Powered by EHA Article OPEN ACCESS Next-generation Sequencing in Bone Marrow Failure Syndromes and Isolated Cytopenias: Experience of the Spanish Network on Bone Marrow Failure Syndromes Eva Gálvez1,2, Elena Vallespín3,4, Elena G. Arias-Salgado4, Carmen Sánchez-Valdepeñas1, Yari Giménez2,5, Susana Navarro2,5, Paula Río2,5, Massimo Bogliolo2,6,7, Roser Pujol2,6,7, Montserrat Peiró2,6,7, Julián Nevado2,3,8, Josune Zubicaray1, Elena Sebastián1, Albert Catalá2,9,10, Cristina Beléndez2,10,11, Cristina Díaz de Heredia10,12,13, Ana Galera10,14, Isabel Badell2,15, Luis Madero1, Rosario Perona2,4, Leandro Sastre2,4, Jordi Surrallés2,6,7,16, Juan Bueren2,5, Pablo Lapunzina2,3,8, Julián Sevilla1,2,10 Correspondence: Eva Gálvez (eva.galvez@salud.madrid.org). Abstract Inherited bone marrow failure syndromes (IBMFSs) are a group of congenital rare diseases characterized by bone marrow failure, congenital anomalies, high genetic heterogeneity, and predisposition to cancer. Appropriate treatment and cancer surveillance ideally depend on the identification of the mutated gene. A next-generation sequencing (NGS) panel of genes could be 1 initial genetic screening test to be carried out in a comprehensive study of IBMFSs, allowing molecular detection in affected patients. We designed 2 NGS panels of IBMFS genes: version 1 included 129 genes and version 2 involved 145 genes. The cohort included a total of 204 patients with suspected IBMFSs without molecular diagnosis. Capture-based targeted sequencing covered > 99% of the target regions of 145 genes, with more than 20 independent reads. No differences were seen between the 2 versions of the panel. The NGS tool allowed a total of 91 patients to be diagnosed, with an overall molecular diagnostic rate of 44%. Among the 167 patients with classified IBMFSs, 81 patients (48%) were diagnosed. Unclassified IBMFSs involved a total of 37 patients, of whom 9 patients (24%) were diagnosed. The preexisting diagnosis of 6 clinically classified patients (6%) was amended, implying a change of therapy for some of them. Our NGS IBMFS gene panel assay is a useful tool in the molecular diagnosis of IBMFSs and a reasonable option as the first tier genetic test in these disorders. Introduction and Shwachman-Diamond syndrome (SDS) being among the Inherited bone marrow failure syndromes (IBMFSs) are a most common in this group of syndromes.3 group of congenital rare diseases with high genetic heterogene- The correct genetic diagnosis of IBMFSs is crucial to pre- ity that are mainly characterized by bone marrow failure, phe- dict the disease course, to provide genetic counseling, and to notypic findings, and cancer predisposition.1,2 A large number of select the most appropriate treatment, including hematopoietic IBMFSs have been described so far, with Fanconi anemia (FA), stem cell transplantation (HSCT) from healthy donors. Many dyskeratosis congenita (DC), Diamond-Blackfan anemia (DBA), patients with IBMFSs remain underdiagnosed, mainly because 1 Servicio de Hematología y Oncología Pediátrica, Fundación de Investigación 13 Vall d’Hebron Institut de Recerca, Barcelona, Spain Biomédica, Hospital Infantil Universitario Niño Jesús, Madrid, Spain 14 Hospital Universitario Virgen de la Arrixaca, Murcia, Spain 2 Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), 15 Unidad Pediátrica de Hematología, Oncología y TPH, Hospital de la Santa Creu ISCIII, Madrid, Spain i Sant Pau, Barcelona, Universidad Autònoma de Barcelona, Institut de Recerca 3 Institute of Medical and Molecular Genetics (INGEMM), Hospital Universitario La del Hospital Sant Pau, Barcelona, Spain Paz, IDIPAZ, Madrid, Spain Servicio de Genética, Hospital de la Santa Creu i Sant Pau, Barcelona, 16 4 Instituto de Investigaciones Biomédicas CSIC/UAM, IDIPaz, Madrid, Spain Universidad Autònoma de Barcelona, Institut de Recerca del Hospital Sant Pau, 5 Hematopoietic Innovative Therapies Division, Centro de Investigaciones Barcelona, Spain. Energéticas, Medioambientales y Tecnológicas, Madrid, Spain 6 Unidad de Medicina Genómica, Instituto de Investigaciones Biomédicas, Supplemental digital content is available for this article. Hospital de la Santa Creu i Sant Pau, Barcelona, Spain Copyright © 2021 the Author(s). Published by Wolters Kluwer Health, Inc. 7 Departamento de Genética y Microbiología, Universidad Autonoma de on behalf of the European Hematology Association. This is an open-access Barcelona, Spain article distributed under the terms of the Creative Commons Attribution-Non 8 ITHACA, European Reference Network Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible 9 Servicio de Hematología y Oncología, Hospital Sant Joan de Déu, Barcelona, Spain to download and share the work provided it is properly cited. The work cannot be 10 Grupo insuficiencias medulares de la SEHOP, Spain changed in any way or used commercially without permission from the journal. 