Date of Award

1-1-2018

Document Type

Thesis

Degree Name

Medical Doctor (MD)

Department

Medicine

First Advisor

Dennis D. Spencer

Second Advisor

Maya B. Lodish

Abstract

Abstract

Historical collections of biological specimens can be a rich resource for molecular genetics studies. In this paper, we test four commercial DNA extraction kits to assess the feasibility of extracting amplifiable genomic DNA from brains fixed in formalin for over a century from neurosurgeon Harvey Cushing’s Tumor collection. From a cohort of 24 patients with pituitary adenomas operated on by neurosurgeon Dr. Harvey Cushing (1869-1939) between 1913-1932 whose brains and tumors are housed at the Yale School of Medicine, we identified nine with clinical and histological signs of acromegaly, Carney complex (CNC), and/or Cushing’s syndrome using surgical charts and records from the Peter Bent Brigham Hospital and neuropathologist Louise Eisenhardt’s (1891-1967) “Black Book”. Tissue samples were taken from the hypothalamus, thalamus, or pituitary tumor of relevant patients. Following DNA extraction, sequencing for mutations in genes of interest associated with acromegaly, Carney complex, and/or Cushing’s syndrome, PRKAR1A, AIP, USP8, GNAS1 and GPR101, was attempted in order to explore the possibility that mutations could be identified. Focusing on brain tissue from individuals with secreting pituitary adenomas, we extracted DNA under different conditions, altering variables such as tissue digestion time, RNase treatment, and extraction buffer composition. A nested PCR approach was used to amplify and sequence DNA. The ZymoResearch formalin-fixed, paraffin embedded (FFPE) kit resulted in amplifiable DNA that could be Sanger sequenced. The quality of the DNA was poor in most samples, thus rendering the full sequencing of large genes and gene dosage analysis challenging. Nevertheless, here we also present the case of GBS (1879-1914), first reported by Dr. Cushing, whose clinical symptoms we now realize fit the diagnostic criteria of CNC. GBS was found to carry a novel p.Arg74His mutation in the PRKAR1A gene, in a codon that was previously described as mutated in CNC, but with a different amino acid change. Patient GBS is the oldest known molecularly confirmed individual with CNC. Our study, thus, demonstrates the power of modern genetics in studying archived tissues, the feasibility of extracting DNA of high enough quality from formalin preserved tissue under suboptimal conditions to perform genomic studies using a commercially available kit, and the importance of recording detailed clinical notes in the diagnosis of disease.

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