Targeted next generation sequencing panel based screening and diagnosis of bone marrow failure cohort reveals diverse genomic landscape of pathogenic germline variants

doi:10.21203/rs.3.rs-2229099/v1

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Targeted next generation sequencing panel based screening and diagnosis of bone marrow failure cohort reveals diverse genomic landscape of pathogenic germline variants

https://doi.org/10.21203/rs.3.rs-2229099/v1

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Introduction: Current study is a 4 year experience in diagnosis and screening of bone marrow failure cases using a targeted sequencing panel.

Methods: A total of 171 cases underwent targeted NGS and were categorized as suspected inherited bone marrow failure syndrome (IBMFS) group (106/62%) and idiopathic aplastic anemia (IAA) group (65/38%) based on clinical and laboratory criteria.

Results: 110 (64%) were pediatric (0-12 years) and 61 (36%) adolescent and adults (13-47 years). In suspected IBMFS group, 47 (44%) were positive for a pathogenic variation while in IAA group, 8 (12%) revealed a silent germline pathogenic variation. Whole exome sequencing performed in 15/61 suspected IBMFS group cases, revealed a clinically important variation in 3 (20%) cases. The targeted panel helped in establishing a diagnosis in 44% (27/61) of phenotypically heterogenous unclassified bone marrow failure syndrome cases and also led to amendment of clinical diagnosis in 5 (4.7%) of genotyped cases.

Conclusion: Overall targeted NGS panel yield was comparable to western data, however, this was achievable at 1/5th the cost, making it a cost-effective panel for resource constraint settings.

Health sciences/Diseases/Haematological diseases

Health sciences/Molecular medicine

IBMFS

IAA

BMF

Pediatric

Adult

NGS

Bone marrow failure (BMF) can be inherited (IBMFS) or acquired (commonly-Idiopathic aplastic anemia/IAA), knowledge of which is pivotal in determining the treatment modality. Hematopoietic stem cell transplant (HSCT) is the treatment of choice for IBMFS in contrast to immunosuppressive therapy (IST) given for acquired aplastic anemia. Timely and correct diagnosis of an IBMFS allows focused disease surveillance, anticipatory guidance, and avoidance of treatment-related toxicities. Identifying the underlying genetic mutation helps in selecting healthy sibling donors and planning the appropriate transplant conditioning regimen to avoid toxicity. It also enables proper monitoring for future cancer risk.(1–3)

Next-generation sequencing (NGS) techniques like targeted gene sequencing (panels), whole-exome sequencing (WES), and whole-genome sequencing (WGS) are now readily available for identifying pathogenic/likely pathogenic variations. Amongst these, targeted gene sequencing is a relatively inexpensive/cost-effective approach linked with rapid data analysis and is currently being increasingly used in the diagnosis of IBMFSs globally.(4–7) In fact, advancements in NGS technologies have led to the identification of a rapidly increasing number of genes related to IBMFS including newer candidates like SAMD9, SAMD9L, MECOM and ERCC6L2.(4–6) Moreover, it also helps in uncovering germline variants in IBMFS genes. Upto 5% of patients who were otherwise labelled as idiopathic aplastic anemia cases may be reclassified as IBMFS based on NGS testing. (8) Therefore NGS is increasingly being incorporated in the current diagnostic algorithms of pediatric aplastic anemia.(9) Our group had recently published a limited data on role of NGS in pediatric marrow failure and identified silent germline variations in up to 13.3% of IAA cohort tested.(3) Current study is an extension of above analysis, wherein we have comprehensively reviewed and analyzed our BMF data over 4 years, including adult cases as well. This analysis is expected to elucidate the genetic landscape of bone marrow failure in our population cohort. The study is also likely to help us formulate guidelines for routine incorporation of NGS testing in both pediatric and adult (< 45 years) IAA cases.

A retrospective analysis of all cases (pediatric 0–12 years and adult > 12 years) of marrow failure who were referred to the genetic laboratory by the clinicians for targeted next generation sequencing, over last 4 years (2018–2022), was done. Samples were run after written and informed consent of parents/legal guardians and study was approved by Institute’s ethics committee and Departmental review board. Clinical and investigational details were recorded and analyzed as two groups based on clinical requisition as:- 1. Suspected inherited bone marrow failure syndrome (IBMFS) group and 2. Idiopathic aplastic anemia group. The suspected IBMFS group was defined based on following clinical and laboratory criteria- i) cases with unilineage/multilineage cytopenias along with documented marrow hypocellularity for age and either associated constitutional abnormalities or a positive family history or anemia with documented erythroblastopenia in marrow and clinical suspicion of Diamond Blackfan anemia due to age of presentation. ii) cases with myelodysplastic syndrome or acute myeloid leukemia based on bone marrow blasts and cytogenetics and history of prior long term unilineage/multilineage cytopenias prior to development of myeloid disorder. iii) cases with cytopenias and hypocellular marrow with positive chromosomal breakage screening results. The idiopathic aplastic anemia (IAA) group included cases with pancytopenia and hypocellular marrow for age with no secondary causes for aplasia and absent constitutional abnormalities. These cases were primarily referred for screening NGS prior to initiation of immunosuppressive therapy or hematopoietic transplant or in case they had suboptimal response to immunosuppressive treatment at 6–9 months post therapy.