11 Servicio de Hematología y Oncología, Hospital Gregorio Marañón, Madrid, Spain HemaSphere (2021) 5:4(e539). http://dx.doi.org/10.1097/ 12 Servicio de Hematología y Oncología Pediátrica, Hospital Vall d’Hebrón, HS9.0000000000000539. Barcelona, Spain Received: 14 September 2020 / Accepted: 11 January 2021 1 Gálvez et al Next-generation Sequencing in BMFS and Isolated Cytopenias they are characterized by a wide range of syndromes and, other IBMFSs. Depending on the clinical suspicion, the patients at the same time, have a high degree of phenotypic overlap. were divided into 2 groups: group 1 included those referred for Consequently, despite having an adequate clinical orientation a suspected classified IBMFS (DBA, FA, DC, SDS, HT, SCN, or based on the patient’s medical history and examination, it is MDS; Table 1) and group 2 included all those referred for unclas- possible that no conclusive diagnosis can be made in many of sified IBMFSs (other cytopenia and other IBMFSs; Table 2). these patients. Until recently, molecular diagnosis in clinical practice was limited to the conventional sequencing of the most frequently NGS gene panel assay mutated genes, using techniques such as Sanger sequencing, DNA was extracted from peripheral blood following rou- based on clinical suspicion. However, nowadays, the large num- tine methods. We designed an NGS assay for a comprehensive ber of genes associated with IBMFSs makes mutation screening panel of 145 IBMFS genes published as of September 2016. We based on conventional techniques difficult, since gene-to-gene devised 2 versions of the panel: version 1 (v1) included a total study strategies are expensive and slow.4 On the other hand, this of 129 IBMFS genes and version 2 (v2) was updated to include approach is not applicable to disorders whose phenotype-based 16 further genes (Supplementary Table S2, http://links.lww.com/ diagnosis is not straightforward and for which there is no obvi- HS/A135). Detailed panel design and bioinformatic analysis are ous candidate gene. presented in Additional file 1: Supplemental Material (http:// The recent advent of next-generation sequencing (NGS) tech- links.lww.com/HS/A135). niques has begun to revolutionize the field of genetic diagnosis and is rapidly becoming established in clinical practice.5 These tools allow thousands of DNA fragments to be obtained in a Strategy used for copy number variant analysis single reaction, making it possible to analyze a high number of genes in a short period of time.6,7 NGS techniques include Copy number variation (CNV) was detected with an in-house targeted gene sequencing (panels), whole-exome sequencing tool (LACONv) that is based on GC-correction followed by a (WES), and whole-genome sequencing (WGS), each of them normalized weighted ratio of depth-of-coverage with respect to with distinct advantages and disadvantages. Compared with controls. The tool was optimized for targeted capture sequenc- WES/WGS, targeted gene sequencing is a relatively inexpensive ing and CNV intervals were determined if “dose” value and its approach for identifying pathogenic mutations and has a rela- z value were above/below some threshold that was adjusted tively higher sequence coverage. experimentally with a reference set. The CNVs of each patient The use of targeted sequencing or gene panels has already were analyzed and those deletions/insertions detected were con- been used in genetically heterogeneous diseases, including firmed using other techniques (multiplex ligation-dependent IBMFSs.8–11 Most of these studies focused on specific syndromes, probe amplification [MLPA] and comparative genomic hybrid- such as FA, DBA, or DC. Nevertheless, there are few published ization [CGH] arrays). studies reporting the overall genetic diagnosis in heterogeneous groups of patients with IBMFSs. With respect to IBMFSs, NGS is emerging as a valuable tool for the molecular diagnosis of Variant analysis and filtering strategy these disorders and is increasingly important as the first-line of The variant analysis and filtering strategy followed