Novel germline variants identified in the inner mitochondrial membrane transporter TIMM44 and their role in predisposition to oncocytic thyroid carcinomas

Abstract

Familial Non-Medullary Thyroid Carcinoma (fNMTC) represents 3-7% of all thyroid tumours and is associated with some of the highest familial risks among all cancers, with an inheritance pattern compatible with an autosomal dominant model with reduced penetrance. We previously mapped a predisposing locus, TCO (Thyroid tumour with Cell Oxyphilia) on chromosome 19p13.2, for a particular form of thyroid tumour characterised by cells with an abnormal proliferation of mitochondria (oxyphilic or oncocytic cells). In the present work, we report the systematic screening of 14 candidate genes mapping to the region of linkage in affected TCO members, that led us to identify two novel variants respectively in exon 9 and exon 13 of TIMM44, a mitochondrial inner membrane translocase for the import in the mitochondria of nuclear-encoded proteins. These variants were co-segregating with the TCO phenotype, were not present in a large group of controls and were predicted to negatively affect the protein (exon 9 change) or the transcript (exon 13 change). Functional analysis was performed in vitro for both changes and although no dramatic loss of function effects were identified for the mutant alleles, subtler effects might still be present that could alter Timm44 function and thus promote oncocytic tumour development. Thus we suggest that TIMM44 should be considered for further studies in independent samples of affected individuals with TCO.

Figure 1

TIMM44 conservation through species. Alignment of Timm44 proteins in different species generated using ClustalW (www.ebi.ac.uk/clustalw): the regions highly conserved are shown in black shades. The arrowhead shows the aa position corresponding to human P308.

Figure 2

Risk haplotype on chromosome 19. Pedigree and haplotype reconstruction across 19p13.2 in the family carrying the change C925A (P308Q) in TIMM44. In bold it is shown the location of the exon 9 variants in TIMM44.

Figure 3

Co-immunoprecipitation experiments for Timm44 P308Q variant. Western blot analysis for V5 tag specific for recombinant Timm44 of cell lysates (lanes 1–3) and immunoprecipitates (lanes 4–6) for GRP75 of HEK293T cells transfected with the plasmid carrying either the wild type (lanes 1 and 4) or the P308Q form (lanes 2 and 5) of Timm44 and the control HEK293 T not transfected (lanes 3 and 6).

Figure 4

Splicing analysis for TIMM44 exon 13 variant. (A) P1 pAltermax plasmid structure, showing the two synthetic exons interrupetd by an intronic sequence carrying the multiple cloning site. Abbreviation: P1-F and P1-R primers forward and reverse specific for the synthetic exons of the vector. In the box it is shown the genomic sequence including exon 13 inserted in the P1 vector. In bold it is shown the change G1276A; underlined it is shown the sequence of the reverse primer specific for human TIMM44 encompassing the TGA stop codon; underlined and hatched it is shown the sequence of primers used for cloning, with the restriction sites shaded in grey. (B) RT–PCR results using the P1-F primer and the human primer specific for TIMM44 on the cDNA from COS7 cells not transfected (lanes 1 and 5) transfected with the empty P1 vector (lanes 2 and 6), allele G-containing vector (lanes 3 and 7), allele A-containing vector (lanes 4 and 8). Lanes 1–4: no superscript in the cDNA reaction mix; lanes 5–8 superscript present in the cDNA reaction mix. The PCR products are correctly visible only in the RT+ lanes of cells transfected with the vectors carrying the two alleles (lanes 7 and 8).