(11) In contrast, a loss of SDHA
staining in tumor cells has been observed only in SDHA
(a) SDS-PAGE and Western blot analysis of mitochondrial respiratory chain complexes in BAT and liver homogenates by polyclonal antibodies against mGPDH and VDAC and monoclonal antibodies against representative subunits of NDH (subunit NDUFA9), SDH (subunit SDHA
), complex III (subunit UCQRC2), and actin.
The membrane was incubated with antibodies targeted against NDUFS4, Core 2, COX2, ATP5A1, and SDHA
. C1-C10 are normal controls.
Mitochondrial complexes II-V (I (subunit NDUFA9), II (subunit SDHA
), III (subunit UQCRC2), IV (subunit IV), and V (subunit ATP5A)) were elevated in GK as compared to Wistar SMCs, yet only complex V differed significantly (p < 0.05, Figure 2(c)).
Mutations involve exons 2, 3, 5, 6, 7, 8, 9, 10, 11, 13, 14 of SDHA
; exons 1, 2, 3, 4, 6, 7 of SDHB; exons 1, 4, 5 of SDHC; and exons 4 and 6 of SDHD.
Paragangliomas associated with a germline SDHA
mutation show negative staining with SDHA
as well as SDHB.
Germline mutations in SDHA
, SDHB, SDHC, and SDHD occur in some, but not all, patients with SDH-deficient GIST.
(1,3,19,20,32-36) While the value of systematic genetic screening for "sporadic" cases remains controversial, clinical features including family history, along with the biochemical and morphologic features (multifocality, adrenal medullary hyperplasia, thick capsule, clear cell morphology) and immunoprofile (loss of SDHB and SDHA
expression) (36,37) (see notes B, F, I and J), can provide important insight to determine which gene(s) should be screened preferentially in patients with pheochromocytomas and/or extra-adrenal paragangliomas.
SDH, also known as mitochondrial complex 2, is composed of several subunits (SDHA
, SDHB, SDHC, SDHD, and SDHAF2).
The primer sequences were as follows: PPAR[gamma] (Peroxisome proliferator-activated receptor gamma) forward primer: 5'- AGTGGGGATGTCTCATAATGCC-3', reverse primer: 5'- AGGTCAGCGGACTCTGGATTC-3'; 18S forward primer: 5'- TCCCCCAACTTCTTAGAGG-3', reverse primer: 5'-CTTATGACCCGCACTTACTG-3'; HPRT1 (Hypoxanthine Phosphoribosyltransferase1) forward primer: 5'-GGCGTCGTGATTAGTGATG-3', reverse primer: 5'- CAGAGGGCTACAATGTGATG-3 '; RPLP0 (Ribosomal protein lateral stalk subunit P0) forward primer: 5'- AACATCTCCCCCTTCTCCT-3', reverse primer: 5'- ACTCGTTTGTACCCGTTGAT-3'; SDHA
(Succinate dehydrogenase complex flavoprotein subunit A) forward primer: 5'- CCACCACTGCATCAAATTCATG-3', reverse primer: 5'-TGGGAACAAGAGGGCATCTG-3'.
Succinate dehydrogenase consists of four protein subunits: SDHA
to SDHD that are localized in the inner mitochondrial membrane and act at the interphase of the tricarboxylic acid cycle and electron transport chain.
The tumour cells showed loss of expression of SDHB (Figure 1) and retention of SDHA
expression suggesting an underlying SDHB, SDHC or SDHD mutation.