the guide- the molecular diagnosis.12,13 lines of the American College of Medical Genetics and Genomics Our hypothesis was that a panel of IBMFS genes could be (ACMG)14 and the publications previously reported.12,13,15 used as an initial screening test to provide precise and clini- The variants that were previously reported to be pathogenic cally relevant molecular diagnoses in a timely manner and at a or disease causing in public databases (Supplementary Table reduced cost. S3, http://links.lww.com/HS/A135). These variants were clas- Herein, we report the design and implementation of a NGS sified according to the ACMG guidelines into 5 groups: likely platform for the genetic diagnosis in 204 patients with sus- benign, benign, variants of unknown significance (VUS), likely pected IBMFSs. The application of this approach has improved pathogenic, and pathogenic. Those variants classified as benign our diagnostic process, resulting in a molecular diagnosis in the or possibly benign were not considered as causative. The other largest series of patients with IBMFSs in Spain. variants classified as VUS, likely pathogenic, and pathogenic were discussed with physicians who were experts in that par- Materials and methods ticular disease area, and genetic diagnoses were made based on the mode of inheritance of each disease, type of mutation, and Patients patient’s clinical findings. All variants detected were validated by Sanger sequencing. family studies were performed to determine A total of 214 consecutive patient samples from 47 Spanish the segregation or “de novo” origin of the detected variants. The centers were received between July 2015 and July 2018. Written algorithm used to filter variants is described in Figure 1. informed consent for genetic investigation and research was provided by either the patients or their legal guardians. The sample collection process is described in Supplementary Figure Results S1 (http://links.lww.com/HS/A135). All patients who were referred for suspected IBMFSs, accord- Quality parameter analysis and variant detection ing to the criteria of the reference specialist, were included. Clinical data was received from each patient. These clinical data Capture-based targeted sequencing covered > 99% of the tar- included the clinical suspicion, description of cytopenia/s, clin- get regions of 145 genes, with more than 20 independent reads. ical findings, family history, bone marrow investigations, and DDX11 and RPS17 did not reach the quality criteria, because other investigations. The criteria used to define the suspicion of the capture-based targeted sequencing of these genes covered an IBMFS are described in Supplementary Table S1 (http://links. < 80%. The average read depth was 367x (0–637). In addition, lww.com/HS/A135). 95% of the targeted regions were covered > 50x. Variants identi- Clinical diagnostic suspicions included DBA, FA, DC, SDS, fied in v1 included in group A and those considered as probable congenital amegakaryocytic thrombocytopenia (CAMT), hered- causative variants included in group B were validated through itary thrombocytopenia (HT), severe congenital neutropenia Sanger sequencing. Among the 214 patient’s cohort, 10 were (SCN), myelodysplastic syndrome (MDS), other cytopenia, and positive controls (Supplementary Table S4, http://links.lww. 2 (2021) 5:4 www.hemaspherejournal.com Table 1 Genotyping Patients With classified IBMFSs (Group 1). No. Patients No. Patients Mutated No. Cases With No. Mutations Novel Mutations Clinical Diagnosis Tested Genotyped Genes Mutations in This Gene in This Gene in This Gene Diamond-Blackfan anemia 45 31 RPL11 8 8 4 RPL5 6 6 6 RPS19 6 6 0 RPS24 1 1 1 RPS26 5 5 2 RPS29 2 2 2 RPS7 2 2 2 Del 15q 1 1 0 Dyskeratosis congenita 12 8 DKC1 3 3 1 RTEL1 3 4 2 TERT 2 2 1 Fanconi anemia 28 16 FANCA 13 19 3 FANCB 1 1 1 FANCG 2 3 0 Hereditary thrombocytopenia 38 16 ANKRD26 1 1 1 CYCS 2 2 2 GP1BA 2 2 2 ITGA2B 1 1 1 ITGB3 2 2 2 MYH9 5 5 0 RBM8A 1 2 1 TUBB1 1 1 1 WAS 1 1 1 Severe congenital neutropenia 25 3 ELANE 1 1 0 JAGN1 1 1 0 TCIRG1 1 1 1 Myelodysplastic syndrome 5 3 RPL11 1 1 0 TERT 1 1 0 CEBPA 1 1 1 Shwachman-Diamond syndrome 6 1 SBDS 1 2 0 Congenital amegakaryocytic thrombocytopenia 5 2 MPL 1 1 1 TERT 1 1 0 Other congenital anemia 3 1 KLF1 1 1 1 IBMFS = inherited bone marrow failure syndrome. Table 2 Genotyping Patients With Unclassified IBMFSs (Group 2). No. Patients No. Patients Mutated No. Cases With Mutations No. Mutations Novel Mutations Clinical