol/l and methionine was 18μmol/l after five years of treatment. Addition of betaine(3g twice per day) lowered homocysteine to 18μmol/l which remained <50μmol for years.

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. NM_000071.2(CBS):c.1058C>T (p.Thr353Met), comp het

ClinVar 1. 120, 2. 131

gnomAD 1. MAF=0.001432(European(non-Finnish)), no homozygotes, 2. MAF=0.0002916(African), no homozygotes

CaseAddInfo Patient carries a common 68bp insertion, 88ins68, variant that does not cause hyperhomocysteinemia. (PMID:9156316): Additionally is heterozygous for a c.699C>T(Y233Y) synonymous variant as well as homozygous c.1080T>C(A360A) synonymous variants.

CasePMIDs 9156316

Case24 6yo diagnosis, Anglo-Celtic

CasePresentingHPOs HP:0001083

(Lens dislocation)

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

CaseNotHPOs HP:0001249

(Intellectual disability)

NotHPOsFreeText n/a

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

CaseMethod1 PCR

CaseMethod2 Sanger

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

Variant 1. NM_000071.2(CBS):c.904G>A (p.Glu302Lys), 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. 496864, 2. 122; 3. 189088

gnomAD 1. MAF=0.00006153(African), no homozygotes 2. MAF=0.00009799(South Asian), no homozygotes, 3. MAF=0.00005341(African), no homozygotes

CaseAddInfo n/a

CasePMIDs n/a

CaseBII-2: N/A

CasePresentingHPOs: N/A

HPOsFreeText: Pretreatment phenylalanine level at 270 umol/L

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: N/A

Variant1: NM_000277.3(PAH):c.688G>A (p.Val230Ile)

ClinVar: 102784 https://www.ncbi.nlm.nih.gov/clinvar/variation/102784/

gnomAD: 0.0009025, https://gnomad.broadinstitute.org/variant/12-102855154-C-T?dataset=gnomad_r3

Variant2: NM_000277.3(PAH):c.1241A>G (p.Tyr414Cys)

ClinVar: 593 https://www.ncbi.nlm.nih.gov/clinvar/variation/593/

gnomAD: 0.0005264, https://gnomad.broadinstitute.org/variant/12-102840474-T-C?dataset=gnomad_r3

Case#: Patient 14, N/A (onset), China

CasePresentingHPOs:

HPOsFreeText: Classic 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.713delC (homozygous)

CAID: CA658820836

gnomAD: Not in gnomAD

VariantEvidence: Predicted LOF

CaseAddInfo: Parents tested.

Case#: Patient 14, 1 month (onset), China

CasePresentingHPOs: HP:0001328, HP:0008344

HPOsFreeText: Classic 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.372_377del (homozygous)

CAID: CA658820835

gnomAD: Not in gnomAD

VariantEvidence: In frame deletion

CaseAddInfo: Parents tested.

Case#: Case report, 8 year old (report), male

CaseHPOs: See free text

CaseHPOsFreeText: In the newborn period – hypotonia, torticollis, right inguinal hernia (surgically repaired). Concern about delays at 4 months of age. First evaluated at 19 months. No NAA or NAAG signal on neurospectroscopy (at 2 years 3 months, and 3 years 6 months). At 8 years, “profound neurological dysfunction”, can walk unaided for short distances, ataxic gait, no speech but can make sounds, behavior abnormalities, microcephaly, profound intellectual impairment (<12 month level), generalized seizures beginning at 5 year 9 months of age with 9 episodes of status epilepticus, tendency to self mutilation if left alone, drools.

CaseNotHPOs: No dysmorphic features. Normal vision and hearing. Normal brain anatomy on MRI.

CasePreviousTesting: MR spectra of urine, plasma, and CSF were unremarkable (3 years 10 months, 5 years 6 months). The latter samples were analyzed by capillary electrophoresis (CE) and revealed no detectable NAA in CSF (this is also seen in controls, and therefore normal). No detectable NAAG (usually in the uM range in controls). CE of urine – NAA 21.0 umol/mmol creatinine and NAAG 1.9 umol/mmol creatinine (within the normal range). Normal plasma amino acids, urine organic acids, oligosaccharides, mucopolysaccharides. No evidence for mitochondrial, organic acid, amino acid, or peroxisomal disorder. CSF normal for cell count, glucose, protein, biogenic amines, folates, and pterins (on two occasions).

CaseMethod1: PCR

CaseMethod2: Sequencing

CaseGenotypingMethod: The three exons of NAT8L were amplified and sequenced.

Variant: NM_178557.4(NAT8L):c.212_230del (p.Gly71fs)

ClinVar: 30849

gnomAD: Not in gnomAD

VariantEvidence: Expression of cDNA containing the variant in HEK-293 cells resulted in no enzyme activity and no cross-reactive material.

CaseAddInfo: “Born in an Eastern European country”, adopted by a family is Switzerland. Exposure to cigarettes and alcohol during the pregnancy.

CasePMIDs: 15328569, 16802720

Case#: 3 years (report), male, Pakistan

CasePresentingHPOs: HP:0008947, HP:0001270, HP:0003700, HP:0000486, HP:0008315, HP:0011936

HPOFreeText: Became increasingly floppy in 2nd year of life, able to walk with support at 3 years, normal MRI (2 years), urinary organic acid consistently showed 2-methylbutyrylblycinuria with excretion of trace amounts of isobutyrylglycine. NBS blood spot showed normal acylcarnitine profile.

CasePreviousTesting: Total RNA extracted from fibroblasts, cDNA synthesized, PCR amplification of entire coding region of SBCAD cDNA and ACAD-8 cDNA, bidirectional sequencing. Enzyme activity of ACAD toward 2-methylbutyryl-CoA in fibroblasts was 10% of controls (0.016 nmol/min/mg protein vs 0.157 nmol/min/mg protein).

CaseGenotypingMethod: PCR and sequencing of ACADSB cDNA, followed by genomic DNA.

Variant: NM_001609.3(ACADSB):c.1228G>A (p.Gly410Ser)

ClinVar: 9199

gnomAD: 0.00002949 overall (no homozygotes)

VariantEvidence: The 1228G>A variant is in the last nucleotide in the skippped exon 10. cDNA analysis from the patient showed no normal-sized ACADSB mRNA. Northern blot of total RNA from fibroblasts showed normal levels of mRNA.

CaseAddInfo: Patient also heterozygous for the SCAD A625 polymoprhism (Corydon, 1996). Healthy father and siblings were heterozygous for the deletion, while mother was also homozygous and found to excrete 2-methylbutyrylglycine in her urine.

