Carnitine biosynthesis and homeostasis in humans. Carnitine is synthesized in four enzymatic steps. After release of TML by lysosomal protein degradation, this compound is hydroxylated by TMLD, producing HTML. HTML is cleaved by HTML aldolase (HTMLA) into TMABA and glycine. Subsequently, TMABA is oxidized by TMABA-dehydroxygenase (TMABA-DH) to form 4-N-trimethylaminobutyrate, also named γ-butyrobetaine (γBB). Finally, γBB is hydroxylated by γBBD, yielding l-carnitine. Because TMLD is located in mitochondria, TML needs to be transported out of the lysosome and across the inner mitochondrial membrane into the mitochondrial matrix by means of transporters, which are unknown at this time. Depending on the subcellular localization of the HTLMA (also uncertain, likely the cytosol), HTML or γBB needs to be transported back to the cytosol (where γBBD is located). In cells that do not contain γBBD, γBB is exported from the cell and imported into tissues (liver, kidney, and brain in humans) that do express γBBD by means of at least one specific transporter, presumably SLC6A13. Carnitine is transported by OCTN2 and other lower affinity transporters (not shown).

Exon 2-containing deletions found in TMLHE and structure of TMLHE. (A) Array CGH showing eight exonic deletions in TMLHE, with the relative location of TMLHE exons 2–6 aligned above the array CGH plots (GRCh37/hg19 assembly; http://genome.ucsc.edu). The horizontal axis shows chromosome position, and the vertical axis shows the log₂ ratio of array signal. Semitransparent filled boxes on CGH plots highlight the regions of aberration; all samples have deletions involving exon 2, and most have a separate deletion in intron 1 (red circle). All samples are autism probands with identifiers found in Tables S1, S4, and S5. (B) Twenty-four unrelated individuals with deletions involving exon 2 of TMLHE are mapped. Deletion coordinates were determined by array CGH unless specified by PCR assay. White arrowheads indicate the continuation of the deletion for SSC 13928.p1. NA 12003 is an unaffected individual whose deletion is published (38) and was better characterized in this study. BPR 664 is an unaffected individual. (C) Diagram of gene structure. Large open arrows represent near-identical inverted repeats. Fa, father; P1, proband; HI, AGRE individuals; #, individual first described by Celestino-Soper et al. (10).

Genetic and enzymatic characterization of hemizygous deletion of exon 2. (A) TMLD activity measured in lymphoblast homogenates of three families with exon 2 deletion. (B) PCR assay results for the AU 0177 family showing the deletion in the two affected brothers (1, 2) and in the mother (3) but not in the father (4), unaffected maternal half-brother (5), or unaffected controls (C1 and C2). There is bias of amplification in the mother, such that the normal band is faint. dl, deletion; nl, normal. (C) (Upper) TMLD activity and Western blot analysis of 2 individuals with exon 2 deletion (P1, HI0690; P2, BPR664) and three controls (C1–C3). Purified TMLD (pTMLD) is used as a positive control. (Lower) Western blot analysis of 2 individuals (P1 and P2) with (+) or without (−) addition of pTMLD, showing the complete absence of protein in cases of exon 2 deletion and confirmation of the identity of the immunoreactive material as TMLD. The upper band in the Western blot is an irrelevant protein. (D) (Upper) TMLD activity measured in lymphoblast homogenates from several autism males. *,TMLHE exon 2 deletion; #, E287K; d, deletion in intron 1 in 13 individuals; −, no deletion in intron 1 in 9 individuals. SSC 12353.p1 was not tested for the presence of intron 1 deletion. (Lower) TMLD activity measured in lymphoblast homogenates from male controls. BPR indicates local unaffected controls, and NA 12003 is an unaffected individual. SSC 12353.fa was not tested for presence of intron 1 deletion. There was no apparent correlation of the level of enzyme activity with the presence or absence of the intronic deletion. For A, C, and D, assays were run in duplicate and the average is plotted without error bars. fa, father; mo, mother; p1, proband.

Increased TML and decreased HTML and γBB in patients with TMLHE exon 2 deletions. (A) Bar diagram of concentrations (mean ± SD) of carnitine biosynthesis intermediates in urine of two patients with exon 2 deletion (1, HI0690; 2, HI0691) compared with controls. (B) Box and whisker plot of carnitine biosynthesis intermediates in plasma of seven patients with exon 2 deletion (HI0690, HI0691, SSC 13928.p1, SSC 13489.p1, SSC 11000.p1, SSC 11229.p1, and SSC 11680.p1) compared with controls. (C) Box and whisker plot showing the diagnostic potential of the (HTML + γBB)/HTML ratio as an indicator of TMLHE deficiency. All seven patients have a very low ratio. The black square represents the mean of the controls, and whiskers show the minimum and maximum values of the control group.