Identification of GNAS Variants in Circulating Cell‐Free DNA from Patients with Fibrous Dysplasia/McCune Albright Syndrome

Abstract

Fibrous dysplasia/McCune-Albright syndrome (FD/MAS) is a rare mosaic bone and endocrine disorder. Although most variants affect the GNAS R201 codon, obtaining a genetic diagnosis is difficult because not all cells harbor the variant, and an invasive biopsy may be required. We explored the presence of GNAS p.R201 variants in blood circulating cell free DNA (ccfDNA) using sensitive techniques of digital droplet polymerase chain reaction (PCR) (ddPCR) and competitive allele-specific TaqMan PCR (castPCR) in an effort to improve the genetic diagnosis of FD/MAS. We isolated ccfDNA from the plasma of 66 patients with a wide range of disease severity and performed both ddPCR and castPCR mutation analysis to search for GNAS p.R201H or R201C variants. We detected R201 variants in ccfDNA samples of 41 of 66 (62.1%) patients by either castPCR or ddPCR, and 45 of 66 (68.2%) of patients if the techniques were combined. Variant detection was more likely in patients with more severe disease. Skeletal disease burden score (SBS) was significantly higher in patients who had detectable variants, and SBS was a predictor of variant allele frequency. By ddPCR analysis, patients aged ≤30 years had higher detection rates, and higher variant allele frequencies, independent of disease burden. We detected variant DNA in only one patient with monostotic FD by ddPCR only. In summary, we have demonstrated that ccfDNA containing variant GNAS can be isolated from the plasma of patients with FD/MAS and that ddPCR and castPCR methods have similar variant detection rates. This methodology represents an important potential advancement in diagnosis for patients with FD/MAS, especially those younger than 30 years or with more severe disease. Published 2023. This article is a U.S. Government work and is in the public domain in the USA.

Diagnostic journey for individuals with fibrous dysplasia / McCune albright syndrome (FD/MAS)

“…Contributing factors may include non-specific symptoms presenting to secondary care, lack of clinical knowledge, unclear referral pathways to expert centres and limited diagnostic laboratory tests for FD/MAS. The detection of the GNAS p.R201 variants in blood circulating cell free DNA has been shown to be effective for diagnosis of FD/MAS for those with endocrinopathy or high skeletal burden score [ 21 ]. However, the diagnostic uncertainty is higher in those with monostotic or craniofacial lesions, where this test is less sensitive.…”

Choosing the Best Tissue and Technique to Detect Mosaicism in Fibrous Dysplasia/McCune–Albright Syndrome (FD/MAS)

“…Genetically, FD/MAS is mainly associated with variants occurring in the Arginine in position 201 (R201) of the Gsα protein [18], which can be substituted by two different amino acids: Cysteine (NM_000516:c.601C>T, p.R201C) or Histidine (NM_000516:c.602G>A, p.R201H) [19]. A mutation in the 227 codon replacing Glutamine with Lysine (NM_000516:c.680A>T, p.Q227L) [19] has been identified in approximately 5% of patients [20].…”

“…In this case, specific variants are interrogated and can detect even very-low-percentage mosaics [27]. This has been used to identify mosaicism in several diseases [36][37][38], including in FD/MAS [19,39]. In other rare diseases, quantitative real-time PCR (qRT-PCR) has also been used for the detection of mosaicism [40].…”

Aplicabilidade da reação em cadeia da polimerase digital em gotas e do sequenciamento paralelo em larga escala no diagnóstico genético da Síndrome de McCune-Albright

“…Apesar da taxa de detecção da mutação no GNAS ser maior quando se estuda o DNA de tecidos acometidos (45), a disponibilidade destas amostras é limitada. A detecção da mutação no GNAS em DNA de leucócitos periféricos tornou-se uma realidade a partir do desenvolvimento de técnicas mais sensíveis, como a ddPCR e o SPLE (48,49,51).…”

“…No entanto o valor foi próximo (gotas totais 9.456), apresentando gotas positivas com boa amplitude de sinal. Alguns estudos consideram a amostra inválida somente quando a formação de gotas é abaixo de 8.000 (51).…”