CaseG1 Female, 17mo report, Turkish

CasePresentingHPOs HP:0003270, HP:0002013, HP:0000737, HP:0011169, HP:0010818, HP:0001336, HP:0011182

(Abdominal distension, Vomiting, Irritability, Clonic seizures, Generalized tonic seizures, Myoclonic jerks, Epileptiform EEG discharges)

HPOsFreeText born at 40wks gestation. (neonatal, pre-treatment:) abdominal distension, vomiting, irritability, 3wks onset of clonic and generalized tonic fits as well as myoclonic jerks, epileptiform discharges with normal background activity, resistance to ACD's. Pyridoxine trial introduced at 9wks, trial dose/route of 10mg/kg/d p.o., complete cessation of seizures, but experienced hypotonia/sleepiness, no apnoea or hypotension, initial maintenance daily dose=10mg/kg. Withdrew form trial at 7mo, seizures recurred after 17 days.

CaseNotHPOs HP:0001557, HP:0025116, HP:0001941, HP:0002098, HP:0001290, HP:0010851, HP:0002521, HP:0000486, HP:0000750

(Abnormal intrauterine movements, Fetal distress, Acidosis, Respiratory distress, Hypotonia, EEG with burst suppression, Hypsarrhythmia, Squint, Speech delay)

NotHPOsFreeText (neonatal, pre-treatment:) acidosis, respiratory distress, hypotonia, burst suppression on EEG, hypsarrythmia. (infancy/childhood:) recurrence of seizures with febrile illness, squint, motor problems, speech delay/difficulties, MRI changes

CasePreviousTesting Administration of pyridoxine relieved patient's seizures. CSF pipecolic acid: 0.6(nv<0.15μM), plasma pipecolic acid: 5.5, 4.9(nv<2.5μM), plasma AASA: 4.1(nv<0.2μM), urine AASA: 53(nv<1 mmol/mol creatinine).

Variant NM_001182.4(ALDH7A1):c.596C>T (p.Ala199Val), homozygous

ClinVar 17998

gnomAD not in gnomAD

CaseAddInfo 1st cousin parents

CasePMIDs n/a

CaseAC014_II-1: Female, 3 yrs report, Lebanon

CasePresentingHPOs: HP:0001992, HP:0025356 (Organic aciduria, Psychomotor retardation)

HPOsFreeText: N/A

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Parents are consanguineous, Proband is confirmed homozygous for this variant via parental testing, Siblings are healthy

CasePreviousTesting: Identified by the assessment of urinary organic acids by gas chromatography–mass spectrometry; Accumulation of N-acetylated amino acids was much less pronounced than in previously reported patients

CaseGenotypingMethod: Genomic DNA was extracted either from peripheral blood or EBV-transformed lymphocytes. PCR-amplification and bidirectional sequencing of all exons and exon-intron boundaries of ACY1 was performed.

Variant: NM_000666.3(ACY1):c.1132C>T (p.Arg378Trp)

ClinVar: 493363, https://www.ncbi.nlm.nih.gov/clinvar/variation/493363/

gnomAD: 0.00001499 in ExAC, https://gnomad.broadinstitute.org/variant/3-52022996-C-T?dataset=exac

Case# P1, Pakistan,

CasePresentingHPOs reduced DHPR activity

CaseMethod1 PCR

CaseGenotypingMethod PCR

Variant NM_000320.2:c.570del

CAID CA658795210

VariantEvidence null

Case7: Female, Japan, Asian

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

HPOsFreeText: Atrophic myofibers

CaseNotHPOs: HP:0001627 (Cardiac abnormality)

NotHPOsFreeText: N/A

CaseAddInfo: Severe congenital myopathy, Alive at 1 yr 7 months

CasePreviousTesting: N/A

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

Variant1: NM_198271.4:c.349C>T (p.Gln117*)

CAID: CA353477271 http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/allele?hgvs=NM_198271.4%3Ac.349C%3ET

gnomAD: Not in gnomAD

Variant2: NM_198271.4:c.1218delA (p.Lys406Asnfs*11)

CAID: CA909806380 http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/allele?hgvs=NM_198271.4%3Ac.1218delA

gnomAD: Not in gnomAD

Case11b: Male, Sweden

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.

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

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

gnomAD: Not in gnomAD

Case1b: Female, Algeria

CasePresentingHPOs: HP:0001290, HP:0003324, HP:0002093, HP:0011968, HP:0001283, HP:0001622 (Generalized hypotonia, Generalized muscle weakness, Respiratory insufficiency, Feeding difficulties, Bulbar weakness, Preterm delivery)

HPOsFreeText: N/A

CaseNotHPOs: HP:0001627 (Cardiac abnormality)

NotHPOsFreeText: N/A

CaseAddInfo: Severe congenital myopathy, Deceased in neonatal period, Parents are consanguineous

CasePreviousTesting: N/A

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

Variant: NM_198271.4(LMOD3):c.138dup (p.Ser47Glnfs)

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

Case#: Male, 3 days (onset)

CasePresentingHPOs: HP:0001250, HP:0002791, HP:0002240, HP:0001943, HP:0001942, HP:0002151, HP:0011968, HP:0003128, HP:0000648

HPOFreeText: 3 days: seizures, hypoventilation, circulatory failure, hepatomegaly, hypoglycemia, metabolic acidosis, elevated lactate. 2 months: poor feeding, somnolence, liver was normal. Developed lactic acidosis in hospital. Increased alanine, alpha-aminobutyrate, leucine, isoleucine, valine, glycine, threonine, methionine, mildy increased phenylalanine, cystathionine, cysteine, histidine. Urine analysis: increased lactic, pyruvic, and alpha-keto- and/or alpha-hydroxy-isovaleric, isocaproic, butyric, and glutaric acids. 15 months: head small (7th percentile), delayed development, progressive optic atrophy. Died at 5.5 years following acute illness.

CaseNotHPOs:

CasePreviousTesting: 3 days: metabolic acidosis (pH 7.05), and elevated lactate (12.8 mM, normal: 0.6–2.5). 2 months: lactic acidosis (10-16 mM). Increased alanine (1348 μM; normal: 132–455), α-aminobutyrate (106 μM; normal: 4–24), leucine (300 μM; normal: 61–183), isoleucine (120 μM; normal: 27–80), valine (345 μM; normal: 78–142), glycine (553 μM; normal 106–254), threonine (236 μM; normal: 65–147), methionine (151 μM; normal: 6–36).

CaseMethod1:

CaseMethod2:

CaseGenotypingMethod: E3 cDNA prepared by RT-PCR from fibroblasts of proband and parents. For proband's E3 cDNA anlaysis, 1.5kb fragment was isolated from agarose gel and transformed into E. coli, followed by sequencing of the entire E3 cDNA. Direct sequencing was performed with genomic DNA to confirm variants in proband and parents.

