Case3:

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 0.25 nM/ml (ref 0.33). Additional testing was performed on patients identified by NBS: all either had elevation of 2-methylbutyrlglycine in urine (with no intermediates consistent with isovaleric acidemia), or increase in 2-methylbutyrylcarnitine in culture from fibroblasts.

CaseGenotypingMethod: Bidirectional sequence analysis of genomic DNA.

Variant: 1. NM_001609.3(ACADSB):c.621G>A (p.Trp207Ter) 2. unknown

ClinVar: 299075

gnomAD:

VariantEvidence: Genomic DNA showed heterozygosity for this variant, but cDNA indicated homozygosity. The variant on the second allele must result in loss of transcription, abnormal splicing, unstable RNA, or deletion containing exon 5.

CaseAddInfo:

CasePMIDs:

PMID: 20547083

Gene: ACADSB

Disease: 2-methylbutyrylglycinuria

MonDO: MONDO:0012392

InheritancePattern: AR

Case2: female, Turkish, neonatal screening

CasePresentingHPOs: HP:0000403

HPOFreeText: Neonatal screening suggestive of MBD deficiency. Normal free carnitine. No treatment, but normal at 6 years. Adenotomy and bilateral paracentesis at 5.5 years.

CaseNotHPOs:

CasePreviousTesting: Neonatal screening: elevated C5 acylcarnitine (0.75 umol/l, ref <0.31). Urinary organic acids showed 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.443C>T (p.Thr148Ile) (homozygous)

ClinVar: 9202

gnomAD: South Asian 0.002678; Overall 0.0008097, no homozygotes

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

CaseAddInfo: Consanguinity. 7 year old brother also found to have enzyme deficiency. No symptoms.

CasePMIDs:

Case4: female, Turkish, neonatal screening

CasePresentingHPOs:

HPOFreeText: Neonatal screening suggestive of MBD deficiency. Treatment of 100 mg L-carnitine/kg body weight and has shown normal development until 3 years.

CaseNotHPOs:

CasePreviousTesting: Neonatal screening: elevated C5 acylcarnitine (0.68 umol/l and 2.08 umol/l , ref <0.31). Urinary organic acids showed 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.1159G>A (p.Glu387Lys) (homozygous)

ClinVar: 9203

gnomAD: Latino 0.0004797, Overall 0.0002404, no homozygotes

VariantEvidence: In vitro expression in E. coli shows no protein on Western blot, reduced enzymatic activity to 2% and defects in protein folding and stability (PMID: 20547083).

CaseAddInfo:

CasePMIDs:

Case: 4 years (Dx), male, Somali

CasePresentingHPOs: HP:0001250, HP:0000750, HP:0000817, HP:0008762

HPOFreeText: 5 months: seizure-like movements, stopped at 8 months with antieplieptic drug. Delayed speech. Protein restricted diet for 5 months with no improvement.

CaseNotHPOs: Normal growth and motor development. 5 years: no dysmorphic features. Normal muscle tone, strength, coordination and deep tendon reflexes. Eye exam, hearing test, EEG, EKG, and MRI normal.

CasePreviousTesting: Blood glucose, urea, creatinine, and liver transaminases normal. Normal chromosome analysis. Southern blot for Angelman syndrome negative. Urine analysis showed large amounts of 2-methylbutyryl glycine. Increased plasma level of C5-acylcarnitine (1.43 micromoles/L, reference range 0.05–0.38) and an increased C5/C3-acylcarnitines ratio of 2.83 (reference range 0.16–0.73).

CaseGenotypingMethod: PCR of all exons and flanking intron sequences of genomic DNA. Mother was also sequenced.

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

ClinVar: 448980

gnomAD:

VariantEvidence: Functional studies demonstrate that the c.303+3A>G variant causes complete exon skipping of exon 3 (Madsen et al., 2006).

CaseAddInfo:

CasePMIDs:

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.

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.