Diagnosis Tested Genotyped Genes in This Gene in This Gene in This Gene Final Diagnosis Aplastic anemia 2 2 FANCA 1 2 1 Fanconi anemia TINF2 1 1 0 Dyskeratosis congenita Bilineage cytopenia 2 2 RECQL4 1 2 0 Rothmund-Thomson syndrome MPL 1 2 2 CAMT Bone marrow failure 21 4 AP3B1 1 1 1 Hermansky-Pudlak syndrome type 2 MPL 1 1 1 CAMT PUS1 1 1 1 Sideroblastic anemia G6PC3 1 1 1 Dursun syndrome Trilineage cytopenia 3 1 TERT 1 1 1 Dyskeratosis congenita CAMT = congenital amegakaryocytic thrombocytopenia; IBMFS = inherited bone marrow failure syndrome. com/HS/A135). All the positive control’s variants were detected missense, 30 were indels, 14 were splicing variants, and 10 by the NGS assay. were nonsense. We identified 104 variants in 90 patients with no previous molecular diagnosis. Using v1, we detected 58 variants in 47 patients, and using v2, we detected 46 variants in 43 patients. Genetic diagnosis by targeted sequencing in the Among the 104 detected variants, 56 variants had previ- total group ously been reported and 48 variants were novel. The variants were classified according to the ACMG guidelines. We detected Of the 204 patients with IBMFSs with no molecular diag- 11 pathogenic variants, 12 likely pathogenic variants, and 25 nosis, we detected pathogenic variants in 90 patients (44%). VUS. Regarding the mutation type of all variants, 50 were We included a total of 104 samples in v1 and 100 samples in 3 Gálvez et al Next-generation Sequencing in BMFS and Isolated Cytopenias Figure 1. Algorithm summarizing analysis and filtering processes (American College of Medical Genetics and Genomics [ACMG]). v2, with diagnostic rates of 45% (47/104) and 43% (43/100), The second case (patient 10002) involved a patient with respectively. We detected pathogenic variants in 35 genes. thrombocytopenia and bilateral malformations of the radii FANCA was the most frequently recognized gene with patho- at birth. In the NGS study, *6C>G on the RBM8A gene was genic variants, which was identified in 13 patients. detected, with an allelic frequency of 80%. The presence of this mutation in homozygosis was confirmed by Sanger sequencing (Supplementary Figure S7, http://links.lww.com/HS/A135). The Genetic diagnosis by targeted sequencing in the study of CNVs performed by NGS therefore showed a signifi- classified IBMFS group (group 1) cant deletion in chromosome 1 that included the RBM8A gene (Supplementary Figure S8, http://links.lww.com/HS/A135). This group included 167 patients with a suspected classi- After performing the single-nucleotide polymorphism microar- fied IBMFS and no molecular diagnosis (Supplementary Table ray study, the presence of a microdeletion on chromosome 1 S5, http://links.lww.com/HS/A135). We calculated genetic was demonstrated, from position 145395440 to 146089268 diagnostic rates through our targeted sequencing pipeline, (Supplementary Figure S9, http://links.lww.com/HS/A135). The taking into account the estimated mode of inheritance. The same study was subsequently carried out on the patient’s parents genetic diagnosis of patients with a clinical diagnosis of DBA using NGS, and it was observed that the *6C>G change on the (31/45, 68%), DC (8/12, 66%), MDS (3/5, 60%), and FA RBM8A gene was present in the patient’s father (Supplementary (16/28, 57%) was achieved in more than half of the patients. Figure S10, http://links.lww.com/HS/A135). The CNV study The genetic diagnostic rates for other categories were as fol- showed a deletion in chromosome 1 in the patient’s mother, lows: SCN (3/25, 12%), HT (16/38, 45%), SDS (1/6, 16%), which included the RBM8A gene (Supplementary Figure S11, CAMT (2/5, 40%), and other congenital anemias (1/3, 33%). http://links.lww.com/HS/A135). Finally, patient 10002 was In total, our targeted sequencing pipeline diagnosed 81 of diagnosed with thrombocytopenia-absent radius syndrome 167 patients (48%) in the classified IBMFS group. Regarding associated with pathogenic variants of the RBM8A gene.16 the panel version, in the classified IBMFS group, we achieved a diagnostic rate of 56% (47/84) in the v1 panel and 47% (39/83) in the v2 panel. Genetic diagnosis by targeted sequencing in the In this group, we identified copy number aberrations in 2 unclassified IBMFS group (group 2) patients. Patient 02021 had a positive chromosomal breakage test with no mutation detected by the NGS panel assay. A sig- We included 37 patients with unclassified IBMFSs nificant deletion was detected by the CNVs analysis