Variant: 1. NM_000108.4(DLD):c.1123G>A (p.Glu375Lys); 2. NM_000108.4:c.405_407del (p.Gly136del)

ClinVar: 1. 11971

CAID: 2. CA891842420

gnomAD: 1. MAF (Ashkenazi Jewish): 0.002219 (23/10366); 2. Not in gnomAD

VariantEvidence: In vitro and in vivo functional studies for the missense variant show reduced enzyme activity compared to wild type (Ambrus et al., 2011; Vaubel et al., 2011).

CaseAddInfo: E3 activity in blood lymphocytes and fibroblasts were 9% ad 3% respectively; PDC activity was 13% of controls; KGDC activity was 6% of controls. Protein levels were not changed.

CaseWG2098: 3 yrs onset

CasePresentingHPOs: HP:0001251, HP:0004302, HP:0001260, HP:0002313, HP:0012120 (Ataxia, Functional motor deficit, Dysarthria, Spastic paraparesis, Methylmalonic aciduria)

HPOsFreeText: N/A

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Proband has normal 14C-propionate incorporation, suggesting normal MCEE protein activity level; The high gnomAD frequency and homozygotes suggests this is likely a benign polymorphism

CasePreviousTesting: Normal MRI; Propionate Incorporation w/o hydroxycobalamin at 7.0 nmols/mg protein/18hr; Propionate Incorporation with hydroxycobalamin at 8.2 nmols/mg protein/18hr; Adenosylcobalamin synthesis percentage at 12.7%; Elevated methylmalonic values of 621 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.178A>C (p.Lys60Gln)

ClinVar: 203812, https://www.ncbi.nlm.nih.gov/clinvar/variation/203812/

gnomAD: 0.01463 (10 homozygotes), https://gnomad.broadinstitute.org/variant/2-71351536-T-G

Case21 Female, 8.8yo report, Ethnicity: Italian

CasePresentingHPOs HP:0003571, HP:0012523, HP:0002013, HP:0008947, HP:0001259, HP:0002789

(propionicacidemia, oral aversion, vomiting, infantile hypotonia, coma, seizures, tachypnea)

HPOsFreeText feeding refusal, vomiting, hypotonia, coma, seizures, tachypnea, exanthema (14do). 55 IQ on Snijders-Omen Test.

CaseNotHPOs n/a

CasePreviousTesting Diagnosed by selective metabolic screen. PCC activity in patient fibroblasts was 1.1% of control.

Variant NM_000532.4(PCCB):c.562G>A (p.Gly188Arg), homozygous

ClinVar 558327

gnomAD MAF= 0.00006152(African), no homozygotes

CaseAddInfo no parental consanguinity

CasePMIDs n/a

FamilyLabel Basinger_Family1

Family1PresentingHPOs HP:0003150, HP:0011471

(Glutaricaciduria, Gastronomy tube feeding in infancy)

HPOsFreeText Urine glutaric acid was highly elevated and 3-hydroxyglutaric acid was elevated. Feedings by both mouth and gastronomy tube were noted. Patients were diet restricted in lysine and tryptophan with per individual adjustments to maintain normal plasma amino acid levels. Treatment also included supplemental carnitine and riboflavin.

FamilyNotHPOs n/a

NotHPOsFreeText n/a

CountryOrigin USA

Ethnicity Lumbee

Race n/a

Family1PreviousTesting Affected individuals were diagnosed with newborn screen.

FamilyMethod1 PCR

FamilyMethod2 Sanger

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

ProbandLabel Case 1

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

ClinVar 167133

gnomAD MAF=0.0002017(African), no homozygotes

FamilyAddInfo nonconsanguineous

FamilyPMIDs n/a

Variant NM_000709.3:c.1267C>T(p.Gln423Ter)

CAID CA406015497

CaseDOIII-3: Female, 12 yrs report, White

CasePresentingHPOs: HP:0001531, HP:0002020, HP:0001942, HP:0001944, HP:0002789, HP:0002013, HP:0002014, HP:0002919, HP:0012120 (Failure to thrive in infancy, Gastroesophageal reflux, Metabolic acidosis, Dehydration, Tachypnea, Vomiting, Diarrhea, Ketonuria, Methylmalonic aciduria)

HPOsFreeText: Elevated methylcitric acid

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Parents are first-cousin consanguineous, Son is healthy but second daughter shares phenotypes with the proband and shows clear methylmalonic aciduria, Proband and her sister are confirmed homozygous for this variant via parental testing

CasePreviousTesting: Serum methylmalonic acid was 1314 ng/ml (normal < 32); Vitamin B12 levels were normal (927 pg/ml, normal 200–1100); Vitamin B12 injections improved patient condition; At 12.5 yrs carnitine, plasma AAs, blood counts, and basic metabolic panels are normal but methylmalonic acid remains high at 180–1456 mmol/mol creatinine; Fibroblasts showed normal AdoCbl synthesis, contrary to expected from MMAA or MMAB variants; MUT and MMAB were sequenced with no variation found

CaseGenotypingMethod: Genomic DNA was isolated and PCR-amplified before directly sequencing all exons and flanking intronic sequences of the MCEE gene. Restriction enzyme digest confirmed the phase of the variant in the proband and the parents.

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

Case: Patient 2/ BIODEF 319, female, Caucasian, 10 days

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

HPOtext: Conceived by IVF. HyperPhe detected by neonatal screen, 240umol/L. Plasma Phe levels at 10 days olf was 585 umol.L. BH4 loading test normalized Phe levels. Urine levels showed high neopterin and low biopterin. PCD levels in patient's blood sample were normal. Urine reevalutation showed increased primapterin, again indicating PCD deficiency. cofactor replacement therapy was started, which improved growth trajectories on a normal diet. At 2 years old is still on diet and developing normally.

CaseNOTHPOs:

CasePreviousTesting: n/a

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

Variant 1: NM_000281.3(PCBD1):c.289G>A (p.Glu97Lys)

ClinVar ID 1: 91907

Variant 2: NM_000281.3(PCBD1):c.292C>T (p.Gln98Ter)

ClinVar ID 2: 16799

compound heterozygous

gnomAD variant 1: MAF (Euro-NF)= 2.320E-5 in hets only, no homozygous. http://gnomad.broadinstitute.org/variant/10-72643733-C-T

gnomAD variant 2 n/a

AddCaseInfo: Father carries E97K variant, mother carrier of the Q98X variant, neither are symptomatic.

VariantEvidence: Expression of recombinant protein in bacteria revealed that both the E97K and Q98X variants are present in the insoluble fraction compared to wildtype which is found in the soluble. the little amount retained of each in the soluble fraction showed that they retained activity.

