Table 4.
Results of genetic and enzymatic analyses in 75 true-positive cases and 5 false-negative cases.
| ID | Enzyme | Condition | Reference Sequence | Allele 1 | ACMG | Allele 2 | ACMG |
|---|---|---|---|---|---|---|---|
| Organic acidurias | |||||||
| 1 | F Cbl a | MMA | NM_052845.3(MMAB) | c.291–1G > A (splice defect) | 5 | c.571C > T (p.Arg191Trp) | 5 |
| 2 | F MUT b | MMA | NM_000255.4(MMUT) | c.675_677delTAT (p.Phe225_Met226delinsLeu) | 5 | c.1106G > A (p.Arg369His) | 5 |
| 3 | NP | MMA | NM_000255.4(MMUT) | c.1655C > T (p.Ala552Val) | 5 | c.1677-1G > A (splice defect) | 5 |
| 4 | NP | Cbl C | NM_015506.2(MMACHC) | c.271dupA (p.Arg91Lysfs*14) | 5 | c.271dupA (p.Arg91Lysfs*14) | 5 |
| 5 | F c | PA | NM_000532.4(PCCB) | c.319G > A (p.Val107Met) | 4 | c.1281_1282delCA (p.Thr428Serfs*12) | 5 |
| 6 | NP | PA | NM_000532.4(PCCB) | c.331C > T (p.Arg111 *) | 5 | c.838dup (p.Leu280Profs*11) | 5 |
| 7 | NP | PA | NM_000532.4(PCCB) | c.1498 + 2T>C (splice defect) | 5 | c.1498 + 2T>C (splice defect) | 5 |
| 8 | L d | IVA | NM_002225.3(IVD) | c.208G>T (p.Glu70 *) | 5 | c.941C>T (p.Ala314Val) | 5 |
| 9 | NP | IVA | NM_002225.3(IVD) | c.296-2A>G (splice defect) | 4 | c.296-2A > G (splice defect) | 4 |
| 10 | NP | GA-I | NM_000159.2(GCDH) | c.572T>C (p.Met191Thr) | 5 | c.1045G>A (p.Ala349Thr) | 5 |
| 11 | NP | GA-I | NM_000159.3(GCDH) | c.1045G>A (p.Ala349Thr) | 5 | c.1204C>T (p.Arg402Trp) | 5 |
| 12 | NP | GA-I | NM_000159.3(GCDH) | c.1240G>A (p.Glu414Lys) | 5 | c.1240G>A (p.Glu414Lys) | 5 |
| 13 | NP | GA-I | NM_000159.3(GCDH) | c.1240G>A (p.Glu414Lys) | 5 | c.1240G>A (p.Glu414Lys) | 5 |
| 14 | NP | HCS | NM_000411.6(HLCS) | c.1519 + 5G > A(splice defect) | 5 | c.1993C > T (p.Arg665 *) | 5 |
| Aminoacidopathies | |||||||
| 15 | 4.5% e | MSUD | ND | ND | |||
| 16 | NP | MSUD | NM_000709.3(BCKDHA) | c.375 + 648_484 + 520del p.Gly126Valfs*3 (ref below) | 5 | c.375 + 648_484 + 520del p.Gly126Valfs*3 (ref below) | 5 |
| 17 | NP | MSUD Ɨ | NM_001918.3(DBT) | c.901C>T (p.Arg301Cys) | 5 | c.1291C>T (p.Arg 431 *) | 5 |
| 18 | NP | MSUD Ɨ | NM_001918.3(DBT) | c.901C>T (p.Arg301Cys) | 5 | c.1291C>T (p.Arg 431 *) | 5 |
| 19 | P f | CBS | NM_000071.2(CBS) | c.451 + 2T>G (splice defect) | 4 | c.833T>C (p.Ile278Thr) | 5 |
| 20 | NP | CBS | NM_000071.2(CBS) | c.728A>G (p.Gln243Arg) | 4 | c.728A>G (p.Gln243Arg) | 4 |
| 21 | NP | TYR-I | NM_000137.2(FAH) | c.554-1G>T (splice defect) | 5 | c.1062 + 5G>A (splice defect) | 5 |
| 22 | NP | TYR-I | NM_000137.2(FAH) | c.742delG (p.Pro249Hisfs*55) | 5 | c.1062 + 5G>A (splice defect) | 5 |
| 23 | NP | TYR-I | NM_000137.2(FAH) | c.742delG (p.Pro249Hisfs*55) | 5 | c.1062 + 5G>A (splice defect) | 5 |
| 24 | NP | TYR-I | NM_000137.2(FAH) | c.1008C>G (p.Asn336Lys) | 4 | c.1062 + 5G>A (splice defect) | 5 |