Briefly, peripheral blood mononuclear cells were extracted from peripheral blood EDTA sample (3-4ml) using Ficoll Hypaque (Sigma-Aldrich) followed by DNA extraction (Qiagen DNA blood mini kit) on QIAcube. DNA quality and quantity was assessed by nano-quant plate (Tecan) and Qubit analyser (ThermoFischer Scientific) before NGS run. A NGS panel of 33 genes was custom designed on IonAmpliseq designer studio (hg38; 400 bp chemistry). The panel genes were curated from literature studies and OMIM database. The panel covers the most important and common genes related with IBMFS (FA, DBA, DC, CAMT, TAR, SBDS, Congenital Neutropenia) and 11 genes (including newer IBMFS genes) that are known to predispose to myeloid disorders (RUNX1, ETV6, DDX41, ANKRD26, GATA-2, SRP72, SAMD9, SAMD9L, DNACJ21, ERCC6L2, MECOM) (Fig. 1). The panel has a total of 494 amplicons covering 98.25% of the coding region of all 33 genes. NGS libraries were prepared using IonAmpliseq Library Plus kit (Invitrogen; ThermoFisher Scientific) followed by template generation (OT2-IonChef 520/530 kit) and chip loading (530 chip) on Ion Chef. The sample loaded chips were run on Ion Torrent S5 studio (mean 250-300x coverage) and analyzed by Ion Reporter and in-house germline bioinformatics pipeline. Clinically important variants were confirmed by Sanger sequencing and reported as per American College of Medical Genetics (ACMG) criteria. In a limited number of cases, with variant of unknown significance (VUS)/heterozygous single gene variation or negative result on targeted NGS panel, whole exome sequencing (WES) was performed on NovaSeq 6000 (Illumina Inc. USA) to look for the second variant. The cost of targeted NGS averaged 100 USD per patient.

All IAA cases were classified as per modified Cammitta criteria and were started on equine anti-thymocyte globulin (ATG) and cyclosporine if no matched donor transplant option was available.

Statistical Analysis

Statistical analysis was performed using GraphPad Prism version 5 software. Categorical data is represented as ratio/proportions and quantitative data as range and mean+-SD. Means were compared using t-test and associations between categorical variables by Fisher’s exact test or Chi- Square test as applicable. A P-value < 0.05 was considered statistically significant.

Clinical and Hematological characteristics

106 (62%) patients from suspected IBMFS group and 65 (38%) from IAA group were subjected to this panel analysis (Flow chart Fig. 1). Among suspected IBMFS group, 79 (74.5%) were pediatric (0-12 years) and 27 (25.5%) adolescents and adults and among IAA group, 31 (48%) pediatric and 34 (52%) adolescents and adult patients. The clinical and hematological characteristics of the two groups are highlighted in Table 1. Briefly, the mean age of presentation in pediatric sub-group in suspected IBMFS group was significantly lower than in IAA group (4.4 y vs. 8.3 y; p-<0.001), while for adult sub-group no difference was observed (23 y vs. 23.7 y; p-0.67). In adult sub-group, males were more commonly affected than females in both the groups. No difference was observed in pediatric sub-groups (12.5:1 and 7.5:1 vs. 1.5:1 and 1:1; p-0.002 and p-0005). Most common hematological phenotypes in pediatric sub-group in group 1 were anemia with erythroblastopenia (21/79; 27%), followed by pancytopenia with hypocellular marrow (<10% cellularity for age) (19;24%) and myeloid disorder diagnosis (12;15%). In adult sub-group (suspected IBMFS), predominant hematological phenotype was pancytopenia and hypocellular marrow (11; 41%) followed by myeloid disorder diagnosis (7;26%). In IAA group, predominant hematological phenotype was severe aplastic anemia (SAA) in both pediatric (27;87%) and adult (33;97%) cases. A total of 79 constitutional abnormalities (CA) were noted (pediatric-61; adult-18) in suspected IBMFS group in 53 (50%) cases (pediatric-40/79; adult-13/27) (p-0.82). A family history of cytopenia and or hematological/solid malignancy was noted in 14/106 (13%) cases in suspected IBMFS group. Consanguinity was present in only a single case.

Genetic variation data in suspected IBMFS group and IAA group

Targeted NGS using a custom panel of 33 genes related to IBMFS was performed in all 171 cases. 63/171 (37%) patients (pediatric-44/40% and adult-19/31%) revealed a clinically important genetic variation in at least one of the 24/33 genes covered in the panel. In 55/63 (87%) cases, variants were pathogenic/likely pathogenic as per ACMG criteria, while 8 were VUS (Supplementary Table 1) primarily due to inability to identify a second variant related with the disease phenotype. The details of all pathogenic/likely pathogenic variations along with clinical phenotype in both suspected IBMFS group and IAA group have been highlighted in Tables 2 and 3 respectively.

In suspected IBMFS group, overall diagnostic yield of the targeted panel was 44% (47/106), with 40.5% (32/79) in pediatric and 55.5% (15/27) in adult subgroups. Pathogenic/Likely pathogenic variations in RPS19 gene were identified in the greatest number of cases (6), followed by RPL5 and SAMD9 in 5 cases each and TERT and FANCA in 4 cases each (Fig 2). However, the most common IBMFS diagnosis was Diamond Blackfan Anemia (DBA) (14/47;30%) followed by Dyskeratosis congenita (DC) (10/47;21%), Fanconi anemia (FA) (5/47;11%), SAMD9 related marrow failure (5/47;11%), Congenital neutropenia (4/47;8.5%), MECOM related and SRP72 related marrow failure syndromes (2 cases each or 4/47; 8.5%), Congenital or cyclical thrombocytopenia (2/47;4%) and other rare forms (GATA-2, THPO and SBDS 1 case each or 3/47;6%). In IAA group, the overall positive yield for a pathogenic/likely pathogenic germline variation was 12% (8/65), with 19% (6/31) for pediatric and 6% (2/34) for adult cases. Pathogenic/Likely pathogenic variations in FANC genes were most common and noted in 3 cases, followed by SAMD9 and DKC1 with 2 cases each (Fig 2). One case had likely pathogenic variation in MECOM gene.

