Analysis of the chromosome X exome in patients with autism spectrum disorders identified novel candidate genes, including TMLHE

Abstract

The striking excess of affected males in autism spectrum disorders (ASD) suggests that genes located on chromosome X contribute to the etiology of these disorders. To identify new X-linked genes associated with ASD, we analyzed the entire chromosome X exome by next-generation sequencing in 12 unrelated families with two affected males. Thirty-six possibly deleterious variants in 33 candidate genes were found, including PHF8 and HUWE1, previously implicated in intellectual disability (ID). A nonsense mutation in TMLHE, which encodes the ɛ-N-trimethyllysine hydroxylase catalyzing the first step of carnitine biosynthesis, was identified in two brothers with autism and ID. By screening the TMLHE coding sequence in 501 male patients with ASD, we identified two additional missense substitutions not found in controls and not reported in databases. Functional analyses confirmed that the mutations were associated with a loss-of-function and led to an increase in trimethyllysine, the precursor of carnitine biosynthesis, in the plasma of patients. This study supports the hypothesis that rare variants on the X chromosome are involved in the etiology of ASD and contribute to the sex-ratio disequilibrium.

Figures

Figure 1

Strategy used for the selection of rare and possibly deleterious variants. Data from NGS and single nucleotide polymorphism (SNP) arrays were combined to conserve only variants located in X regions shared by the affected sibs (families 1–11). Further filters included a minor allele frequency (MAF) in silico predictions compatible with an effect of the variant on the gene or the protein (nonsense variants, missense variants with at least one prediction in silico by SIFT (scale-invariant feature transform) or Polyphen-2 that it is deleterious and synonymous, intronic or 5–3′UTR variants with possible effects on splice sites or promoters using Alamutv2.1/AlamutHT). For variants present in at least two index cases, only those that segregated in all affected members of all families were conserved. For one family (family 12), microarray data were unavailable for the affected uncle; segregation of variants found in the index case was performed at a later time.

Figure 2

Identification of variants in PHF8 and HUWE1 in families 8 and 4.(a) Pedigree of family 8 and segregation analysis of the p.Ser969del variant inPHF8. The arrow indicates the index case. (b) Sequence electropherograms showing the presence of the p.Ser969del variant at the hemizygous state in the two affected brothers and at the heterozygous state in their mother. (c) Alignment of the region flanking the variant in orthologous proteins, showing the high conservation of Serine 969. (d) Pedigree of family 4 and haplotypes reconstructed from eight informative single nucleotide polymorphisms (SNPs) adjacent to HUWE1 (genotypes of these SNPs were obtained from Illumina cytoSNP-12 arrays analysis), showing that the same maternal haplotype was transmitted to the affected brothers with and without the p.Val950Asp mutation. The arrow indicates the index case. (e) Sequence electropherograms showing the presence of p.Val950Asp in the index case and its absence in the affected brother and in the mother. These results are consistent with the de novo occurrence of p.Val950Asp in the index case. (f) Alignment of the region flanking the variant in orthologous proteins, showing the high conservation of valine 950.

Figure 3

Identification of TMLHE mutations in three families. (a) Pedigrees and segregation analysis of the TMLHE mutations in families 9, PED-804 and AU-205. The arrows indicate the index cases. (b) Sequence electropherograms of the mutations at the hemizygous state in the index cases (835–03 in family 9, 804–03 and 205–03) and the affected brother of family 9 (835–04), and at the heterozygous state in the mothers (835–02, 804–02 and 205–02). (c) Analysis of TMLHE mRNA in lymphoblasts from members of family 9 and schematic representations of the splicing isoforms detected in subjects with the p.Arg77X mutation in exon 3. Reverse transcriptase–PCR products using primer pairs in exons 2 and 4, run on 2% agarose gels, showed two mRNA isoforms in the index case (835–03), his affected brother (835–04) and his mother (835–02) and a single isoform in a control subject (c). Sequence analysis confirmed that the long isoform contains the premature termination codon in exon 3 and that exon 3 was skipped in the short isoform, probably as a consequence of nonsense-associated alternative splicing. (d) Alignment of the region flanking the two missense variants in orthologous proteins showing the conservation of the altered amino acids. (e) Quantification of TMLHE mRNA expression in fibroblasts (F) and lymphoblasts (L) from members of family 9 by quantitative real-time PCR, using primer pairs in exons 7 and 8. TMLHE mRNA was expressed 10 times less in patients compared with healthy controls (green bars). Overnight treatment with 10 μg ml−1 emetin (blue bars), an inhibitor of nonsense-mediated decay, restored the expression of the TMLHE mRNA. (f) Assay of free carnitine by UPLC (ultra performance liquid chromatography) chromatographic and TQD (tandem quadrupole detector) mass spectrometry in the plasma of patients. (g) Assay of trimethyllysine (TML) by UPLC chromatographic and TQD mass spectrometry showing a 2–3-fold increase in the plasma of patients.