In monoclonal gammopathies, patients are characterized by a unique M-protein, produced by a B cell or a plasma-cell clone, that can be used as a specific tumor biomarker and can be involved in organ damage pathogenesis. In our laboratory, was recently established a novel Next Generation Sequencing (NGS)-based technique, named Single Molecule Real-Time Sequencing of the M protein (SMaRT M-Seq), which enables unbiased sequencing of the full-length variable region of the expressed M proteins in a high throughput manner. Since its publication in 2022, the cohort of sequenced patients has extensively expanded and actually accounts for 431 patients with monoclonal gammopathies, mostly diagnosed with AL amyloidosis (n=259) and multiple myeloma (MM, n=148), but including also few other monoclonal gammopathies (n=24), such as monoclonal gammopathy of undetermined significance (MGUS), Waldenström's macroglobulinemia (WM), IgM-related neuropathy, light chain deposition disease (LCDD), and crystal cryoglobulinemia. A total of 519 samples were subjected to SMaRT M-Seq sequencing: among these, 334 samples were derived from bone marrow aspirates (BM), 9 samples were derived from peripheral blood (PB), and 88 samples from matched BM and PB samples. Initially designed for light chain sequencing, SMaRT M-Seq was later employed to also study heavy chains, obtaining a total of 462 sequences from 519 samples: 234 sequences for λ and 194 sequences for κ light chains, 23 sequences for γ, 8 sequences for µ, and 3 sequences for α heavy chains. Validation of the sequence accuracy was performed on 235 patients, integrating SMaRT M-Seq data with Sanger sequencing, biochemical and mass spectrometry (MS) analyses on biological samples from patients with monoclonal gammopathies, including BM-MNC, fat biopsies, urines, and sera. In all except three patients, a clearly dominant unique light chain sequence was identified. SMaRT M-Seq in PB, eventually combined with MS-based analysis of the urinary proteome, allows the identification of the full-length clonal light chain sequence in 98% of patients with monoclonal gammopathies without the need of BM studies. The clonal immunoglobulin light chain sequences obtained through SMaRT M-Seq were used also to investigate the presence of the N-glycosylation in κ light chains, and in silico prediction of the light chain toxicity. This analysis showed a significantly higher rate of N-glycosylation in κ light chains for patients with AL amyloidosis compared to patients with MM, with a preferential localization of the predicted N-glycosylation site in the DE loop of the framework region 3. These observations confirmed the existence of an N-glycosylation hotspot in κ light chain associated with AL amyloidosis, which was recently described by our laboratory. In conclusion, sequencing methods providing the knowledge of the full-length sequence of the M protein.
Le gammapatie monoclonali sono caratterizzate dalla presenza di un’immunoglobulina unica, paziente-specifica, chiamata componente monoclonale, prodotta da un clone di linfociti B o plasmacellule, che può essere utilizzata come biomarcatore tumorale specifico e può essere coinvolta nella patogenesi del danno d'organo. Nel nostro laboratorio, è stata recentemente sviluppata una tecnica basata sul sequenziamento di nuova generazione (Next Generation Sequencing, NGS), denominata Single-Molecule Real-Time Sequencing of the M protein (SMaRT M-Seq), per identificare in modo inequivocabile l’intera sequenza variabile della catena leggera delle immunoglobuline clonali con elevato throughput. Dalla pubblicazione originale di questa metodica nel 2022, la coorte di pazienti sequenziati mediante SmaRT M-Seq è notevolmente aumentata e comprende 431 pazienti, principalmente pazienti con diagnosi di amiloidosi AL (n=259), mieloma multiplo (MM, n=148), ma anche pazienti affetti da altre gammapatie monoclonali (n=24), quali gammapatia monoclonale di significato indeterminato (Monoclonal gammopathy of undetermined significance, MGUS), macroglobulinemia di Waldenström (WM), neuropatia associata a IgM, malattia da deposito delle catene leggere (Light Chain Deposition Disease, LCDD) e cristalcrioglobulinemia. Un totale di 519 campioni sono stati sottoposti al sequenziamento con SMaRT M-Seq: tra questi, 334 campioni di aspirato midollare (BM), 9 campioni da sangue periferico (PB) e 88 coppie di campioni di midollo osseo e sangue periferico. Inizialmente progettato per il sequenziamento delle catene leggere, SMaRT M-Seq è stato successivamente impiegato anche per studiare le catene pesanti delle immunoglobuline, ottenendo un totale di 462 sequenze dai 519 campioni analizzati. La validazione dell'accuratezza delle sequenze ottenute da SMaRT M-Seq è stata effettuata su 235 pazienti, integrando i dati ottenuti con SMaRT M-Seq con dati ottenuti da sequenziamento con metodica Sanger, da analisi biochimiche e di