We also performed whole exome sequencing (WES) in a limited number of 15 cases (3/8 cases with single heterozygous variation or variants of unknown significance and 11/53 suspected IBMFS group cases negative on targeted panel). WES revealed a definitive diagnosis in additional 3/15 (20%) cases with variations in FANCF, PARN and ADA2 genes related with Fanconi anemia, Dyskeratosis Congenita and Deficiency of adenosine deaminase 2 (DADA2) syndrome respectively. Second clinically important variation could also not be identified in any of the 3/8 VUS cases on WES.

Germline variations in genes predisposing to myeloid disorders including newer IBMFS genes

A total of 15 pathogenic/likely pathogenic variations were noted in 5/11 myeloid disorder predisposing genes covered in the panel. Of these, 12 were in suspected IBMFS group with 10 being germline (SAMD9-5; MECOM and SRP72- 2 cases each; GATA-2- 1 each) and 2 somatic in nature as confirmed on remission sample testing (RUNX1- 2). IAA group had 3 cases with germline pathogenic/likely pathogenic variations (SAMD9- 2; MECOM- 1). Hence, overall frequency of pathogenic/likely pathogenic germline variations in myeloid disorder predisposing genes was 13/171 (7.6%), with 9.4% (10/106) in suspected IBMFS group and 4.6% (3/65) in IAA group.

A total of 7 patients were noted to have SAMD9 related IBMFS. Two of the cases had constitutional abnormalities in form of neurological issues in 1 and lymphedema and vitiligo in second. 5 cases had pancytopenia and hypocellular marrow and 1 case had bicytopenia with hypocellular marrow. One of these developed Hodgkin’s lymphoma in adult age while two received immunosuppressive therapy as clinical suspicion of IAA and showed partial response at 8-9 months. One additional case presented with myelodysplastic syndrome due to additional somatic RUNX1 hit along with monosomy 7 and 20q deletion on fluorescent in situ hybridization (FISH) study. Three patients had MECOM related IBMFS. One patient had AML with clinodactyly and thenar atrophy and another presented with pancytopenia and facial dysmorphism. The third patient was being treated as IAA and showed no response to immunosuppressive therapy at 8 months.

Conventionally, diagnosis of IBMFS is usually based upon recognition of uni or multi-lineage haematological manifestations, specific clinical phenotypes or constitutional abnormalities and results of syndrome-specific screening tests. With recent advances in genetic sequencing coupled with an exhaustive list of probable IBMFS causative genes, next generation sequencing has become an important tool for accurate diagnosis. However, despite the improved diagnostic yield with NGS, cost, technical expertise, and clinical validation, limit its application in lower-middle income countries. Targeted NGS panels are being increasingly used to establish molecular diagnosis, however there is a paucity of literature on IBMFS in lower-middle income countries owing to lack of clinical registries. In resource constraint settings, while developing targeted panels, it is essential to strike a balance between the depth, breadth of sequencing and the cost of testing. Our goal was to develop a small, cost- and time-effective NGS gene panel to target diverse IBMFS that could be easily integrated in the evaluation of patients with bone marrow failure. In this study we evaluated the effectiveness of a custom designed NGS panel (33 genes) in detection of IBMFS. It was done at 1/5th of the cost (USD 90–100 per sample) of other larger targeted next generation sequencing panels for IBMFS genes reported in the literature.(4) The target panel overall diagnostic yield in suspected IBMFS group was 44% (47/106) and screening yield was 12% (8/65) in IAA group. The diagnostic yield is similar to many studies reported till date however ranging from 43–59%.(4, 5, 7) In addition, we also performed whole exome sequencing in a limited number of suspected IBMFS group cases (15/61) with variants of unknown significance or negative targeted panel results. However this could aid in further diagnostic yield in only 3/15 (20%) cases, similar to frequency reported in literature.(5, 10) We also screened 65 IAA cases as pre-immunosuppressive therapy (41;63%) or hematopoietic transplant (5;8%) screen or those partially (6;9%) or completely unresponsive (13;20%) to immunosuppressive therapy at 6–9 months, to detect any pathogenic/likely pathogenic germline variations in IBMFS genes. In our setting, with not many patients affording hematopoietic transplant due to lack of suitable donor or financial reasons, immunosuppressive therapy remains the main treatment modality offered for idiopathic aplastic anemia. In such situations, the discrimination between acquired and inherited cause becomes crucial as to avoid undue toxicity with immunosuppressive regimen in a child with an underlying inherited bone marrow failure syndrome. Our results demonstrate the routine incorporation of targeted panel based screening for IAA group as silent germline pathogenic/likely pathogenic variations were noted in 12% of children/adults. The pick-up rate was much higher in pediatric cohort (19%) compared to adult cohort (6%). 50% of the positive cases were either partially or non-responsive to immunosuppressive therapy and rest 50% were picked up at pre-initiation of immunosuppressive therapy screen. Studies report variable frequency of germline variations in IAA cohort ranging from 5–30%.(4, 8, 11, 12) Our study also helped in establishing diagnosis in 44% (27/61) of phenotypically heterogenous basket of unclassified bone marrow failure syndrome cases, in line with a previous study from our centre, though on a smaller cohort.(3) The diagnostic utility of targeted panel in this heterogenous group was however higher than in studies reported till date.(4, 7, 13) This finding is probably due to lack of access/unavailability of few commonly used screening tests (telomere length analysis, pancreatic function tests etc.) for IBMFS at our centre.