Patient12: 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_001159508.1:c.870_871insG

CAID: CA913184835, http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA913184835

gnomAD: n/a

CaseAHN15.1: Australia, White

CasePresentingHPOs: HP:0008353 (Neutral hyperaminoaciduria)

HPOsFreeText: N/A

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Variants segregate with the disease

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

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

Variant1: NM_001003841.3(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.850G>A (p.Gly284Arg)

CAID: CA3182918, http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA3182918

gnomAD: 0.00005440, https://gnomad.broadinstitute.org/variant/5-1214143-G-A

Thinking this needs to be written differently to get into Allele registry, don't know where to start.

"position is located before the beginning of the transcript" error in Allele registry

Is this a variant that only ClinVar would be able to register at this time?

FamilyPA1: United Kingdom

FamilyPresentingHPOs: HP:0000518, HP:0002493, HP:0004322, HP:0001263, HP:0011842, HP:0002013 (Cataract, Upper motor neuron dysfunction, Short stature, Global developmental delay, Abnormality of skeletal morphology, Vomiting)

HPOsFreeText: N/A

FamilyNotHPOs: N/A

NotHPOsFreeText: N/A

FamilyGenotypingMethod: ALDH18A1 was directly sequenced upon investigation for a causal role in SPG9, due to being within the linked region 10q23.3-q24.2, not having been discarded as a candidate gene, and mutations having been known to cause cutis laxa. This revealed one mutation in each pedigree that strictly co-segregated with the disease. Exome NGS sequencing within the interval showed only two variants were unknown in ExAC with the MYOF substitution unlikely to be disease causing due to poor conservation.

FamilyPreviousTesting: N/A

FamilyAddInfo: Family shows autosomal dominant segregation

FamilyPMIDs: 8779323

Variant: NM_002860.4(ALDH18A1):c.755G>A (p.Arg252Gln)

ClinVar: 217117 https://www.ncbi.nlm.nih.gov/clinvar/variation/217117/

gnomAD: Not in gnomAD

Affected: 7

Unaffected: 3

Segregations: 6

AddSegregationInfo: Candidate Gene Sequencing, Pedigree in Family 1

Case# Pat4, F, Chinese, 5day onset

CasePresentingHPOs HP:0008344, HP:0002013, HP:0011968, HP:0001250, HP:0001276, HP:0001279

CasePreviousTesting Tested 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.491T>C

CAID CA406004676(c.178), CA406010453(c.491)

CaseAddInfo mutations confirmed in parents by Sanger sequencing, Her elder brother was healthy. This patient presented with lethargy and feeding difficulty when she was 5 days old. She was admitted because of unconsciousness at the age of 5 months, and she was diagnosed with MSUD accompanied by metabolic acidosis, liver damage and hypermyotonia. Cerebral MRI showed abnormal signals in the left and right globus pallidus. Clinical improvement as well as improvement in the laboratory test results was observed after treatment. She is now 3 years and 11 months old with severe psychomotor retardation. She is still unable to walk or speak.

Case#: 3, female, 12 days (onset), 4 y (current- this differs than what is in table 1), Chinese

CasePresentingHPOs: HP: 0008344, HP: 0012088, HP:0011968, HP: 0002013, HP:0011344, HP: 0002543, HP: 0012751 (elevated A keto acids in urine, abnormal urinary odor, feeding difficulties, vomiting, severe global developmental delay, abnormal globus pallidus, abnormal basal ganglia)

HPOsFreeText: Bilateral damage to capsula interna and externa, Blood ammonia: 205 uM, Hemoglobin: 118 g/L, Leucin: 891.90 uM, Valine: 502.50 uM, Isoleucine: 165.99 uM, Alloisoleucine: 90.12 uM

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: 2 different variants, unsure of cis or trans

CasePreviousTesting: n/a

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant: NM_000709.3(BCKDHA):c.178G>T (p.Glu60Ter), NM_000709.3(BCKDHA):c.1214_1219dupCCAACC (p.Asn406Leu407insProGln)

ClinVar: n/a

CAID: CA406004676 , CA658820662

gnomAD: neither in gnomAD

GroupPresentingHPOs: HP:0008344 (elevated A keto acids in urine)

HPOsFreeText:

GroupNotHPOs:

NotHPOsFreeText:

NumberMales: 4

NumberFemales: 4

CountryOrigin: mainland China

Ethnicity:

Race: Chinese

AgeRange: 2 y 3 mo- 4 y 3 mo (present age)

TotalNumber: 8

VariantCount: 8 (subunits of BKDC: either BCKDHA, BCKDHB, or DBT)

NonVariant: 0

OtherVariant: 0

GroupPreviousTesting: n/a

GroupMethod1: PCR

GroupMethod2: Multiple sequence alignments

GroupGenotypingMethod: genomic DNA extracted from peripheral blood lymphocytes of patients and parents using TIANamp Blood DNA Kit. Exons and flanking intronic regions of BCKDHA, BCKDHB, and DBT were amplified with PCR and sequenced. Results compared with reference sequences in UCSC genome (BCKDHA (NM_000709), BCKDHB (NM_183050), DLD (NM_000108), DBT (NM_001918)), and compared with integrated set of variants from 1000 Genome Project to identify mutations. PolyPhen-2 and Mutationtaster program predicted the impact of missense alterations on protein function. Multiple sequence alignments obtained using BLAST.

Case#: 6, male, 18 m (onset), 2 y 6 m (current), Chinese

CasePresentingHPOs: HP: 0008344, HP; 0012088, HP: 0011342, HP: 0001332, HP:0002543, HP: 0012751, HP:0012747 (elevated A keto acids in urine, abnormal urinary odor, mild global developmental delay, dystonia, abnormal globus pallidus, abnormal basal ganglia, abnormal brainstem MRI signal intensity)

HPOsFreeText: damage to cerebellar dentate nucleus, damage to corpora quadrigemina, damage to frontal lobe, damage to insular cortex, Isoleucine: 167.13 uM, Alloisoleucine: 44.88 uM

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: 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.767A>G (p.Tyr256Cys), NM_183050.3:c.768C>G (p.Tyr256Ter)

ClinVar: neither found

CAID: CA364659935, CA142287564

gnomAD: 0.000008820 (European, non-Finnish), not found

Case#: 8, male, 13 days (onset), 1 y 6 m (current), Chinese

CasePresentingHPOs: HP: 0008344, HP:0012088, HP:0011968, HP:0001254, HP:0002415, HP: 0006989 (elevated A keto acids in urine, abnormal urinary odor, feeding difficulties, lethargy, leukodystrophy, dysplastic corpus callosum)

