Supplementary Material
CDC73 mutations and parafibromin immunohistochemistry in parathyroid
tumors: Clinical correlations in a single-centre patient cohort
Vito Guarnieri, Claudia Battista, Lucia Anna Muscarella, Michele Bisceglia, Danilo de
Martino, Filomena Baorda, Evaristo Maiello, Leonardo D’Agruma, Iacopo Chiodini,
Celeste Clemente, Salvatore Minisola, Elisabetta Romagnoli, Sabrina Corbetta, Raffaella
Viti, Cristina Eller-Vainicher, Anna Spada, Michela Iacobellis, Nazzarena Malavolta,
Massimo Carella, Lucie Canaff, Geoffrey N. Hendy, David E.C. Cole, Alfredo Scillitani
Correspondence: Alfredo Scillitani,
Endocrinology Unit, Ospedale “Casa Sollievo della Sofferenza”, Istituto di Ricovero e Cura a
Carattere Scientifico, 71013 San Giovanni Rotondo (Foggia), Italy
email: alscill@tin.it Tel: ++390882410625; Fax: ++390882410627
Supplementary Fig. 1
Analysis of allelic imbalance (loss of heterozygosity or LOH) at the CDC73 locus. Shown at the
top is a physical map of the locus at 1p31.2, and location of LOH markers.
Supplementary Methods
LOH analysis was performed using the following markers flanking CDC73 [S1-S3]: D1S428,
D1S412, D1S413, D1S2840 and D1S2794, as well as one intragenic intron 10 marker [S4]. PCR
was performed in a 50 L reaction volume containing 5 L 10X PCR Buffer (Eppendorf), 0.25
nM dNTPs, 20 pmol of each primer, 1 U HotMaster Taq (Eppendorf) and 100 ng of DNA.
Cycling conditions consisted of 40 cycles of the LMS2 (ABI Prism Linkage Mapping Set
version 2) method performed on a GeneAmp PCR System 9700 (Applied Biosystems, Foster
City, CA) for all markers.
After capillary array electrophoresis and data reduction (ABI GeneScan and Genotyper
Software 3.7 NT, Applied Biosystems, Foster City, CA), LOH was determined by allelic
imbalance (AI) scoring. The AI was quantitated using the formula: (peak 1 height/peak2 height
in tumour DNA)/(peak 1 height/peak 2 height in normal DNA), which is a more conservative
criterion than sometimes suggested [S5]. LOH was assigned if the AI was 0.5 or less, while an
AI between 0.5 and 0.65 was defined as borderline. ROH = Retention of Heterozygosity; NI =
not informative.
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