from exon (Supplementary Table S6, http://links.lww.com/HS/A135). We 9 to 43 of the FANCA gene (Supplementary Figures S2 and S3, identified 13 variants in 9 patients, with a diagnostic rate of http://links.lww.com/HS/A135). This deletion was confirmed by 24% (9/37) in this group (Table 2). With regard to the panel MLPA in the patient and parents. The mother carried a deletion version, we achieved a diagnostic rate of 17% (5/28) in v1 and from exon 9 to 15 of the FANCA gene, and her father carried a 44% (4/9) in v2. deletion from exon 16 to 43 of the same gene (Supplementary Of the 9 patients in whom pathogenic variants were detected, Figures S4–S6, http://links.lww.com/HS/A135). 5 patients were diagnosed with typical IBMFSs. Patients 12006 4 (2021) 5:4 www.hemaspherejournal.com and 08001 were diagnosed with DC, having mutations in the changes and no molecular diagnosis had been achieved, with TINF2 and TERT genes, respectively. Patients 23001 and IBMFSs still suspected. Of the remaining 30 patients, 3 patients 10003 were diagnosed with CAMT, having MPL gene muta- proceeded to HSCT, 1 of whom died due to complications tions in both cases. Patient 04016 was diagnosed with FA with following the procedure. In 4 cases, a molecular diagnosis of compound heterozygosis of the FANCA gene. IBMFS had been made using the other techniques summarized The other 4 patients were diagnosed with other less com- in Table 3. Three patients had had spontaneous hematological mon syndromes. Two of the successfully genotyped patients recovery without treatment, and the remaining 20 patients had with unclassified IBMFSs had predominant neutropenia. Patient been reclassified as having other noncongenital diseases. The 40004 had a homozygous pathogenic variant of the G6PC3 majority of patients (12 cases) were reclassified as immune gene, which is associated with the development of Dursun syn- cytopenias, and the remaining patients were distributed heter- drome.17 In the other case, patient 20001, the mutation was ogeneously: AA (3 cases), immunodeficiency (2 cases), pyruvate detected in the AP3B1 gene associated with Hermansky-Pudlak kinase deficiency (1 case), myeloproliferative syndrome (1 case), syndrome type 2.18 and mitochondrial disease (1 case). The last 2 patients diagnosed in this group were 11029 and 01015, who were diagnosed with Rothmund-Thomson syn- drome and sideroblastic anemia with pathogenic variants of the Discussion RECQL4 and PUS1 genes, respectively. In recent years, the identification of the underlying molecular pathology has become the cornerstone for establishing a con- Diagnostic amendments clusive diagnosis of IBMFS, leading to better clinical care and follow-up of these patients. Until recently, Sanger sequencing of The diagnosis of 6 clinically classified patients (6%) was candidate genes has been the gold standard tool for the genetic amended after the results of the NGS gene panel assay became diagnosis of IBMFS, resulting in a costly and lengthy technique.4 available. In 2015, NGS techniques emerged as the herald of the revolu- The first example involved 2 patients clinically diagnosed with tion in the genetic diagnosis of IBMFSs.12,13,15,26 aplastic anemia (AA) based on hematological findings. In 1 of In this study, we have reached the molecular diagnosis of them (patient 12006, 4 years old), we found a pathogenic vari- a cohort of patients from hospitals in Spain with suspected ant of TINF2 (c.815G>A),19 which produced a stop codon. In IBMFSs using a NGS assay, for which we developed a molecular view of the results, a telomere length study was carried out and diagnostic system that utilized a targeted sequencing pipeline very short telomeres were detected (C).20,21 The chromosomal breakage test came back variants, which confirmed the strong analytical sensitivity and positive, so the patient was finally diagnosed with FA. reproducibility of the platform. We detected 11 known variants Patients 40001 and 38001 were eventually diagnosed with in 7 genes, including missense and nonsense variants and small DC. Based on their clinical evolution and hematological find- deletions from 10 patients. ings, patients 40001 and 38001 were initially referred for sus- In this series of patients, we identified 104 candidate vari- pected CAMT and for suspected myelodysplasia, respectively. ants in 35 genes, 46% of which were absent from the main However, in both cases, the study showed mutations associated reference databases, thus emphasizing