Abnormal biomarkers predict complex FAS or FADD defects missed by exome sequencing

November 16, 2023

Anne Rensing-Ehl 1, Myriam Ricarda Lorenz 2, Marita Führer 3, Wolfgang Willenbacher 4, Ella Willenbacher 5, Sieghart Sopper 6, Mario Abinun 7, Maria Elena Maccari 8, Christoph König 9, Pauline Haegele 10, Sebastian Fuchs 10, Carla Castro 10, Patrick Kury 10, Olivier Pelle 11, Christian Klemann 10, Maximilian Heeg 10, Julian Thalhammer 12, Oliver Wegehaupt 8, Marco Fischer 8, Sigune Goldacker 10, Björn Schulte 13, Saskia Biskup 13, Philippe Chatelain 13, Volker Schuster 14, Klaus Warnatz 15, Bodo Grimbacher 16, Andrea Meinhardt 17, Dirk Holzinger 18, Prasad Thomas Oommen 19, Tanja Hinze 20, Holger Hebart 21, Karlheinz Seeger 22, Kai Lehmberg 23, Timothy Ronan Leahy 24, Alexander Claviez 25, Simon Vieth 25, Freimut H Schilling 26, Ilka Fuchs 10, Miriam Groß 10, Frederic Rieux-Laucat 11, Aude Magerus 11, Carsten Speckmann 8, Klaus Schwarz 27, Stephan Ehl 10; ALPS study group: Ales Janda, Arnulf Pekrun, Bernd Buchholz, Catharina Schuetz, Charlotte M Niemeyer, Claas Hinze, Fabian Hauck, Carl Friedrich Classen, Gregor Dückers, Hedwig E Deubzer, Helmut Wittkowski, Henner Morbach, Horst von Bernuth, Jörg Leyh, Kaan Botzug, Kathrin Siepermann, Leo Kager, Linnea Schuez-Havupalo, Luis Ignacio Gonzalez Granado, Luis M Allende, Markus G Seidel, Martina Stiefel, Michael Albert, Nora Naumann-Bartsch, Peter Svec, Petr Smisek, Roman Crazzolara, Rosie Hague, Shahrzad Bakhtiar, Simone Storck, Sophie Hambleton, Stefan Schönberger, Thomas Kühne, Ulrich Baumann


Background: Elevated TCRαβ+CD4CD8 double-negative T-cells (DNT) and serum biomarkers help identifying FAS mutant patients with autoimmune lymphoproliferative syndrome (ALPS). However, in some patients with clinical features and biomarkers consistent with ALPS, germline or somatic FAS mutations cannot be identified upon standard exon sequencing (ALPS-undetermined: ALPS-U).

Objective: We aimed to explore whether complex genetic alterations in the FAS gene escaping standard sequencing or mutations in other FAS pathway-related genes could explain these cases.

Methods: Genetic analysis included whole FAS gene sequencing, copy number variation analysis and sequencing of FAS cDNA and other FAS pathway related genes. It was guided by FAS expression analysis on CD57+DNT, which can predict somatic loss-of-heterozygosity (sLOH).

Results: Nine of 16 ALPS-U patients lacked FAS expression on CD57+DNT predicting heterozygous “loss of expression” FAS mutations plus acquired somatic second hits in the FAS gene, enriched in DNT. Indeed, 7/9 analyzed patients carried deep intronic mutations or large deletions in the FAS gene combined with sLOH detectable in DNT, one patient showed a FAS exon duplication. Three patients had reduced FAS expression, two of them harbored mutations in the FAS promoter, which reduced FAS expression in reporter assays. Three of the four ALPS-U patients with normal FAS expression carried heterozygous FADD mutations with sLOH.

Conclusion: A combination of serum biomarkers and DNT phenotyping is an accurate means to identify patients with ALPS who are missed by routine exome sequencing.

Clinical implication: Detection of complex FAS pathway gene alterations by extended genetic analysis allows targeted therapy with sirolimus.

Keywords: ALPS; ALPS-U; DNT; FADD; FAS; double negative T-cells; genetic defect accumulation; loss of heterozygosity; promoter; somatic mutation.