Our panel results led to amendment of clinical diagnoses in 5 (4.7%) of the genotyped cases. This is due to the fact that many of these diseases have a significant phenotypic overlap, and these pitfalls can be overcome only by a comprehensive next generation sequencing panel testing of all IBMFS genes. This finding has been well demonstrated in previous studies, wherein diagnosis was altered in 3.5–9% cases.(4, 5, 7) Molecular characterisation of the disease is theranostic, for example, the amendment of clinical diagnosis of Diamond Blackfan Anemia to Dyskeratosis Congenita in one of the patients indicates a need for HSCT rather than trial of steroids. Similarly in one case diagnosis was changed from a suspected GATA2 associated marrow failure syndrome to SAMD9 related IBMFS post sequencing. This patient may need a longitudinal follow up as SAMD9 may spontaneously regress or may evolve into myelodysplastic syndrome.(14, 15)

Newer IBMFS genes namely SAMD9 and MECOM revealed pathogenic/likely pathogenic variations in overall 6% (10/171) of cases with SAMD9 accounting for 70% of the cases. The SAMD9 germline variation cohort of 7 unrelated patients had a diverse clinical presentation wherein one had neonatal onset pancytopenia and another had prolonged cytopenia history with nodular variant of Hodgkin lymphoma development, two cases presented as IAA, two were unclassified marrow failure syndromes and the other was clinically suspected to have Monomac syndrome. Presentation of germline SAMD9 variations as pancytopenia in early infancy and isolated bone marrow failure in the absence of physical anomalies is well described in the available limited literature.(16) One of our patients with SAMD9 variation had monosomy 7 and additional RUNX1 mutation. Strong enrichment of SAMD9 variations in monosomy 7 is well described.(17) Acquisition of additional variations including RUNX1 increases the risk of progression to high risk MDS/AML.(17) To the best of our knowledge, our cohort highlights the first report of a germline SAMD9 variation associated with Hodgkin’s lymphoma. Cases with MECOM related IBMFS also had diverse presentations, one had AML with clinodactyly, other was a case of IAA and the third was an unclassified bone marrow failure, all of which are described in the available literature.(20) Hence, next generation sequencing not only helped in the diagnosis of these newer IBMFS but also helped in recognition of their expanded phenotypic spectrum.

An occasional case with bone marrow failure phenotype overlap was also identified. This was a case of DADA2 disease where the infant had anemia, erythroblastopenia and was clinically labeled as DBA. The targeted panel sequencing was negative but whole exome sequencing revealed compound heterozygous variation in ADA2 gene.(21, 22) Few had atypical presentations associated with specific genotype, for example, we had a case of HAX1 variation presenting with cyclic neutropenia and a case of MPL variation presenting with cyclic thrombocytopenia.(23, 24)

Our results demonstrate fairly good diagnostic yield (44%) of a smaller (33 gene) targeted next generation sequencing panel and highlight it’s utility as a cost-effective diagnostic tool for inherited bone marrow failure syndromes in clinical practice, especially in resource constraint settings. The study results further add substantial evidence to incorporate routine screening of idiopathic aplastic anemia cases, both pediatric and adult (< 45 years), since a high proportion of these cases (12%) may harbour silent germline inherited marrow failure variations. Further the data adds novel phenotypes in relation to SAMD9 and MECOM related newer IBMFS and stresses that SAMD9 or MECOM germline pathogenic variants are common in otherwise classical IAA cases (38%) and these need to be longitudinally followed up to assess myelodysplastic syndrome risk.

Data Availability Statement: The raw data required for non-commercial purposes can be requested from authors by email at [email protected]

Conflict of Interest Statement: The authors have no conflict of interests to declare. None of the authors is in recipient of any commercial affiliations, consultancies or any other financial assistance from commercial sources.

Ethics Approval: The study has been approved by Institute Ethics Committee vide approval No. PGI/2018/11277-319 and Departmental Review Board Committee.

Patient consent statement: An informed and written consent had been taken from all participants and or their legal guardians/parents for inclusion in the study.

Permission to reproduce material from other sources: No material from other sources has been used in the current study that would require permission.

Funding sources: None

Acknowledgements: None

Author Contributions: SP, GS, PB- Analyzed the data and wrote the manuscript draft; PB, AT- Conceptualised and designed the study; PC, AK, AT, AJ, DB, RJ, SP, SM, AJ, DS, PM: Clinicians involved from Pediatric and adult specialties responsible for enrolment and referral of cases for eugenic testing and for treatment and follow up of these cases; MS, PS, RT: Performed genetic testing; PB: Bioinformatic analysis and reporting of genetic data; SS, RD, NV: Reporting of bone marrow specimens and chromosomal breakage studies.

ACMG-American College of Medical Genetics and Genomics

ADA-Adenosine deaminase

AML-Acute Myeloid leukemia

ATG-Antithymocyte Globulin

BMF- Bone marrow Failure

CA-Constitutional Abnormality

CAMT-Congenital amegakaryocytic thrombocytopenia

CBS-Chromosomal breakage study

CN-Congenital neutropenia

DADA2-Deficiency of ADA 2

DBA-Diamond Blackfan anemia

DC-Dyskeratosis congenita

EDTA- Ethylenediaminetetraacetic acid

ERCC6L2- ERCC Excision Repair 6 Like 2

FA-Fanconi Anemia

HSCT-Hematopoietic stem cell transplant

IAA-Idiopathic Aplastic anemia

IBMFS- Inherited Bone Marrow Failure Syndrome

IST-Immunosuppressive therapy

LMICs-Low middle income countries

MDS-Myelodysplastic syndrome

MDS-P-MDS predisposing

MECOM- MDS1 and EVI1 complex locus protein EVI1

MPAL-Mixed phenotypic acute leukemia

NGS-Next generation sequencing

NLPHL- Nodular lymphocyte-predominant Hodgkin lymphoma

PARN- Poly(A)-Specific Ribonuclease

PBMC- A peripheral blood mononuclear cell

SAA-Severe Aplastic Anemia

SAMD9- Sterile alpha motif domain-containing protein 9

SAMD9L- Sterile α motif domain-containing protein 9-like

SDS- Shwachman Diamond Syndrome

UBMFS- Unclassified bone marrow failure syndrome

VUS-Variant of uncertain significance

WES-Whole exome Sequencing

WGS-Whole Genome sequencing

1. David G Nathan, Stuart H Orkin. Nathan and Oski’s Hematology and oncology of infants and childhood Ch. 7 (Elsevier Saunders, 2015).