the great heterogeneity with the development of DC on the TERT gene (c.2225G>A and of the molecular pathology underlying IBMFSs. Appropriate c.1234C>T, respectively). Both mutations have been previously interpretation of the pathogenicity of candidate genetic vari- described in the literature.22,23 DC was confirmed by a telomere ants found by NGS remains a major challenge, especially for length study of these patients, with both cases presenting with novel variants, even if present in well-established IBMFS genes. telomere shortening below the first percentile for their age. To prevent misinterpretation, the use of consensus guidelines is The last 2 patients included in this group were eventually highly recommended, although there is significant discordance diagnosed with DBA. Patient 04006, aged 7 years, was stud- between laboratories.14,27 Following established guidelines and ied for an episode of transient thrombocytopenia; however, studies carried out by other groups, we have designed our own a c.164C>T pathogenic variant was detected on the RPS19 variant-filtering strategy carrying out the same analysis for each gene, which was associated with the development of DBA.24 variant.12–14 Finally, of the 104 identified candidate variants, we Reviewing the clinical history of patient 04006, it seems that, classified 23% and 25% as pathogenic and likely pathogenic, in early childhood, he had an episode of severe anemia but pro- respectively. Approximately half of the variants were classified gressively recovered until reaching his present level of 10 g/dL of as VUS. The high proportion of VUS supposes 1 of the major hemoglobin. Patient 10013, aged 23 years, had been studied for limitations of the study, since we did not have the possibility to mild anemia from childhood with features of myelodysplasia in carry out functional studies. However, through the family study the bone marrow study performed in adulthood. This patient and after evaluating each of the cases with the responsible spe- had a deletion on the RPL11 gene, which has already been cialist, we analyzed the clinical picture of the patient, the mode described as causing DBA.25 of inheritance of each disease, and the characteristics of the variants. Therefore, as changes are compatible with the clinical Analysis of undiagnosed patients picture of the patients, we interpret that these variants could have an important role in the development of the disease. These Of the 204 patients analyzed with NGS, a total of 113 patients results indicate the great complexity to interpret variants in this were not diagnosed, corresponding to 55% of the total sample. type of disease and, at the same time, reflect the need to deepen Two years after the recruitment of these patients, the doctors the knowledge of the molecular bases of IBMFSs to be able to responsible for monitoring the clinical evolution of the patients assess their involvement in the development of the disease. were questioned again. Among these 113 patients, it was pos- Overall, our NGS approach enabled a molecular diagnosis in sible to record the follow-up of 105 patients (Figure 2); 70% 44% of patients. The diagnostic rate of our targeted sequencing of these patients (75/105) presented with no significant clinical platform was similar to those obtained by other groups. The 5 Gálvez et al Next-generation Sequencing in BMFS and Isolated Cytopenias Figure 2. Description of clinical evolution in undiagnosed patients. HSCT = hematopoietic stem cell transplantation. previous studies published by Ghemlas et al12 and Muramatsu rate is acceptable; nevertheless, for patients with more complex et al,13 which involved similar approaches, studied patients with phenotypes, diagnosis using a NGS gene panel may be insuffi- suspected IBMFSs using a NGS assay and analyzed the diagnos- cient. This is supported by 1 of the first papers published in this tic yield. The first work published by Ghemlas et al12 diagnosed field. Zhang et al15 used a NGS panel of 85 genes to study a 61 of the 158 patients studied using a panel of 72 genes, with total of 71 patients affected by IBMFSs with no typical clinical an overall diagnostic rate of 38%. The report by the Japanese characteristics. The author described the molecular basis of 8 of group studied diagnostic rates in a population of patients using the 71 patients, with a diagnostic rate of 11%. These 8 patients a NGS panel and WES. With targeted sequencing, this group presented pathogenic