spettrometria di massa effettuate su cellule mononucleate derivate da midollo osseo, biopsie di grasso periumbelicale, campioni di urine e siero. In tutti i pazienti, eccetto tre, è stata identificata un’unica sequenza di catena leggera immunoglobulinica chiaramente dominante. SMaRT M-Seq effettuato su campioni di sangue periferico, eventualmente combinato con l'analisi del proteoma urinario tramite spettrometria di massa, consente l'identificazione della sequenza completa della catena leggera dell’immunoglobulina clonale nel 98% dei pazienti con gammapatie monoclonali senza la necessità di studi sul midollo osseo. Le sequenze clonali delle catene leggere immunoglobuliniche ottenute tramite SMaRT M-Seq sono state inoltre impiegate per indagare la presenza della N-glicosilazione nelle catene leggere κ, e in studi in silico per la predizione di tossicità della sequenza della catena leggera. Questa analisi ha mostrato un tasso significativamente più elevato della presenza della N-glicosilazione nelle catene leggere κ dei pazienti affetti da amiloidosi AL rispetto a quelli con mieloma multiplo, con una localizzazione preferenziale del sito di N-glicosilazione nel braccio DE della regione framework region 3 (FR3). In conclusione, metodi di sequenziamento che consentono di conoscere la sequenza intera della componente monoclonale.
The Pavia Amyloidosis Research and Treatment Center’s experience in clonal immunoglobulin gene sequencing through SMaRT M-Seq
PANNO, GIADA
2023/2024
Abstract
In monoclonal gammopathies, patients are characterized by a unique M-protein, produced by a B cell or a plasma-cell clone, that can be used as a specific tumor biomarker and can be involved in organ damage pathogenesis. In our laboratory, was recently established a novel Next Generation Sequencing (NGS)-based technique, named Single Molecule Real-Time Sequencing of the M protein (SMaRT M-Seq), which enables unbiased sequencing of the full-length variable region of the expressed M proteins in a high throughput manner. Since its publication in 2022, the cohort of sequenced patients has extensively expanded and actually accounts for 431 patients with monoclonal gammopathies, mostly diagnosed with AL amyloidosis (n=259) and multiple myeloma (MM, n=148), but including also few other monoclonal gammopathies (n=24), such as monoclonal gammopathy of undetermined significance (MGUS), Waldenström's macroglobulinemia (WM), IgM-related neuropathy, light chain deposition disease (LCDD), and crystal cryoglobulinemia. A total of 519 samples were subjected to SMaRT M-Seq sequencing: among these, 334 samples were derived from bone marrow aspirates (BM), 9 samples were derived from peripheral blood (PB), and 88 samples from matched BM and PB samples. Initially designed for light chain sequencing, SMaRT M-Seq was later employed to also study heavy chains, obtaining a total of 462 sequences from 519 samples: 234 sequences for λ and 194 sequences for κ light chains, 23 sequences for γ, 8 sequences for µ, and 3 sequences for α heavy chains. Validation of the sequence accuracy was performed on 235 patients, integrating SMaRT M-Seq data with Sanger sequencing, biochemical and mass spectrometry (MS) analyses on biological samples from patients with monoclonal gammopathies, including BM-MNC, fat biopsies, urines, and sera. In all except three patients, a clearly dominant unique light chain sequence was identified. SMaRT M-Seq in PB, eventually combined with MS-based analysis of the urinary proteome, allows the identification of the full-length clonal light chain sequence in 98% of patients with monoclonal gammopathies without the need of BM studies. The clonal immunoglobulin light chain sequences obtained through SMaRT M-Seq were used also to investigate the presence of the N-glycosylation in κ light chains, and in silico prediction of the light chain toxicity. This analysis showed a significantly higher rate of N-glycosylation in κ light chains for patients with AL amyloidosis compared to patients with MM, with a preferential localization of the predicted N-glycosylation site in the DE loop of the framework region 3. These observations confirmed the existence of an N-glycosylation hotspot in κ light chain associated with AL amyloidosis, which was recently described by our laboratory. In conclusion, sequencing methods providing the knowledge of the full-length sequence of the M protein.File | Dimensione | Formato | |
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Thesis Panno Giada.pdf
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Descrizione: We aimed to perform a comprehensive analysis of the Pavia Amyloidosis Center's experience in sequencing clonal light and heavy chains from BM and PB samples using SMaRT M-Seq, describing the data of a cohort of 431 monoclonal gammopathies patients.
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https://hdl.handle.net/20.500.14239/28297