Kinga Németh1, Ottó Darvasi2, István Likó2, Nikolette Szücs1, Sándor Czirják3, Lilla Reiniger4, Borbála Szabó5, Péter Igaz1, Attila Patócs2,5, Henriett Butz2,5
1 2nd Department of Medicine, Faculty of Medicine, Semmelweis University, Budapest
2 MTA-SE „Lendulet” Hereditary Endocrine Tumors Research Group, Hungarian Academy of Sciences and Semmelweis University, Budapest
3 National Institute of Clinical Neurosciences, Budapest
4 1st Department of Pathology and Experimental Cancer Research, Semmelweis University, Budapest
5 Department of Laboratory Medicine, Semmelweis University, Budapest
Mitochondrial genome variants have been assessed only in pituitary oncocytomas but not in other subtypes of pituitary tumors. Mitochondria sequencing can be challenging because of multiple copies in a single cell (called heteroplasmy) and the presence of different mitochondrial pseudogenes encoded in the nuclear genome). Recently next generation sequencing (NGS) offers a new opportunity in investigation of mitochondrial genome variants. Due to high coverage the relative low copies can also be detected.
To apply NGS technology in detection of mitochondrial genome variants in various pituitary adenoma tumours.
22 gonadotroph, 11 growth hormone producing, and 11 hormone-immunonegative pituitary adenoma samples were analysed. After DNA isolation, sequencing library was prepared using VariantPro™ Amplicon Mitochondrion Panel kit and total mitochondrial genome was sequenced on Illumina MiSeq Instrument. Complex bioinformatical and statistical analysis followed by Sanger sequencing validation was carried out.
High total coverage and coverage depth (630 ±370 reads; avg±SE) was achieved. Parallel runs of the same samples showed high correlation. 496 mitochondrial variants were identified. The heteroplasmy was 7.22%. No adenoma type specific and no variants associating with Ki-67 index or recurrent-nonrecurrent status was identified. Four variants were more common in hormone-immunonegative than in gonadotroph adenomas. ChrM_14798, chrM_4216, chrM_15452 were non-synonymous polymorphisms leading to amino acid change in MT-CYB (mitochondrially encoded cytochrome b) and in MT-ND1 (mitochondrially encoded NADH dehydrogenase 1) genes. ChrM_16189 was however a non-protein coding variant which apeared in 40% of nonrecurrent adenomas compared to recurrent ones (6/15 vs. 0/11) (p=0.02).
We were able to validate chrM_14798 and chrM_4216 variants by Sanger sequencing with 100% sensitivity and specificity on 44 samples.
NGS is a reliable method for investigating mitochondrial genome mutations in pituitary tumours. Low level of heteroplasmy and poor associations between variants and clinico-pathological features suggests that mitochondrial genome alterations may not play a central role in pathogenesis of pituitary adenomas.
Doctoral school: Clinical Medicine
Program: Hormonal Regulations
Supervisor: Henriett Butz
Email address: firstname.lastname@example.org