| 25 | NP | TYR-I | NM_000137.2(FAH) | c.1062 + 5G>A (splice defect) | 5 | c.1062 + 5G>A (splice defect) | 5 |
| 26 | S16% g | BD | NM_000060.2(BTD) | c.235C>T (p.Arg79Cys) | 5 | c.1330G>C (p.Asp444His) | 5 |
| 27 | S22% g | BD | NM_000060.2(BTD) | c.278A>G (p.Tyr93Cys) | 5 | c.1330G>C (p.Asp444His) | 5 |
| 8 | S2% g | BD | NM_000060.2(BTD) | c.424C>A (p.Pro142Thr) | 4 | c.424C>A (p.Pro142Thr) | 4 |
| 29 | S11% g | BD | NM_000060.3(BTD) | c.470G>A (p.Arg157His) | 5 | c.470G>A (p.Arg157His) | 5 |
| 30 | S18% g | BD | NM_000060.3(BTD) | c.470G>A (p.Arg157His) | 5 | c.1330G>C (p.Asp444His) | 5 |
| 31 | S6% g | BD | NM_000060.2(BTD) | c.470G>A (p.Arg157His) | 5 | c.1333G>A (p.Gly445Arg) | 5 |
| 32 | S23% g | BD | NM_000060.3(BTD) | c.511G>A (p.Ala171Thr) | 5 | c.1330G>C (p.Asp444His) | 5 |
| 33 | S15% g | BD | NM_000060.3(BTD) | c.511G>A (p.Ala171Thr) | 5 | c.1330G>C (p.Asp444His) | 5 |
| 34 | S11% g | BD | NM_000060.3(BTD) | c.605A>T (p.Asn202Ile) | 5 | c.605A>T (p.Asn202Ile) | 5 |
| 35 | S7% g | BD | NM_000060.2(BTD) | c.1006C > T (p.Gln336 *) | 5 | c.1006C > T (p.Gln336 *) | 5 |
| 36 | S < 1% g | BD | NM_000060.2(BTD) | c.1006C > T (p.Gln336 *) | 5 | c.1006C > T (p.Gln336 *) | 5 |
| 37 | S27% g | BD | NM_000060.3(BTD) | c.1330G>C (p.Asp444His) | 5 | c.1368A>C (p.Gln456His) | 5 |
| 38 | S8% g | BD | NM_000060.2(BTD) | c.626G>A (p.Arg209His) | 5 | c.1595C>T (p.Thr532Met) | 5 |
| Fatty acid oxidation defects | |||||||
| 39 | NP | CTD | NM_003060.3(SLC22A5) | c.51C>G (p.Phe17Leu) | 5 | c.136C>T (P.Pro46Ser) | 5 |
| 40 | F12% h | CTD Ɨ | NM_003060.3(SLC22A5) | c.136C>T (p.Pro46Ser) | 5 | c.136C>T (p.Pro46Ser) | 5 |
| 41 | NP | CTD Ɨ | NM_003060.3(SLC22A5) | c.136C>T (p.Pro46Ser) | 5 | c.136C>T (p.Pro46Ser) | 5 |
| 42 | NP | CTD | NM_003060.3(SLC22A5) | c.136C>T (p.Pro46Ser) | 5 | c.844C>T (p.Arg282 *) | 5 |
| 43 | NP | CTD | NM_003060.3(SLC22A5) | c.847T>A (p.Trp283Arg) | 4 | c.847T>A (p.Trp283Arg) | 4 |
| 44 | F i | CPT-IA | NM_001031847.2(CPT1A) | c.619C > T (p.Gln207 *) | 5 | c.2215A > G (p.Lys739Glu) | 4 |
| 45 | 14% j | MCADD | NM_000016.4(ACADM) | c.250C>T (p.Leu84Phe) | 5 | c.985A>G (p.Lys329Glu) | 5 |
| 46 | 11% j | MCADD | NM_000016.4(ACADM) | c.250C>T (p.Leu84Phe) | 5 | c.985A >G (p.Lys329Glu) | 5 |
| 47 | <1% j | MCADD | NM_000016.4(ACADM) | c.362C>T (p.Thr121Ile) | 5 | c.362C>T (p.Thr121Ile) | 5 |
| 48 | 6% j | MCADD | NM_000016.4(ACADM) | c.388-19T > A (intronic) | 5 | c.985A>G (p.Lys329Glu) | 5 |
| 49 | NP | MCADD | NM_000016.4(ACADM) | c.985A>G (p.Lys329Glu) | 5 | c.985A>G (p.Lys329Glu) | 5 |
| 50 | NP | MCADD | NM_000016.4(ACADM) | c.985A>G (p.Lys329Glu) | 5 | c.985A>G (p.Lys329Glu) | 5 |
