Table 3. Variants in the SLC22A5 gene in patients referred for possible primary carnitine.
(A) Molecular analysis of the SLC22A5 gene in our patients identified 133 variants, with 33 being novel (*). Of the 133 variants identified, 90 missense variants and 2 in-frame deletions were expressed in CHO cells to evaluate carnitine transport (functional activity). Carnitine transport was expressed as percent of the activity in CHO cells expressing the wild-type OCTN2 carnitine transporter (100% activity). Carnitine transport was reduced to less than 20% of wild-type OCTN2 by 73/92 variants. (B) Table 3B shows the frequency in normal individuals in the ExAC Browser Beta and in the gnomAD of the alleles found in our patients along with the number of normal individual homozygous for a specific allele. 1:221 alleles contained either one of the missense mutations expressed in this study or a mutation causing the premature insertion of a stop codon or affecting splicing in the SLC22A5 gene. (C) The 90 missense variants and 2 in-frame deletions whose carnitine transport was tested in CHO cells were evaluated for pathogenicity using the PolyPhen-2 v2.2.2r398 (http://genetics.bwh.harvard.edu/pph2/index.shtml, using default parameters, defining damaging any variant with a score >0.15) and SIFT (http://sift.jcvi.org/www/SIFT_enst_submit.html, using default parameters and assuming as damaging any change with a score of <0.05) software with the predicted effects indicated. Actual values for each amino acid substitution are reported in Supplemental Table S2. Variants were submitted to ClinVar https://www.ncbi.nlm.nih.gov/clinvar/?term=SLC22A5%5Bgene%5D.
| A | B | C | ||||||
|---|---|---|---|---|---|---|---|---|
| Exon | cDNA | PROTEIN | Transport activity % wild-type OCTN2 | Reference(s) | Allele Frequency (ExAC) | Homozygous (ExAC) | Polyphen-2 | SIFT |
| OCTN2 deletion | (Li, et al., 2010) | |||||||
| 1 | c.-91_22del | (Nezu, et al., 1999) | ||||||
| 1 | c.3G>T | p.Met1Leu | 0.16 | (Dobrowolski, et al., 2005) | 3.23E-05 | 0 | damaging | damaging |
| 1 | c.4_5insC | p.Arg2Profs*136 | (Nezu, et al., 1999) | |||||
| 1 | c.12C>G | p.Tyr4* | (Wang, et al., 2001) | 0.0000173 | 0 | |||
| 1 | c.34G>A | p.Gly12Ser | 51.7 | (Li, et al., 2010) | 0.0007152 | 1 | damaging | damaging |
| 1 | c.43G>T | p.Gly15Trp | 11.05 | (El-Hattab, et al., 2010) | 0.0001028 | 0 | damaging | damaging |
| 1 | c.47C>T | p.Pro16Leu | 0.00 | * | benign | benign | ||
| 1 | c.51C>G | p.Phe17Leu | 10.74 | (Lee, et al., 2010; Urban, et al., 2006) | 0.0000601 | 0 | damaging | damaging |
| 1 | c.56G>C | p.Arg19Pro | 2.03 | (Wang, et al., 2001) | damaging | benign | ||