variants in the GATA2, RUNX1, DKC1, achieved a genetic diagnosis rate of 53 out of 121 (44%). and LIG4 genes but did not present the typical phenotypic In our study, we analyzed the results according to the groups characteristics associated with these genes. This illustrates the into which the patients were divided. The percentage of diagno- fact that the molecular diagnosis of unclassified IBMFSs using ses achieved in the classified IBMFS group was 48% (81/167) a NGS gene panel does not exceed 20%, suggesting that this compared with 24% (9/37) in the unclassified IBMFS group. group could benefit from other approaches. Data provided by Remarkably, the diagnostic rate was higher for patients present- Bluteau et al28 support this idea. These authors studied a total ing with a well-defined clinical phenotype indicative of a partic- of 179 samples from patients with suspected IBMFSs without ular type of IBMFS. The Canadian group of Ghemlas et al12 also a defined clinical phenotype, excluding those with characteris- used this division, observing a similar disparity, with a molec- tics of FA, DBA, DC, SDS, and SCN. For all the samples, WES ular diagnosis rate of 59% in the case of classified IBMFSs, of the DNA extracted from fibroblasts was undertaken, so all compared with 18% for unclassified IBMFSs. This difference samples corresponded to the germ line. Finally, a total of 86 suggests that, for cases of clinically well-defined diseases with patients were diagnosed, with a diagnostic rate of 48%. This specific screening analysis (such as chromosomal breakage test, data exceeds the results obtained using NGS panels, which telomere length, or biochemical characteristics), the diagnostic seems to indicate that WES of germline samples is an interesting 6 (2021) 5:4 www.hemaspherejournal.com Table 3 follow-up to monitor the onset of bone marrow failure, as well Patients Diagnosed by Other Molecular Techniques. as the development of pulmonary or hepatic fibrosis or the appearance of cancer. Another example was the patient under Clinical Suspicion Gene/CHR Affected Technique Final diagnosis study for mild thrombocytopenia who was finally diagnosed DBA Chr 15q deletion CGH array DBA with DBA. This finding implied the convenience of performing SDS DNAJC21 mutation WES SDS close monitoring of the patient due to the increased risk of can- IBMFS SAMD9 mutation WES MDS cer in patients with DBA. Therefore, in addition to the specific FA FANCA deletion MLPA FA treatment that the development of anemia would require, it is advisable that these patients should follow an early detection CGH = comparative genomic hybridization; Chr = chromosome; DBA = Diamond-Blackfan anemia; program in adulthood. FA = Fanconi anemia; IBMFS = inherited bone marrow failure syndrome; MDS = myelodysplastic syndrome; MLPA = multiplex ligation-dependent probe amplification; SDS = Shwachman-Diamond In this work, there was no particular selection of the patient syndrome; WES = whole-exome sequencing. cohort. The selection criteria were less strict than for other groups, so it would be expected that a percentage of the patients were not affected by a congenital disease. Based on this premise, tool to be considered for patients who do not present with typi- the reference specialist doctors responsible for monitoring the cal clinical characteristics. clinical evolution of the patients were questioned 2 years after Regarding to specific groups, in this series, the diagnostic their patients had been included in this study. After this consul- yield in FA group had been 57%, being lower than expected. tation, 24 patients did not meet the clinical criteria for a IBMFS, This could be explained by patient selection. All referred and some experienced spontaneous hematological remission. patients were included in this study and, in some cases, the Therefore, it seems reasonable to establish a series of minimum chromosomal breakage test had not been performed. Therefore, criteria, both clinical and analytical, before applying this type of probably some patients were affected by another syndrome, no diagnostic approach. FA. However, 87% (14/16) patients with positive chromosomal Using this approach, we have detected variants potentially breakage test were diagnosed using the NGS platform, being responsible of the phenotype in only half of the patients. This the diagnostic rate of this approach higher in patients with a could be due to the technical limitations inherent in this