FreeTextHPOs: blood ammonia: 96 uM, hemoglobin: 98 g/L, leucin: 1047.24 uM, isoleucine: 261.09 uM, alloisoleucine: 127.05 uM

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: homozygous mutation

CasePreviousTesting: same as group

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant: NM_001918.3:c.1A>G (p.Met1Val)

ClinVar: not found

CAID: CA969877

gnomAD: not found

Case9 sex unknown, Netherlands, Dutch

CasePresentingHPOs HP:0001250, HP:0001263, HP:0000750

(Seizures, Psychomotor delay, Speech delay)

HPOsFreeText Seizures ceased upon administration of pyridoxine. (PMID:16159904:) 1st seizure at 3do, first administered pyridoxine(and age at trial) 8mo, initial dose:50mg, maintenance dose:50mg/day, psychomotor delay, poor language 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 9.6(nv <1mmol/mol cr), α‐AASA plasma 0.8(nv <0.2μmol/l), pipecolic acid urine 2.0(nv 0.55-24.1mmol/mol cr <6mo, 0.01–1.54mmol/mol cr >6 mo) pipecolic plasma 22(nv 3.75-10.8μmol/l <1wk, 0.7-2.46μmol/l >1wk)

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

ClinVar 17994

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

CaseAddInfo n/a

CasePMIDs 17088338 , 16159904

CaseCA3: Male, 6 months diagnosis

CasePresentingHPOs: HP:0001644, HP:0001875, HP:0001508, HP:0003535, HP:0003756, HP:0030319, HP:0003551 (Dilated cardiomyopathy, Neutropenia, Failure to thrive, 3-Methylglutaconic aciduria, Skeletal myopathy, Facial weakness, Difficulty climbing stairs)

HPOsFreeText: N/A

CaseNotHPOs: HP:0003107 (Abnormal circulating cholesterol concentration)

NotHPOsFreeText: N/A

CaseAddInfo: Cardiac transplant 11 months after presentation; Variant also found in mother and grandmother

CasePreviousTesting: N/A

CaseGenotypingMethod: The TAZ gene was PCR amplified in three fragments and directly sequenced in the mother and proband.

Variant: NM_000116.5(TAZ):c.589G>A (p.Gly197Arg)

ClinVar: 11105, https://www.ncbi.nlm.nih.gov/clinvar/variation/11105/

gnomAD: Not in gnomAD

Case#: Hou 2016 Proband, M, 7 days (onset), Taiwan (Amis Tribe)

CasePresentingHPOs: HP:0004396, HP:0030842, HP:0040181, HP:0002045 (continue)

HPOsFreeText:

CaseNotHPOs: .

CasePreviousTesting: Hemoglobin 1.36 mM (normal 1.4-2.17 mM); 613x10^9 platelets/L (normal 150-400x10^9); glucose 2.16 mM (normal 3.3-5.5 mM). Valine 1026 uM (normal 84.07-354 uM); leucine/isoleucine 829.41 uM (normal 56.82-287 uM).

CaseGenotypingMethod: Direct sequencing and duplex PCR of DBT.

Variant: 1. NM_001918.3(DBT):c.1282-4142_*(434_435)del; . NM_001918.3:c.650dup

ClinVar: 1. 11951

CAID: 2. CA658820832

gnomAD: 1. Not in gnomAD; Not in gnomAD

VariantEvidence: The deletion includes the 3' half of intron 10, the entire coding region of the terminal exon 11, and the 5' part of the 3' UTR of DBT.

CaseAddInfo: The 4.7 kb deletion had been previously reported in Paiwan tribe in Taiwan.

Patient5: Male, 11mo, sibling to patient 4

CasePresentingHPOs:

HPOsFreeText: Asymptomatic, C5 (DBS) = 6.0 μmol l−1, IVA incorp (%) = 64, IVG in urine mmol permol creatinine = 955, Ammonium (μmol l−1) = 65, pH = 7.41, Protein intake (g kg−1 per day) = 1.5, treated with carnitine 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 with NBS at 10 days old

CasePMIDs:

CasePreviousTesting: same as group

CaseGenotypingMethod: same as group

Variant1: NM_001159508.1:c.1214T>C

CAID: CA268781818, http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA268781818

gnomAD: 0.000003977, http://gnomad.broadinstitute.org/variant/15-40710485-T-C

Variant2: NM_001159508.1:c.500T>C

CAID: CA391717541, http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA391717541

gnomAD: n/a

CaseGuA: Male, 6 weeks onset

CasePresentingHPOs: HP:0200134, HP:0020217, HP:0002069, HP:0010852, HP:0012012, HP:0012017, HP:0012018, HP:0010841, HP:0025152, HP:0030211, HP:0008936, HP:0002061, HP:0006986, HP:0002187, HP:0011344, HP:0001344, HP:0012448, HP:0002283, HP:0002079, HP:0012696, HP:0002500, HP:0012751 (Epileptic encephalopathy, Focal aware motor seizure, Bilateral tonic-clonic seizure, EEG with parietal focal spike waves, EEG with parietal focal spikes, EEG with temporal focal spikes, Multifocal epileptiform discharges, Poor visual behavior for age, Slow pupillary light response, Muscular hypotonia of the trunk, Lower limb spasticity, Upper limb spasticity, Profound intellectual disability, Severe global developmental delay, Absent speech, Delayed myelination, Global brain atrophy, Thin corpus callosum, Abnormal thalamic MRI signal intensity, White matter abnormalities, Abnormal basal ganglia MRI signal intensity)

HPOsFreeText: Dyskinetic movements of arms, In ability to sit unsupported, Abnormal MRI signal in putamen and caudate

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; Capture of right arm jerks and chin twitching showed no EEG correlate; Head circumference was 45.1 cm at 8 months (50%), 52 cm at 5 years (50th percentile); Partial response to phenobarbital, levetiracetam, and clobazam; Longest seizure-free period was 16 months

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.244G>A (p.Gly82Arg)

ClinVar: 438736, https://www.ncbi.nlm.nih.gov/clinvar/variation/438736/

gnomAD: Not in gnomAD

Case5 age/gender unknown, Caucasian, Italy

CasePresentingHPOs HP:0001250

(Seizures)

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

CaseNotHPOs n/a

CasePreviousTesting post treatment testing: Pipecolic acid in plasma 7.8(nv 0.4-4.9µmol/liter), AASA in plasma 5.8(nv<0.2µmol/liter), urine AASA 28(nv<1mmol/molcreatinine). Normal urine PA after pyridoxine treatment.