| 1 | c.59T>A | p.Leu20H | 46.77 | * | 0.0013610 | 1 | damaging | benign |
| 1 | c.64_66del | p.Phe23del | 0.46 | (Lamhonwah, et al., 2002) | 0.0000259 | 0 | / | damaging |
| 1 | c.77G>A | p.Ser26Asn | 5.51 | (Rose, et al., 2012) | 0.0000262 | 0 | damaging | benign |
| 1 | c.83G>T | p.Ser28Ile | 1.35 | (Rahbeeni, et al., 2002) | damaging | benign | ||
| 1 | c.95A>G | p.Asn32Ser | 0.88 | (Lamhonwah, et al., 2002; Rasmussen, et al., 2014b) | 0.0000268 | 0 | damaging | benign |
| 1 | c.131C>T | p.Ala44Val | 8.22 | * | 0.0000197 | 0 | damaging | damaging |
| 1 | c.136C>T | p.Pro46Ser | 2.47 | (Schimmenti, et al., 2007) | 0.0005515 | 0 | damaging | damaging |
| 1 | c.137C>T | p.Pro46Leu | 4.10 | * | 0.0000202 | 0 | damaging | damaging |
| 1 | c.149G>A | p.Cys50Tyr | 0.00 | * | damaging | damaging | ||
| 1 | c.196A>C | p.Thr66Pro | 1.80 | (Li, et al., 2010) | damaging | damaging | ||
| 1 | c.224G>C | p.Arg75Pro | 1.93 | (Li, et al., 2010) | 0.0000177 | 0 | benign | benign |
| 1 | c.232delC | p.His79Thrfs*51 | (Amat di San Filippo, et al., 2006) | 0.0000181 | 0 | |||
| 1 | c.248G>T | p.Arg83Leu | 0.14 | (Makhseed, et al., 2004) | 0.0004323 | 0 | damaging | damaging |
| 1 | c.254_264dup | p.Ile89Glyfs*45 | (Lamhonwah, et al., 2002; Wang, et al., 2001) | 0.0000385 | 0 | |||
| 1 | c.278C>G | p.Ser93Trp | 0.00 | * | damaging | damaging | ||
| 1 | c.283C>G | p.Leu95Val | 28.92 | * | benign | benign | ||
| 1 | c.287G>C | p.Gly96Ala | 19.65 | (Li, et al., 2010) | 0.0003927 | 0 | damaging | damaging |
| 1 | c.344A>G | p.Asp115Gly | 4.04 | * | damaging | damaging | ||
| 1 | c.350G>A | p.Trp117* | (Li, et al., 2010) | 0.0000042 | 0 | |||
| 1 | c.368T>G | p.Val123Gly | 13.96 | (Li, et al., 2010) | damaging | damaging | ||
| 1 | c.393G>C | p.Glu131Asp | 32.84 | * | damaging | damaging | ||
| IVS1 | c.393+5G>A | (Han, et al., 2014) | ||||||
| IVS1 | c.394-16T>A | (Rose, et al., 2012) | ||||||
| 2 | c.396G>A | p.Trp132* | (Koizumi, et al., 1999; Nezu, et al., 1999; Tang, et al., 1999) | 0.0000082 | 0 | |||
| 2 | c.419G>A | p.Trp140* | * | |||||
| 2 | c.424G>T | p.Ala142Ser | 25.68 | (Amat di San Filippo, et al., 2006) | 0.0000494 | 0 | benign | benign |
| 2 | c.428C>T | p.Pro143Leu | 1.18 | (Li, et al., 2010) | 0.0000081 | 0 | damaging | damaging |
| 2 | c.433dupA | p. Thr145Asnfs*50 | (Han, et al., 2014) | |||||
| 2 | c.451G>A | p.Val151Met | 65.93 | * | 0.0000330 | 0 | damaging | benign |
| c.453G>A | p.Val151Val | * | 0.0000433 | 0 | ||||
| 2 | c.458_459del | p.Val153fs*41 | (Dobrowolski, et al., 2005) | |||||
| IVS2 | c.497+1G>T | (Han, et al., 2014) | ||||||
| Exon 3 deletion | p.Phe167Aspfs*61 | * | ||||||