| 51 | NP | MCADD | NM_000016.4(ACADM) | c.985A>G (p.Lys329Glu) | 5 | c.985A>G (p.Lys329Glu) | 5 |
| 52 | NP | MCADD | NM_000016.4(ACADM) | c.985A>G (p.Lys329Glu) | 5 | c.985A>G (p.Lys329Glu) | 5 |
| 53 | <1% j | MCADD | NM_000016.4(ACADM) | c.985A>G (p.Lys329Glu) | 5 | c.985A>G (p.Lys329Glu) | 5 |
| 54 | <1% j | MCADD | NM_000016.4(ACADM) | c.985A>G (p.Lys329Glu) | 5 | c.985A>G (p.Lys329Glu) | 5 |
| 55 | 3% j | MCADD | NM_000016.4(ACADM) | c.985A>G (p.Lys329Glu) | 5 | c.985A>G (p.Lys329Glu) | 5 |
| 56 | <1% j | MCADD | NM_000016.4(ACADM) | c.985A>G (p.Lys329Glu) | 5 | c.985A>G (p.Lys329Glu) | 5 |
| 57 | <1% j | MCADD | NM_000016.4(ACADM) | c.985A>G (p.Lys329Glu) | 5 | c.985A>G (p.Lys329Glu) | 5 |
| 58 | NP | MCADD | NM_000016.4(ACADM) | c.985A>G (p.Lys329Glu) | 5 | c.985A>G (p.Lys329Glu) | 5 |
| 59 | <1% j | MCADD | NM_000016.4(ACADM) | c.985A>G (p.Lys329Glu) | 5 | c.1171A>G (p.Met391Val) | 3+ |
| 60 | NP | MCADD | NM_000016.4(ACADM) | c.244dup (p.Trp82Leufs*23) | 5 | c.244dup (p.Trp82Leufs*23) | 5 |
| 61 | NP | MCADD | NM_000016.4(ACADM) | c.244dup (p.Trp82Leufs*23) | 5 | c.244dup (p.Trp82Leufs*23) | 5 |
| 62 | L k | CPT-II | NM_000098.2(CPT2) | c.149C>A (p.Pro50His) | 5 | c.149C>A (p.Pro50His) | 5 |
| 63 | L k | CPT-II | NM_000098.2(CPT2) | c.149C>A (p.Pro50His) | 5 | c.1369A>T (p.Lys457 *) | 5 |
| 64 | NP | CPT-II Ɨ | NM_000098.2(CPT2) | c.338C>T (p.Ser113Leu) | 5 | c.481C>T (p.Arg161Trp) | 4 |
| 65 | L k | CPT-II | NM_000098.2(CPT2) | c.338C>T (p.Ser113Leu) | 5 | c.1444_1447del (p.Thr482Trpfs*49) | 5 |
| 66 | NP | CPT-II | NM_000098.2(CPT2) | c.1798G>A (p.Gly600Arg) | 4 | c.1798G>A (p.Gly600Arg) | 4 |
| 67 | F l | CACT | NM_000387.5(SLC25A20) | c.82G>T (p.Gly28Cys) | 5 | c.82G>T (p.Gly28Cys) | 5 |
| 68 | 15% m | VLCADD | NM_000018.3(ACADVL) | c.533T>C (p.Leu178Pro) | 5 | c.1066A>G (p.Ile356Val) | 3 |
| 69 | 17% m | VLCADD | NM_000018.3(ACADVL) | c.848T>C (p.Val283Ala) | 5 | c.848T>C (p.Val283Ala) | 5 |
| 70 | 12% m | VLCADD | NM_000018.3(ACADVL) | c.848T>C (p.Val283Ala) | 5 | c.848T>C (p.Val283Ala) | 5 |
| 71 | 7% m | VLCADD | NM_000018.3(ACADVL) | c.848T>C (p.Val283Ala) | 5 | c.865G>A (p.Gly289Arg) | 5 |
| 72 | 9% m | VLCADD | NM_000018.3(ACADVL) | c.848T>C (p.Val283Ala) | 5 | c.1177A>G (p.Thr393Ala) | 3+ |
| 73 | <1% m | VLCADD | NM_000018.3(ACADVL) | c.1837C>T (p.Arg613Trp) | 5 | c.1837C>T (p.Arg613Trp) | 5 |
| 74 | NP | LCHADD | NM_000182.4(HADHA) | c.1528G>C (p.Glu510Gln) | 5 | c.1528G>C (p.Glu510Gln) | 5 |
| 75 | L n | TFP | NM_000182.4(HADHA) | c.1678C>T (p.Arg560 *) | 5 | c.1678C>T (p.Arg560 *) | 5 |
| 76 | NP | TFP | NM_000182.4(HADHA) | c.1678C>T (p.Arg560 *) | 5 | c.1678C>T (p.Arg560 *) | 5 |
| 77 | NP | TFP | NM_000183.2(HADHB) | c.209 + 1G>C (splice defect) | 5 | c.255-1G>A (splice defect) | 5 |