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

ClinVar 17994

gnomAD not in gnomAD

CaseAddInfo n/a

CasePMIDs n/a

CaseSA1: Male, Serbia

CasePresentingHPOs: HP:0025190, HP:0001987, HP:0000825, HP:0001988, HP:0001252, HP:0025502, HP:0001263 (Generalized tonic-clonic seizure, Hyperammonemia, Hyperinsulinemic hypoglycemia, Recurrent hypoglycemia, Muscular hypotonia, Overweight, Global developmental delay)

HPOsFreeText: Born at term with a birth weight of 3850 g, birth length of 51 cm, and an Apgar score of 10

CaseNotHPOs: HP:0002353 (EEG abnormality)

NotHPOsFreeText: N/A

CaseAddInfo: Parents are healthy and non-consanguineous with no family history,

CasePreviousTesting: At 9 weeks blood glucose at 2.0 mmol/L, serum insulin level of 32.4 mIU/L, plasma ammonia level was of 277 μmol/L; No abnormalities in plasma amino acid levels nor in urinary organic acid concentrations

CaseGenotypingMethod: Genetic analysis was perfomed on the GLUD1 gene.

Variant: NM_005271.5(GLUD1):c.1493C>T (p.Ser498Leu)

ClinVar: 16122, https://www.ncbi.nlm.nih.gov/clinvar/variation/16122/

gnomAD: Not in gnomAD

Case#: P15, F, 2 months (onset)

CasePresentingHPOs: HP:0001263, HP:0200134

CasePresentingHPOs (free text): TMS/GC-MS: Leu+Ile- 517, Val- 315. Diagnosed with intermediate form of MSUD.

CasePreviousTesting: Biochemical testing (elevated BCAAs) used to make diagnosis.

CaseMethod1: PCR

CaseMethod2: Sequencing

CaseGenotypingMethod: Genomic DNA extracted, coding regions (and flanking exon-intron boundaries) of BCKDHA, BCKDHB, and DBT were PCR-amplified, followed by bidirectional sequencing.

Variant: NM_001918.3(DBT):c.939G>C (p.Lys313Asn) (homozygous)

ClinVar: 94017

gnomAD: MAF (South Asian) 9.747e-5

VariantEvidence: None

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

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

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

CaseMethod1 Sanger

CaseGenotypingMethod Sanger of CA5A exons

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

ClinVar 127089

CaseAddInfo consanguineous, heterozygous parents checked with qtPCR

NonPatientCell: EFO:0001182 (HEK293 Cells)

GeneFunction: GO:0008233 (Peptidase activity)

GeneAlteration: HEK293 cells were grown and 8 missense mutations were identified and generated by PCR-mutagenesis. The human mutant cDNAs were transfected into the cells using the calcuim-phosphate coprecipitation method and examined.

FunctionalEvidence: When HEK293 cells were transfected with wild-type and mutant human cDNA for PHEX, the resulting cells were subject to immunoblotting to determine the protein levels in both the cell lysate and the media of cells. All the variants and the wild-type showed presence in the lysate at various levels, but C85R, G579R, G579V, S711R, and A720T were all devoid of any secreted protein in the media. Ananlysis of these cells also showed possible rescue by growing transfected cells at 26 C, thus glycosylating and maturing the proteins. The endopeptidase activity of the wild-type and variants were also compared, with D237G and Y317F mutant proteins exhibiting 50–60% of wild-type activity; E581V and S711R also displayed nearly undetectable activity. This displayed that the disease causing mechanism could be related to loss of function of peptidase activity or cellular trafficking. Evidence is found under "Results".

FAVariant1: NM_000444.5:c.253T>C (p.Cys85Arg)

ClinVar: 438549 https://www.ncbi.nlm.nih.gov/clinvar/variation/438549/

FAVariant2: NM_000444.5:c.1735G>A (p.Gly579Arg)

ClinVar: 226119 https://www.ncbi.nlm.nih.gov/clinvar/variation/226119/

FAVariant3: NM_000444.5:c.1736G>T (p.Gly579Val)

ClinVar: 372777 https://www.ncbi.nlm.nih.gov/clinvar/variation/372777/

FAVariant4: NM_000444.5:c.2131A>C (p.Ser711Arg)

CAID: CA412575294 https://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA412575294

FAVariant5: NM_000444.5:c.2158G>A (p.Ala720Thr)

CAID: CA412575364 https://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA412575364

This variant was mentioned as being previously published (PMID: 12559846). Structural changes for this variant were analyzed by calculating RMSD and predicted this variant to result in a severe phenotype. However, this type of prediction is not used by the LD VCEP.

PMID: 18241071

Gene: PAX6

Variant NM_001368894.2:c.-128-2del

Disease: Ocular dysgenesis

InheritancePattern: AD

Disease PAX6-related ocular dysgenesis MONDO:0800183

Case 1: HP:0001639 KLF10 no family history NM_005655.3:c.34G>A (p.Ala 12Thr) gnomAD= 4.108e-6

Case 2: HP:0001639, parents and siblings had neg echos, no family history, HCM was NOT late onset (diagnosed at 15), myectomy performed, NM_005655.3:c.80T>C (p. Met 27Thr), gnomAD=7.91e-5, euro(Nf)= 1.344e-4

Case 6: HP:0001639, HP:0001635, HP:0006510, died at 76 of heart failure and COPD, NM_005655.3:c.674G>C (p. Ser225Asp), gnomAD= 1.118e-3 Euro (NF)= (1.16e-2)

PMID: 28851564

Gene: RAB39B

Disease: early-onset parkinsonism-intellectual disability syndrome

MonDO: MONDO:0010709

Unable to annotate on article.

Case#: Konstantinidis Case 2

DiseaseAssertion: optic atrophy

FamilyInfo: none provided

CasePresentingHPOs: none, tests performed on blastocyst

CaseHPOFreeText:

CaseNotHPOs: none

CaseNotHPOFreeText:

PreviouslyPublished: n/a

Variant: c.182A > G

ClinVar: 511815

CAID: CA2758946

SupplementalData: n/a

Case#: Hudson Proband II:5, 66yo, female, UK

DiseaseAssertion: OA plus

FamilyInfo: Pedigree (Fig 1) shows 6 segregations

CasePresentingHPOs: HP:0007663, HP:0001288, HP:0001730, HP:0000746, HP:0002367, HP:0000648, HP:0000544, HP:0001488, HP:0001349, HP:0002066, HP:0001315, HP:0003236, HP:0033568, HP:0001272, HP:0002120

CaseHPOFreeText: slowly progressive deterioration in her visual acuity, a progressive gait disturbance of 20 years duration, and a 5-year history of deteriorating hearing, acute paranoid delusions, low mood, just able to perceive light, bilateral optic atrophy, plantar responses flexor, elevated serum creatine kinase (417 U/l, normal <190 U/l) with a normal fasting blood glucose and lactate, mild cortical disturbance, prominent small-vessel ischaemic lesions in the cerebral white matter bilaterally. Muscle biopsy of the index case (II:5) revealed features that were diagnostic of mitochondrial myopathy

CaseNotHPOs: HP:0001627, HP:0031983

CaseNotHPOFreeText:

PreviouslyPublished: n/a

PreviousTesting: The entire mitochondrial genome-coding region was amplified and found (m.8839G>A) in the MTATP6 gene. No sequence variants were identified in the coding region or flanking exon–intron boundaries for POLG1, PEO1, POLG2 and SLC25A4.