| 3 | c.505C>T | p.Arg169Trp | 0.01 | (Lamhonwah, et al., 2002; Wang, et al., 2000c) | 0.0000082 | 0 | damaging | damaging |
| 3 | c.506G>C | p.Arg169Pro | 0.24 | * | 0.0000577 | 0 | damaging | damaging |
| 3 | c.506G>A | p.Arg169Gln | 0.22 | (Burwinkel, et al., 1999) | 0.0000447 | 0 | damaging | damaging |
| 3 | c.517delC | p.Leu173Cysfs*3 | (Han, et al., 2014) | |||||
| 3 | c.523G>A | p.Val175Met | 7.03 | * | 0.0000330 | 0 | damaging | benign |
| 3 | c.529A>G | p.Met177Val | 14.40 | (Li, et al., 2010) | 0.0001071 | 0 | damaging | damaging |
| 3 | c.535A>T | p.Met179Leu | 48.13 | (Koizumi, et al., 1999) | benign | benign | ||
| 3 | c.557T>C | p.Leu186Pro | 0.00 | (Li, et al., 2010) | 0.0000041 | 0 | damaging | damaging |
| 3 | c.565_568del | p.Phe189Argfs*13 | * | |||||
| 3 | c.573delG | p.Asn192Ilefs*12 | (Li, et al., 2010) | 0.0000494 | 0 | |||
| 3 | c.614T>G | p.Met205Arg | 0.00 | * | damaging | damaging | ||
| 3 | c.629A>G | p.Asn210Ser | 0.13 | * | 0.0000203 | 0 | damaging | benign |
| 3 | c.632A>G | p.Tyr211Cys | 0.00 | (Vaz, et al., 1999) | 0.0000081 | 0 | damaging | damaging |
| 3 | c.641C>T | p.Ala214Val | 32.56 | * | 0.0007989 | 2 | damaging | damaging |
| IVS3 | c.652+1G>A | (Lamhonwah, et al., 2002) | 0.0000122 | 0 | ||||
| IVS3 | c.653-2A>C | * | ||||||
| 4 | c.656C>A | p.Thr219Lys | 28.25 | * | damaging | damaging | ||
| 4 | c.674C>T | p.Ser225Leu | 11.34 | * | damaging | benign | ||
| 4 | c.680G>A | p.Arg227His | 7.25 | (Li, et al., 2010) | 0.0000577 | 0 | damaging | damaging |
| 4 | c.688T>C | p.Phe230Leu | 0.80 | (Li, et al., 2010) | 0.0000108 | 0 | damaging | damaging |
| 4 | c.692C>T | p.Ser231Phe | 0.00 | * | damaging | damaging | ||
| 4 | c.695C>T | p.Thr232Met | 19.14 | (Dobrowolski, et al., 2005) | 0.0001318 | 0 | damaging | damaging |
| 4 | c.718G>A | p.Ala240Thr | 2.06 | (Li, et al., 2010) | damaging | benign | ||
| 4 | c.725G>T | p.Gly242Val | 0.00 | (Wang, et al., 2000c) | damaging | damaging | ||
| 4 | c.740C>G | p.Pro247Arg | 0.07 | * | damaging | damaging | ||
| 4 | c.745_748del | p.Phe249Leufs*13 | (Han, et al., 2014) | |||||
| 4 | c.760C>T | p.Arg254* | (Tang, et al., 2002) | 0.0001071 | 0 | |||
| 4 | c.761G>A | p.Arg254Gln | 34.24 | * | 0.0001236 | 0 | damaging | damaging |
| 4 | c.768G>A | p.Trp256* | (Amat di San Filippo, et al., 2006) | |||||
| 4 | c.769C>T | p.Arg257Trp | 7.65 | (Li, et al., 2010) | 0.0000906 | 0 | damaging | damaging |
| 4 | c.791C>T | p.Thr264Met | 46.00 | (Amat di San Filippo, et al., 2008) | 0.0000330 | 0 | damaging | damaging |
| 4 | c.791C>G | p.Thr264Arg | 2.48 | (Li, et al., 2010) | 0.0000406 | 0 | damaging | damaging |