type of positive chromosomal breakage test. diagnostic approach, such as mutations in deep intronic areas In SDS group, only 1 patient was diagnosed by NGS. The or genes for which there was not sufficient depth of reading. explanation could lie in the previous genetic studies performed Additionally, problems of bioinformatic tools determining the before they were included in the study. When this NGS panel pathogenicity of rare variants or incomplete knowledge of the was designed, the only SBDS gene had been associated with the IBMFSs may have accounted for these limitations. NGS can development of this disease, so of the 5 patients without molecu- miss large deletions or duplications; CNVs involving > 1000 bp; lar diagnosis, 3 of them were negative for this gene. Throughout or large structural chromosomal variants, translocations, and the development of this project, new genes associated with aneuploidy unless they have been specifically designed for such this disease have been described, specifically DNAJC2129,30 a purpose.29 Thus, for successful molecular diagnosis of certain and EFL131. In 1 of the patients in whom no result was found, cases, the NGS assay should be combined with other molecular WES was subsequently performed, detecting DNAJC21 gene approaches, such as CGH array, quantitative polymerase chain mutated. Therefore, we could say that those patients highly sug- reaction, or MLPA. gestive of SDS negatives for the SBDS gene could not benefit Possibly the greatest limitation for detecting molecular from approaches using NGS panels that are not updated. alterations using a NGS panel of already defined genes is the In this work, 2 versions of the same panel were used, with progressive discovery of new IBMFS genes. Indeed, since the 129 genes included in v1 and 145 genes included in v2. The design of this panel, new genes have been discovered. In this percentage of molecular diagnosis achieved for the 2 versions work, there are 2 patients who have been diagnosed by WES did not differ substantially (45% in v1 versus 43% in v2), which for genes not included in this panel, such as DNAJC21 and may suggest that the number of genes included per panel was SAMD9, published in 2017.14,30,31,33 The best option could be to not decisive in the percentage of diagnoses achieved by this use an updated panel as initial screening and subsequently select tool. One possible explanation is that the panels that include a those patients who might be candidates for WES or even WGS. greater number of genes are those that have been designed more However, even with WES and WGS techniques, a percentage of recently, including recently discovered genes that have been patients are still not diagnosed. described with a lower frequency. Since this approach is some- In summary, our NGS gene panel assay constituted a useful what similar to screening, it is likely that, for the vast majority tool for the genetic diagnosis of IBMFSs. The correct classifi- of patients, the mutations fall within the group of genes most cation of IBMFSs using NGS facilitates more accurate medical frequently detected in patients with IBMFSs. However, gene management of these complex conditions. Specifically, our gene panels require periodic updating to ensure they cover as many panels were able to identify the genetic alterations in a high per- genes as possible, thus optimizing molecular diagnosis. centage of patients and constituted a reasonable option for the In most patients diagnosed, the clinical suspicion coincided genetic screening of IBMFSs prior to the application of more with the molecular diagnosis. However, it is important to high- complex studies, including WES and WGS, which might facili- light the fact that 6 of the 91 patients diagnosed (6%) were tate the discovery of new genes. reclassified, implying a change in their clinical management. In this work, the rate of diagnostic amendments is somewhat lower than that published for other series, where the rate is around Disclosures 10%–26%.12,13,32 However, even if the number is not high, it has The authors have no conflicts of interest to disclose. very important clinical implications, such as avoiding unneces- sary treatments or giving adequate genetic counseling. In 1 case, the clinical diagnosis of AA was amended to FA, meaning the References convenience of entering into a bone marrow transplant proce- 1. Dror Y. Inherited bone marrow failure syndromes. 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