2. Gluckman E, Wagner JE. Hematopoietic stem cell transplantation in childhood inherited bone marrow failure syndrome. Bone Marrow Transplant 41, 127-132 (2008).

3. Chhabra P et al. Pediatric bone marrow failure: Clinical, hematological and targeted next generation sequencing data. Blood Cells, Molecules, and Diseases 87, 102510 (2021).

4. Ghemlas I et al. Improving diagnostic precision, care and syndrome definitions using comprehensive next-generation sequencing for the inherited bone marrow failure syndromes. J Med Genet 52, 575-584 (2015).

5. Muramatsu H et al. Clinical utility of next-generation sequencing for inherited bone marrow failure syndromes. Genetics in Medicine 19, 796-802 (2017).

6. Shimamura A. Aplastic anemia and clonal evolution: germ line and somatic genetics. Hematology 1, 74-82 (2016).

7. Gálvez E et al. Next-generation Sequencing in Bone Marrow Failure Syndromes and Isolated Cytopenias: Experience of the Spanish Network on Bone Marrow Failure Syndromes. HemaSphere 5, e539 (2021).

8. Keel SB et al. Genetic features of myelodysplastic syndrome and aplastic anemia in pediatric and young adult patients. Haematologica 101, 1343-1350 (2016).

9. Samarasinghe S, Veys P, Vora A, Wynn R. Paediatric amendment to adult BSH Guidelines for aplastic anaemia. Br J Haematol 180, 201-205 (2018).

10. Hamada M et al. Diagnostic Whole Exome Sequencing for 166 Patients with Inherited Bone Marrow Failure Syndrome. Blood 136 (Supplement 1), 9-9 (2020).

11. Zhang MY et al. Genomic analysis of bone marrow failure and myelodysplastic syndromes reveals phenotypic and diagnostic complexity. Haematologica 100, 42-48 (2015).

12. An W et al. The Significance of the Next Generation Targeted Sequencing in the Precise Diagnosis of Pediatric Acquire Aplastic Anemia and Inherited Bone Marrow Failure Syndromes. Blood 128, 3908-3908 (2016).

13. Teo JT et al. Clinical and Genetic Analysis of Unclassifiable Inherited Bone Marrow Failure Syndromes. Pediatrics 122, 139-148 (2008).

14. Bluteau O et al. A landscape of germ line mutations in a cohort of inherited bone marrow failure patients. Blood 131, 717-732 (2018).

15. Davidsson J et al. SAMD9 and SAMD9L in inherited predisposition to ataxia, pancytopenia, and myeloid malignancies. Leukemia 32, 1106-1115 (2018).

16. Wong JC et al. Germline SAMD9 and SAMD9L mutations are associated with extensive genetic evolution and diverse hematologic outcomes. JCI Insight 3, 121086 (2018).

17. Schwartz JR, Wlodarski MW, Klco JM. Role of Genetic Evolution and Germline Mutations in SAMD9 and SAMD9L Genes. Blood 134 (Supplement_1), SCI-33-SCI-33 (2019). N

18. Hakkarainen M et al. Multinational Study on the Clinical and Genetic Features of the ERCC6L2-Disease. Blood 138(Supplement 1), 864-864 (2021).

19. Douglas SPM et al. ERCC6L2 defines a novel entity within inherited acute myeloid leukemia. Blood 133, 2724-2728 (2019).

20. Germeshausen M et al. MECOM-associated syndrome: a heterogeneous inherited bone marrow failure syndrome with amegakaryocytic thrombocytopenia. Blood Advances 2, 5986-596 (2018).

21. Lee PY. Vasculopathy, Immunodeficiency, and Bone Marrow Failure: The Intriguing Syndrome Caused by Deficiency of Adenosine Deaminase 2. Front Pediatr 6, 282 (2018).

22. Özen S et al. A Monogenic Disease with a Variety of Phenotypes: Deficiency of Adenosine Deaminase 2. J Rheumatol 47, 117-125 (2020).

23. Cipe FE, Celiksoy MH, Erturk B, Aydogmus Ç. Cyclic manner of neutropenia in a patient with HAX-1 mutation. Pediatric Hematology and Oncology 35, 181-185 (2018).

24. Zhang H et al. Longitudinal study of 2 patients with cyclic thrombocytopenia, STAT3 , and MPL mutations. Blood Advances (2022); https://doi.org/10.1182/bloodadvances.2021006701.