| 4 | c.806delT | p.Leu269Argfs*27 | (Cederbaum, et al., 2002) | |||||
| 4 | c.806T>C | p.Leu269Pro | 44.86 | * | damaging | damaging | ||
| IVS4 | c.824+13T>C | (Mutlu-Albayrak, et al., 2015) | 0.4346000 | 27668 | ||||
| 5 | c.825G>A | p.Trp275* | (Dobrowolski, et al., 2005) | 0.0000082 | 0 | |||
| 5 | c.839delC | p.Arg282Aspfs*14 | (Lamhonwah, et al., 2002) | 0.0000082 | 0 | |||
| 5 | c.839C>T | p.Ser280Phe | 0.53 | (Amat di San Filippo, et al., 2006) | 0.0000081 | 0 | damaging | damaging |
| 5 | c.844C>T | p.Arg282* | (Burwinkel, et al., 1999; Vaz, et al., 1999);(Wang, et al., 1999) | 0.0000494 | 0 | |||
| 5 | c.844delC | p.Val295* | (Komlosi, et al., 2009) | |||||
| 5 | c.845G>A | p.Arg282Gln | 5.60 | (Amat di San Filippo, et al., 2006) | damaging | benign | ||
| 5 | c.847T>C; T>A | p.Trp283Arg | 0.45 | (Mayatepek, et al., 2000) | damaging | damaging | ||
| 5 | c.849G>T | p.Trp283Cys | 0.00 | (Koizumi, et al., 1999) | damaging | damaging | ||
| 5 | c.865C>T | p.Arg289* | (Dobrowolski, et al., 2005) | 0.0000082 | 0 | |||
| 5 | c.902C>A | p.Ala301Asp | 0.33 | (Wang, et al., 2000c) | damaging | damaging | ||
| 5 | c.934A>G | p.Ile312Val | 67.14 | (Amat di San Filippo, et al., 2008; Li, et al., 2010) | 0.0008072 | 0 | benign | benign |
| 5 | c.949G>A | p.Glu317Lys | 100.00 | (Amat di San Filippo, et al., 2008) | 0.0000082 | 0 | damaging | benign |
| 5 | c.955C>T | p.Gln319* | (Li, et al., 2010) | 0.0000041 | 0 | |||
| 6 | c.1008delA | p.Thr337Profs*12 | (Lamhonwah, et al., 2002) | |||||
| 6 | c.1043T>C | p.Ile348Thr | 57.69 | * | 0.0000496 | 0 | damaging | benign |
| 6 | c.1051T>C | p.Trp351Arg | 0.00 | (Wang, et al., 2000c) | damaging | damaging | ||
| 7 | c.1064C>T | p.Ser355Leu | 1.55 | (Li, et al., 2010) | 0.0000081 | 0 | benign | damaging |
| 7 | c.1072T>A | p.Tyr358Asn | 0.08 | (Li, et al., 2010) | 0.0000082 | 0 | damaging | damaging |
| 7 | c.1088T>C | p.Leu363Pro | 0.00 | (Akpinar, et al., 2010) | 0.0000041 | 0 | damaging | damaging |
| 7 | c.1161T>G | p.Tyr387* | (Tang, et al., 2002) | 0.0000323 | 0 | |||
| 7 | c.1175_1177del | p.Leu394del | 5.29 | * | 0.0000082 | 0 | / | damaging |
| 7 | c.1193C>T | p.Pro398Leu | 0.46 | (Amat di San Filippo, et al., 2006) | 0.0000989 | 0 | damaging | damaging |
| 7 | c.1195C>T | p.Arg399Try | 4.54 | (El-Hattab, et al., 2010) | 0.0000741 | 0 | damaging | damaging |
| 7 | c.1196G>A | p.Arg399Gln | 0.45 | (Wang, et al., 2001) | 0.0000162 | 0 | damaging | damaging |
| 7 | c.1202_1203insA | p.Tyr401* | (Lamhonwah, et al., 2002; Wang, et al., 1999) | 0.0000082 | 0 | |||
| 7 | c.1234A>G | p.Ser412Gly | 107.72 | * | benign | benign | ||