GenotypingMethod: The 30 coding exons and intron–exon junctions of the OPA1 gene were sequenced using a set of 30 primer pairs

Variant: c.1800C>G (p.Ser600Arg)

ClinVar: 30461

CAID: CA10575554

SupplementalData:

Case: 8

DiseaseAssertion: hypertrophic cardiomyopathy, Left ventricular hypertrophy

FamilyInfo: Finnish, no family info

CasePresentingHPOs: HP:0001639, HP:0001712

"hypertrophic cardiomyopathy, Left ventricular hypertrophy"

CaseHPOFreeRext:

CaseNOTHPOs:

CaseNOTHPOFreeText:

CasePreviousTesting: 24 h Holter registration and bicycle ergometer test performed in majority of patients. Cardiac magnetic resonance imaging was performed to verify the diagnosis of HCM

GenotypingMethod: Targeted next-generation sequencing of exons, UTRs and 10 bp flanking regions of 59 cardiomyopathy associated genes, filtered variants confirmed by Sanger sequencing

MultipleGeneVariants:

(1) LAMA4 Variant: c.2593C>G (unknown HGVS descriptor)

ClinVarID: unknown

gnomAD: unknown

(2) MYH7 Variant: NM_000257.4:c.3158G>A

ClinVarID: 155814

gnomAD: http://gnomad.broadinstitute.org/variant/14-23891476-C-T

Total Allele Frequency: 0.0000601 European (Finnish): 0.0006368

PreviouslyPublished:

SupplementalData: Table S3: list of all 382 patients with HCM included in the FinHCM Genetic Study, and the rare variants (GMAF <1%) detected in each patient.

Case: 2

DiseaseAssertion: hypertrophic cardiomyopathy, Left ventricular hypertrophy

FamilyInfo: Finnish, no family info

CasePresentingHPOs: HP:0001639, HP:0001712

"hypertrophic cardiomyopathy, Left ventricular hypertrophy"

CaseHPOFreeRext:

CaseNOTHPOs:

CaseNOTHPOFreeText:

CasePreviousTesting: 24 h Holter registration and bicycle ergometer test performed in majority of patients. Cardiac magnetic resonance imaging was performed to verify the diagnosis of HCM

GenotypingMethod: Targeted next-generation sequencing of exons, UTRs and 10 bp flanking regions of 59 cardiomyopathy associated genes, filtered variants confirmed by Sanger sequencing

MultipleGeneVariants:

(1) MYH7 Variant: NM_000257.4:c.3158G>A

ClinVarID: 155814

gnomAD: http://gnomad.broadinstitute.org/variant/14-23891476-C-T

Total Allele Frequency: 0.00006010 European Finnish: 0.0006368

(2) MYBPC3 Variant: NM_000256.3:c.440C>T

ClinVarID: 181035

gnomAD: http://gnomad.broadinstitute.org/variant/11-47371630-G-A

Total Allele Frequency: 0.0005896 European (Finnish): 0.005563

(3) MYOZ2 Variant: NM_016599.4:c.738A>G

ClinVarID: 30509

gnomAD: http://gnomad.broadinstitute.org/variant/4-120107298-A-G

Total Allele Frequency: 0.0023 European (Finnish): 0.02188

(4) PSEN2 Variant: NM_012486.2:c.211C>T

ClinVarID: 192130

gnomAD: http://gnomad.broadinstitute.org/variant/1-227071475-C-T

Total Allele Frequency: 0.003711 European (Finnish): 0.01791

(5) SCN5A Variant: NM_000335.4:c.1715C>A

ClinVarID: 67683

gnomAD: http://gnomad.broadinstitute.org/variant/3-38645378-G-T

Total Allele Frequency: 0.005175 European (Finnish): 0.03855

PreviouslyPublished:

SupplementalData: Table S3: list of all 382 patients with HCM included in the FinHCM Genetic Study, and the rare variants (GMAF <1%) detected in each patient.

annotating here since this is a PDF

Case#: Yamagishi Case 2, female, 10m at dx

DiseaseAssertion: Hurler

FamilyInfo:

CasePresentingHPOs:

CaseHPOFreeText: mental delay. height=70cm

CaseNotHPOs: HP:0002240

CaseNotHPOFreeText: cardiac involvement

PreviouslyPublished: n/a

Variant: 704ins5/704ins homozygous

ClinVar: 11921

CAID: CA256123

SupplementalData: n/a

Case#: KMP 1, female, 39 yrs, East Slovakia

DiseaseAssertion: HCM

FamilyInfo: n/a

CasePresentingHPOs: n/a

CaseHPOFreeText: symptomatic; LVEF 60%; NYHA I.; ECG; echocardiography

CaseNotHPOs: n/a

CaseNotHPOFreeText: no LVOTO; no ICD

CasePreviousTesting: Additional Sanger sequencing of exons 12, 24, 25 of MYBPC3, exon 12 NEBL, exons 12, 20, 21 of SCN5A, exon 7 of TNNI3, and exon 9 of TNNT2 gene

CaseGenotypingMethod: Sanger sequencing

Variant: NM_000257.4(MYH7):c.2845G>A (p.Glu949Lys)

ClinVar: 14093 https://www.ncbi.nlm.nih.gov/clinvar/variation/14093/

CAID: n/a

gnomAD: absent

Case#: KMP 16, female, 60 yrs, East Slovakia

DiseaseAssertion: HCM

FamilyInfo: n/a

CasePresentingHPOs: n/a

CaseHPOFreeText: symptomatic; LVOTO yes; LVEF 39-40%; NYHA I.; ICD yes; ECG; echocardiography;

CaseNotHPOs: n/a

CaseNotHPOFreeText: n/a

CasePreviousTesting: Additional Sanger sequencing of exons 12, 24, 25 of MYBPC3, exon 12 NEBL, exons 12, 20, 21 of SCN5A, exon 7 of TNNI3, and exon 9 of TNNT2 gene

CaseGenotypingMethod: Sanger sequencing;

Variant: NM_000257.4(MYH7):c.2845G>A (p.Glu949Lys)

ClinVar: 14093 https://www.ncbi.nlm.nih.gov/clinvar/variation/14093/

CAID: n/a

gnomAD: absent

Case#: N5214, F, 62 y.o., Italian

CasePresentingHPOs: HP:0007354, HP:0002015, HP:0001260, HP:0002380, HP:0003690, HP:0003202

CaseNotHPOs: HP:0100543

CaseGenotypingMethod: Sanger sequencing

Variant: NM_006000.3(TUBA4A):c.1045A>T p.Thr349Ser

CAID: CA350723588

CaseAddInfo: While most cases were found to be negative during pre-screening for known mutations, some cases were positive for mutations in SOD1 (n=4), C9orf72 (n=12), TARDBP (n=4), & FUS/TLS (n=1) genes. Whether this case tested positive for any of these is not specified.