S.N	parameters	Suspected IBMFS group (106) 62% Pediatric (79) 74.5% Adult (27) 25.5%		IAA group (65) 38% Pediatric (31) 48% Adult (34) 52%
1	Age	(79) 74.5%; Mean - 4.4 y	(27) 25.5%; Mean - 23 y	(31) 47.7%; Mean - 8.3 y	(34) 52.3%; Mean - 23.7 y
2	Gender	M- 48; F- 31; Ratio 1.5:1	M- 25; F-2; Ratio 12.5:1	M- 15; F- 16; Ratio 1:1	M- 30; F-4; Ratio 7.5:1
3	Hematological presentation and bone marrow findings	Cytopenias i) Isolated anemia:- (29) 37% ii) Isolated neutropenia:-(7) 9% iii) Isolated Thrombocytopenia:-(4) 5% iv) Bicytopenia:-(13) 16% v) Pancytopenia:- (26) 33% Bone Marrow Findings Erythroblastopenia:- (21) 27% Hypocellular marrow:-(34) 43% Granulocytic maturation arrest:-(7) 9% Reduced megakaryocytes:- (4) 5% MDS/AML/MPAL:-(12) 15% vi) B-ALL:-(1) 1%	Cytopenias i) Isolated anemia:- (1) 4% ii) Isolated Thrombocytopenia:-(1) 4% iii) Bicytopenia:-(6) 22% iv) Pancytopenia:- (19) 70% Bone Marrow Findings Hypocellular marrow:-(16) 59% Reduced megakaryocytes:- (1) 4% MDS/AML/MPAL:-(7) 26% B-ALL/TLBL/HL:-(3) 11%	MAA:-(1) 3% SAA:-(27) 87% VSAA:-(3) 10%	MAA:-(1) 3% SAA:-(33) 97% BM Fibrosis:-(2) 6%
4	Extra hematological involvement / constitutional abnormalities (suspected IBMFS group)* or NGS reason (IAA group)	Dys:-(12) 15% SS:-(11) 14% Sk/Thumb:-(13) 16% N/S/H:-(8) 10% CNS:(9) 11% GU:-(1) 1% Others:-(7) 9%	SS:-(2) 7% Sk/Thumb:-(3) 11% N/S/H:-(10) 37% GU:-(1) 4% Others:-(2) 7%	i) Pre-IST screen:-(14) 45% ii) Partial IST response:-(6) 19% iii) No IST response:-(11) 36	i) Pre-IST screen:-(27) 79% ii) No IST response:-(2) 6% iii) Pre-HSCT:-(5) 15%
5	Functional/cytogenetics/FISH studies	CBS Positive:-(3) 4% Monosomy 7:-(2) 2.5%	CBS Positive:-(1) 4% Monosomy 7:-(1) 4%	-	Minor PNH Clone:-(3) 9%
6	Family history/Consanguinity data	Family history Positive:-(6) 8% Consanguinity:-(1) 1%	Family history Positive:-(8) 30%	-	-
7	NGS positivity	Targeted 33 gene panel:-(37) 47%; Whole exome sequencing :-(3) 21%	Targeted 33 gene panel:-(16) 59%	Targeted 33 gene panel:-(7) 22.5%	Targeted 33 gene panel:-(3) 9%

Table 1: Clinical and Hematological characteristics of suspected IBMFS group and IAA group cases with bone marrow failure

Footnote:- IBMFS: Inherited bone marrow failure syndrome; IAA: Idiopathic aplastic anemia; MDS: Myelodysplastic syndrome; AML: Acute myeloid leukemia; MPAL: Mixed phenotype acute leukemia; B-ALL: B-cell acute lymphoblastic leukemia; TLBL: T-cell lymphoblastic lymphoma; HL: Hodgkin’s lymphoma; MAA: Moderate aplastic anemia; SAA: Severe aplastic anemia; VSAA: Very severe aplastic anemia; BM Fib: Bone marrow fibrosis; Dys: Facial dysmorphism; SS: Short stature; Sk/Thumb: Skeletal and or thumb anomalies like polydactyly, clinodactyly, bifid thumb etc; N/S/H: Nail and or skin and or hair changes; CNS:Neurological abnormalities like seizures, microcephaly, dolichocephaly, global developmental delay; GU: Genitourinary anomalies like single or ectopic kidney; IST: Immunosuppressive therapy; HSCT: Hematopoietic stem cell transplant; PNH: Paroxysmal nocturnal hemoglobinuria; CBS: Chromosomal breakage studies; FISH: Fluorescence in situ hybridization; *Some cases had more than one constitutional abnormality.