| IVS7 | c.1267+3_1267+23del | (Dobrowolski, et al., 2005) | ||||||
| 8 | c.1302delG | p.Gly435Alafs*24 | (Wang, et al., 1999) | |||||
| 8 | c.1316T>G | p.Val439Gly | 0.52 | * | damaging | damaging | ||
| 8 | c.1319C>T | p.Thr440Met | 0.00 | (Lamhonwah, et al., 2002) | 0.0000165 | 0 | damaging | damaging |
| 8 | c.1324_1325delGCinsAT | p.Ala442Ile | 14.83 | (El-Hattab, et al., 2010) | damaging | damaging | ||
| 8 | c.1327T>G | p.Phe443Val | 0.34 | (Li, et al., 2010) | damaging | benign | ||
| 8 | c.1336G>T | p.Val446Phe | 0.58 | (Mayatepek, et al., 2000) | 0.0000041 | 0 | damaging | damaging |
| 8 | c.1340A>G | p.Tyr447Cys | 0.00 | (Amat di San Filippo and Longo, 2004; Rahbeeni, et al., 2002) | damaging | damaging | ||
| 8 | c.1342G>T | p.Val448Leu | 10.62 | * | damaging | benign | ||
| 8 | c.1345T>G | p.Tyr449Asp | 16.56 | (Amat di San Filippo and Longo, 2004) | 0.0003247 | 1 | damaging | damaging |
| 8 | c.1354G>A | p.Glu452Lys | 2.14 | (Wang, et al., 2000a) | 0.0000494 | 0 | damaging | damaging |
| 8 | c.1364C>G | p.Pro455Arg | 0.18 | (Li, et al., 2010) | 0.0000072 | 0 | damaging | damaging |
| 8 | c.1372delG | p. Val458* | (Han, et al., 2014) | |||||
| 8 | c.1385G>T | p.Gly462Val | 2.80 | * | damaging | damaging | ||
| 8 | c.1392_1409delinsCA | p.Val465Thrfs*29 | * | |||||
| 8 | c.1400C>G | p.Ser467Cys | 16.56 | (Koizumi, et al., 1999) | 0.0001565 | 0 | damaging | damaging |
| 8 | c.1403C>G | p.Thr468Arg | 0.64 | (Lamhonwah, et al., 2002) | 0.0000081 | 0 | damaging | damaging |
| 8 | c.1409C>T | p.Ser470Phe | 0.00 | (Lamhonwah, et al., 2002) | 0.0000165 | 0 | damaging | damaging |
| 8 | c.1412G>C | p.Arg471Pro | 0.00 | (Filippo, et al., 2011) | damaging | damaging | ||
| 8 | c.1412G>A | p.Arg471His | 1.40 | (Spiekerkoetter, et al., 2003) | damaging | damaging | ||
| 8 | c.1427T>G | p.Leu476Arg | 0.00 | (Mutlu-Albayrak, et al., 2015) | damaging | damaging | ||
| 8 | c.1433C>T | p.Pro478Leu | 0.28 | (Tang, et al., 1999) | damaging | damaging | ||
| 8 | c.1441G>T | p.Val481Phe | 63.00 | (Amat di San Filippo, et al., 2008) | benign | benign | ||
| IVS8 | c.1451-1G>A | (Nezu, et al., 1999) | 0.0004777 | 0 | ||||
| 9 | c.1462C>T | p.Arg488Cys | 9.50 | (Schimmenti, et al., 2007) | 0.0000412 | 0 | damaging | damaging |
| 9 | c.1463G>A | p.Arg488His | 40.19 | (Amat di San Filippo, et al., 2006) | 0.0032620 | 4 | damaging | damaging |
| 9 | c.1520T>C | p.Leu507Ser | 4.85 | (Li, et al., 2010) | 0.0000323 | 0 | damaging | damaging |
| 9 | c.1556_1559dup | p.Ile521Hisfs*3 | (Schimmenti, et al., 2007) | |||||
| 10 | c.1645C>T | p.Pro549Ser | 23.79 | (Amat di San Filippo, et al., 2008; Urban, et al., 2006) | 0.0064990 | 28 | benign | benign |