| 78 | L o | TFP | NM_000182.4(HADHA) | c.180 + 3A>G (splice defect) | 5 | c.180 + 3A>G (splice defect) | 5 |
| 79 | F p | MADD/GA-II | NM_017986.3(SLC52A1) | c.1134 + 11G>A (intronic) | 1 | wild type | |
| 80 | F22% q | MADD/GA-II | NM_000126.3(ETFA) | c.348A>T (splice defect) | 3+ | c.348A>T (splice defect) | 3+ |
New pathogenic DNA variants are shown in bold, and variants are classified according to the ACMG criteria: 5 for pathogenic, 4 for likely pathogenic, and 3 for variant of unknown significance [10]. Ɨ False-negative cases; FCblB a, deficient14C propionate incorporation in fibroblasts (Cbl B complementation group); FMUT b, deficient 14C propionate incorporation in fibroblasts (MUT0 complementation); NP, enzyme analysis not performed; F c, deficient propionyl-CoA carboxylase activity in fibroblasts at 0.02 nmol/min.mg, reference range 0.42–2.6; L d, isovaleryl-CoA dehydrogenase activity in lymphocytes <0.14 nmol/min.mg, reference range 0.89–2.13; F e, decreased decarboxylation rate in fibroblasts of [1–14C] leucine (4.5% of intra-assay control); P f, plasma cystathionine-β-synthase (CBS) activity at 29.3 nmol/L (reference 100–1000), classified in vitro as a possible pyridoxine non-responder with some residual CBS activity; S% g, serum biotinidase enzyme activity as percentage of the reference mean; F h, plasma membrane carnitine transporter (OCTN2) activity in fibroblasts (12% of the reference mean); F i, carnitine palmitoyltransferase I (CPT-I) activity in fibroblasts below limit of quantification of assay (<0.04 nmol/(min.mg protein), reference range 0.75–2.23; L j, medium-chain acyl-CoA-dehydrogenase (MCAD) activity in lymphocytes <1–14% of the mean of reference values; L k, carnitine palmitoyltransferase II (CPT-II) activity in leukocytes <0.5 nmol/mg prot./min, reference range 7–12 nmol/mgprot./min; F l, mitochondrial carnitine/acylcarnitine transporter activity below the limit of the quantification of the assay (<8 pmol/(min.mg protein), reference range 70–274; L m, very long-chain acyl-CoA dehydrogenase activity in lymphocytes <1–17% of the mean of reference values; L n, long-Chain 3- hydroxy-acyl-CoA dehydrogenase (LCHAD) activity in lymphocytes 15% of the reference mean and long-chain 3-ketoacyl-CoA thiolase activity 6% of the reference mean; L o, long-chain 3-hydroxy-acyl-CoA dehydrogenase (LCHAD) activity in lymphocytes at 33% of the reference mean and long-chain 3-ketoacyl-CoA thiolase activity at 27% of the reference mean; F p, normal overall beta oxidation (acylcarnitine profiling) in fibroblasts; F22% q, oleate beta-oxidation activity in fibroblasts (flux assay) at 22% of the activity in controls. Ref ID 16, BCKDHA variant [17].