Table 2: Genetic data of pathogenic/likely pathogenic variation positive cases (suspected IBMFS group; n=50)
Case ID	Age at presentation/Gender	Haematological and Clinical Phenotype	Gene variations on Targeted NGS Panel/WES Result	Codon	Protein change	Nature	Heterozygous (HETRO)/ Homozygous (HOMO) (VAF)	ACMG Classification	Final Diagnosis*
C1	5/M	Anemia with erythroblastopenia in bone marrow; Early greying of hair; Nail dystrophy; congenital pedal oedema	TINF2	c.706C>T	p.Pro236Ser	Missense	HETRO (49.5%)	Likely Pathogenic	Dyskeratosis Congenita¹⁰
C3	1.5/M	Anemia with erythroblastopenia in bone marrow; Dysmorphism; Cleft palate	RPS19	c.71+1C>G	p?	Splice site	HETRO (50.6%)	LPLikely Pathogenic	Diamond Blackfan Anemia¹⁰
C4	2months/M	Anemia with erythroblastopenia in bone marrow; Short stature; Skeletal malformation	RPL5	c.381_382insT	p.Asp128Ter	INDEL	HETRO (52.7%)	Pathogenic	Diamond Blackfan Anemia
C5	6/M	Acute Myeloid Leukemia with myelodysplasia related changes in bone marrow;1.5 year long history of thrombocytopenia	DKC1 +RUNX1	c.264-3CA>AT; c.428G>A	p?; p.Gly143Glu	Splice site and Missense	HEMI+Somatic (29%)	Likely Pathogenic	Dyskeratosis Congenita with acute myeloid leukemia development
C7	2 months/M	Anemia with erythroblastopenia in bone marrow; Short Stature	RPS19	c.184C>T	p.Arg62Trp	Missense	HETRO (54%)	Pathogenic	Diamond Blackfan Anemia¹⁰
C8	8/M	Anemia with hypocellular marrow for age; Bilateral ear lobe abnormalities	TERT	c.1552G>T	p.Ala518Ser	Missense	HETRO (40%)	Likely Pathogenic	Dyskeratosis Congenita
C9	2 months/M	Anemia with erythroblastopenia in bone marrow	RPS19	c.280C>T	p.Arg94Ter	Nonsense	HETRO (40%)	Pathogenic	Diamond Blackfan Anemia¹⁰
C10	8/F	Pancytopenia with hypocellular bone marrow; Short stature; Hypopigmented skin; 42% Ringed sideroblasts; Chromosomal breakage study -Positive	FANCC	c.195delC + c.66G>A	p.Thr66GlnfsTer16; p.Trp22Ter	INDEL and missense	Comp HETRO (53%+50%)	Pathogenic	Fanconi Anemia¹⁰
C11	2/F	Pancytopenia with hypocellular bone marrow; Neurological issues with seizures	SAMD9	c.3176A>G	p.His1059Arg	Missense	HETRO (54%)	Likely Pathogenic	SAMD9 related bone marrow failure ¹⁰
C12	11/M	Pancytopenia with hypocellular bone marrow; Dysmorphism; Early hair greying; Family history with early greying hair in first cousin	TERT	c.2026_2027insG	p.Leu676ArgfsTer30	INDEL	HETRO (42%)	Pathogenic	Dyskeratosis Congenita¹⁰
C13	9/M	Anemia with erythroblastopenia in bone marrow; Sinus pericranii and Family history of similar illness in sibling	RPS29	c.139A>T	p.Ile47Phe	Missense	HETRO (40%)	Likely Pathogenic	Diamond Blackfan Anemia
C14	3 months/F	Anemia progressed to bicytopenia along with hypocellular bone marrow; Abnormal thumb	RPS19	c.392T>G	p.Leu131Arg	Missense	HETRO (48%)	Likely Pathogenic	Diamond Blackfan Anemia¹⁰
C15	1/F	Pancytopenia with hypocellular bone marrow; Dysmorphism; skeletal malformation	RPL5	c.701A>G	p.Asp234Gly	Missense	HETRO (50%)	Pathogenic	Diamond Blackfan Anemia¹⁰
C16	6/M	Bicytopenia with hypocellular bone marrow; Maturation arrest in granulocytic series	ELANE	c.301G>A	p.Val101Met	Missense	HETRO (50%)	Pathogenic	Congenital Neutropenia
C17	7/F	Pancytopenia with hypocellular bone marrow; Dysmorphism; Right ectopic kidney; Chromosomal breakage study- Positive	FANCA	c.3761_3762delAG	p.Glu1254GlyfsTer23	INDEL	HOMO (99%)	Pathogenic	Fanconi Anemia
C20	4 months/M	Anemia with erythroblastopenia in bone marrow; Consanguinity history positive; Elder sibling has cleft palate	RPL5	c.169_172del AACA	p.Asn57fs	INDEL	HETRO (47%)	Pathogenic	Diamond Blackfan Anemia¹⁰
C21	11/M	Neutropenia with granulocytic maturation arrest in bone marrow	HAX1	c.430delG	p.Val144SerfsTer70	INDEL	HOMO (100%)	Pathogenic	Congenital Neutropenia
C22	2 months/M	Anemia with erythroblastopenia in bone marrow; Bifid thumb and Congenital cardiac anomaly	RPS19	c.320_321insT	p.Pro108SerfsTer8	INDEL	HETRO (55%)	Pathogenic	Diamond Blackfan Anemia¹⁰
C23	9/F	Pancytopenia with hypocellular bone marrow; Short Stature; Dysmorphism; Eczema; Microcephaly	CSF3R	c.1254delT	p.Thr420LeufsTer13	INDEL	HOMO (100%)	Likely Pathogenic	Congenital Neutropenia¹⁰
C24	3 months/M	Anemia with erythroblastopenia in bone marrow; Bifid thumb	RPS19	c.411+2T>A	p?	Splice site	HETRO (51%)	Likely Pathogenic	Diamond Blackfan Anemia¹⁰
C25	8/M	Anemia with erythroblastopenia in bone marrow; Eczema	SRP72	c.574C>G	p.Leu192Val	Missense	HET(52%)	Likely Pathogenic	SRP72 related bone marrow failure
C26	1 month/M	Pancytopenia with hypocellular bone marrow; Mild increase immature cells on flow cytometry	SAMD9 + HAX1	c.2509_2510insA + c.676C>T	p.Arg837LysfsTer16; p.Arg226Cys	INDEL and Missense	HETRO (48%)+(51%)	Likely Pathogenic	SAMD9 related bone marrow failure leading to loss of function mutation on other allele and myelodysplasia