Case#: P937, F, Italian

CasePresentingHPOs: HP:0007354

CaseGenotypingMethod: Sanger sequencing

Variant: c.19C>A p.Val7Ile

CaseAddInfo: This allele is not found in the reference NM_006000.2 or NM_006000.3 (CAID can not be created or retrieved because there is a G in this location instead of the C reported here).

CaseAddInfo: While most cases were found to be negative during pre-screening for known mutations, some cases were positive for mutations in SOD1 (n=4), C9orf72 (n=12), TARDBP (n=4), & FUS/TLS (n=1) genes. Whether this case tested positive for any of these is not specified.

NonHumanModel Mus Musculus

GeneAlteration The 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).

ModelPhenotype HP:0004758

HumanPhenotype HP:0004758

ModelEvidence Fig. 1 ECG during stimuli shows VT in only the mutant mice

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

Proband III:15, 47 yr old male, Dx = 37 yrs

HP:0001639, HP:0001681

HCM, chest tightness (angina)

Variant: NM_016599.4(MYOZ2):c.505A>C (p.L169G)

Not in ClinVar or EXAC

Variant NM_005186.3(CAPN1):c.407delC (p.Pro136Argfs), NM_001198868.1(CAPN1):c.1605+5G>A

ClinVar 225767, 225768

gnomAD none, 0.0003171

Case# FamilyC:IV-13, 33 AoO, Female, USA

CasePresentingHPOs HP:0002064, HP:0001258, HP:0002355

CaseMethod1 WES

CaseMethod2 Sanger

Variant NM_005186.3(CAPN1):c.407delC (p.Pro136Argfs), NM_001198868.1(CAPN1):c.1605+5G>A

ClinVar 225767, 225768

gnomAD none, 0.0003171

CaseAddInfo nonconsanguineous family

Family#PresentingHPOs HP:0001258, HP:0002460, HP:0007067, HP:0002395

CountryOrigin Morocco

Race White

Family#Method1 WES

Family#Method2 Sanger

Affected 4

Unaffected 3

Segregations 4

eLOD 1.93

AddSegregationInfo consanguineous

ProbandLabel FamilyB:IV-1 age of onset 35, Male

Variant NM_001198868.1(CAPN1):c.1579C>T (p.Gln527Ter)

ClinVar 225766

FamilyAddInfo Fig 1

Case#: Wang Patient #7, female, 7yo at dx

DiseaseAssertion: Hurler/Scheie

FamilyInfo:

CasePresentingHPOs:

CaseHPOFreeText: IDUA activity=0.3 nmol/h/mg protein (normal range=27.2–52 nmol/h/mg protein.), delayed mental development, cardiac disease, Macrocephaly/joint stiffness

CaseNotHPOs:

CaseNotHPOFreeText: mongolian macula, hernia, airway obstruction

PreviouslyPublished:

Variant: p.A79V; c.1225dupG

ClinVar: 1458769;

CAID: CA356987

SupplementalData:

Case#: Wang Patient #26, female, 3yo at dx

DiseaseAssertion: Hurler

FamilyInfo:

CasePresentingHPOs:

CaseHPOFreeText: IDUA activity=not stated, delayed mental development, cardiac disease, Macrocephaly/joint stiffness/mongolian macula, Hepatosplenomegaly/ hernia, airway obstruction

CaseNotHPOs:

CaseNotHPOFreeText:

PreviouslyPublished:

Variant: p.A79V; 668insGCG

ClinVar: 1458769;

CAID: CA356987

SupplementalData:

FamilyLabel: Van Driest Family

Family#PresentingHPOs: HP:0001639 (hypertrophic cardiomyopathy)

FamilyNotHPOs:

CountryOrigin:

Ethnicity:

Race:

Family#PreviousTesting: Three highly polymorphic markers for each of 9 known HC genes (MYH7, TNNT2, TNNI, TPM1, MYL3, MYL2, MBPC, ACTC, and PRKAG2) were PCR-amplified for haplotype analysis. Only 3 candidate genes, MYL3, TNNT2, and TPM1, were shared by proband, affected siblings, and father.

Family#Method1: (other) PCR amplification of all protein-coding exons and splice junctions of the 3 candidate genes from genomic DNA was analyzed using DHPLC (denaturing high-performance liquid chromatography).

Family#Method2: Cycle sequencing was performed on all DHPLC amplicons with abnormal elution profiles (exon 5 of TPM1).

Family#GenotypingMethod: DHPLC screening followed by cycle sequencing of TPM1 exon 5.

Affected: 4

Unaffected:

Segregations: 3 (Figure 1)

PublishedLOD: N/A

eLOD: N/A; < 4 segregations

AddSegregationInfo:

ProbandLabel: III-1

Variant: NM_001018005.2(TPM1):c.554T>G (p.Leu185Arg)

ClinVar: 31881

CAID: CA019057

gnomAD: 15-63060930-T-G has no data in gnomAD v4.0.0

FamilyAddInfo: TPM1 variant reported as c.T610G (p.L185R), using coding positions relative to GeneBank accession M19713.1?

FamilyPMIDs:

Case#: III-1

CasePresentingHPOs: HP:0001639 (hypertrophic cardiomyopathy); HP:0001645 (sudden cardiac death)

CaseHPOFreeText:

CaseNotHPOs:

CaseNotHPOFreeText:

CasePreviousTesting: None for the proband

CaseMethod1:

CaseMethod2:

CaseGenotypingMethod: DNA extracted from paraffin-embedded spleen tissue, taken at autopsy, was subjected to PCR-amplification of TPM1 exon 5 followed by cycle sequencing to confirm presence of the familial TPM1 variant.

Variant: NM_001018005.2(TPM1):c.554T>G (p.Leu185Arg)

ClinVar: 31881

CAID: CA019057