C28	10/M	Pancytopenia with hypocellular bone marrow; Dysmorphism; Hypoplastic thumbs	FANCG	c.182delC +c.118C>T	p.Pro61LeufsTer11; p.Gln40Ter	INDELand Nonsense	Comp HETRO (53%+50%)	Pathogenic	Fanconi Anemia
C29	8/M	Pancytopenia with hypocellular bone marrow; Dysmorphism	FANCA + FANCD2	c.3239G>A +c.3210T>G	p.Arg1080Gln; p.His1070Gln	Missense and Missense	Comp HETRO (53%+50%)	Likely Pathogenic	Fanconi Anemia
C30	11 months/F	Isolated thrombocytopenia with reduced megakaryocytes on bone marrow	MPL	c.212+5G>A	p?	Splice site	HOMO (100%)	Likely Pathogenic	Congenital amegakaryocytic throwbocytopenia
C31	8/F	Acute Myeloid Leukemia with myelodysplasia related changes in bone marrow; Past history of cytopenias since 4 years	SBDS	c.258+2T>C	p?	Splice site	HOMO (100%)	Pathogenic	Shwachman Diamond syndrome with acute myeloid leukemia development
C32	7/F	Acute Myeloid Leukemia; Clinodactyly; Thenar atrophy	MECOM	c.946C>T	p.Pro316Ser	Missense	HETRO (45%)	Likely Pathogenic	MECOM related bone marrow failure with acute myeloid leukemia
C33	3/M	Anemia with hypocellular bone marrow; Short Stature	RPL5	c.535C>T	p.Arg179Ter	Nonsense	HETRO (47%)	Pathogenic	Diamond Blackfan Anemia
C34	11/M	Acute Myeloid Leukemia with myelodysplasia related changes in bone marrow; Short stature; Monosomy 7 on FISH	GATA-2	c.1082G>A	p.Arg361His	Missense	HETRO (53%)	Likely Pathogenic	GATA-2 related bone marrow failure syndrome with acute myeloid leukemia
C35	2 months/M	Pancytopenia with hypocellular bone marrow; Skin/nail/hair changes; Retinopathy	TINF2	c.845G>A	p.Arg282His	Missense	HET (52%)	Pathogenic	Dyskeratosis Congenita
C36	8/M	Pancytopenia with hypocellular bone marrow; Skin/nail/hair	DKC1	c.1058C>T	p.Ala353Val	Missense	HEMI (100%)	Pathogenic	Dyskeratosis Congenita
C37	6 month/M	Anemia with erythroblastopenia in bone marrow	RPL5	c.190-3T>G	p?	Splice site	HETRO (53%)	Pathogenic	Diamond Blackfan Anemia
C64	4/M	Bicytopenia with hypocellular bone marrow; Lymphoid aggregates on bone marrow biopsy	PARN	c.47_48insATCTTCA	p.Tyr16Ter	INDEL	HOMO (99%)	Pathogenic	Dyskeratosis Congenita
C65	7/M	Pancytopenia with hypocellular bone marrow; Dysmorphism; Short Stature; Abnormal hypopigmentation; Microcephaly; Chromosomal breakage study- Negative	FANCF	c.496C>T+c.388dup	p.Gln166Ter+p.Gln130ProfsTer23	Nonsense and INDEL	Comp HETRO (50%+48%)	Pathogenic	Fanconi Anemia
C66	9 months/M	Anemia with hypocellular bone marrow and skin changes	CERC1/ADA2	c.11O8A>T+c.116T>A	p.Asn370Tyr; p.Leu39Ter	Missense and Nonsense	Comp HETRO (50%+49%)	Pathogenic	Deficiency of adenosine deaminase 2 syndrome (DADA2)
C38	21/M	Pancytopenia with hypocellular bone marrow; Family history of aplastic anemia positive	TERT	c.3158-2CA>A	p?	Splice site	HETRO (45%)	Pathogenic	Dyskeratosis Congenita
C39	21/M	Nodular lymphocyte predominant Hodgkin’s Lymphoma; History of cytopenias and hypocellular marrow	SAMD9	c.2693A>C	p.Glu898Ala	Missense	HETRO (51%)	Likely Pathogenic	SAMD9 related bone marrow failure with Hodgkin lymphoma
C40	25/M	Pancytopenia with hypocellular bone marrow; Nail changes	TERT	c.2452G>A+c.2071C>T	p.Val818Met; p.Arg691Cys	Missense and Missense	Comp HETRO (53%+58%)	Likely Pathogenic	Dyskeratosis Congenita
C41	13/M	Pancytopenia with hypocellular bone marrow; Skin pigmentation; Early hair greying; Leukoplakia; Short Stature; Chromosomal breakage study- Positive	FANCG	c.85-2C>A	p?	Splice site	HOMO (99%)	Pathogenic	Fanconi Anemia
C42	25/M	Pancytopenia with hypocellular bone marrow; Nail dystrophy; Lacy pigmentation; Leukoplakia	DKC1	c.1204G>A	p.Gly402Arg	Missense	HEMI (100%)	Pathogenic	Dyskeratosis Congenita
C43	16/M	T-cell Lymphoblastic Lymphoma; Family history of malignancy positive	TINF2	c.706C>T	p.Pro236Ser	Missense	HETRO (49%)	Pathogenic	Dyskeratosis Congenita
C44	26/F	Acute Myeloid Leukemia with myelodysplasia related changes in bone marrow; Hyperpigmented rashes face	SRP72	c.574C>G	p.Leu192Val	Missense	HETRO (53%)	Likely Pathogenic	SRP72 related bone marrow failure with acute myeloid leukemia
C45	15/M	Acute Myeloid Leukemia; Past significant history of chronic pancytopenia	CSF3R	c.185G>T	p.Try477Leu	Missense	HETRO (42%)	Pathogenic	Congenital Neutropenia leading to acute myeloid leukemia development
C46	16/M	Bicytopenia with hypocellular bone marrow; Non transfusion dependent thalaasemia with recurrent thrombocytopenia	GATA-1	c.647G>A	p.Arg216Gln	Missense	HEMI (100%)	Pathogenic	X-linked thrombocytopenia with thalassemia
C47	34/M	Pancytopenia with hypocellular bone marrow; Family History of aplastic anemia present	THPO	c.482G>A + c.466C>T	p.Arg161His + p.Leu156Phe	Missense and Missense	Comp HETRO (53%+50%)	Pathogenic	THPO related thrombocytopenia leading to marrow failure
C48	23/M	Pancytopenia with hypocellular bone marrow; Clinodactyly; Cafe-au-lait spots	RPS26	c.4-2C>T	p?	Splice site	HETRO (40%)	Likely Pathogenic	Diamond Blackfan Anemia
C49	24/M	Bicytopenia with hypocellular bone marrow; Lymphoedema; Vitiligo	SAMD9	c.3176A>G	p.His1059Arg	Missense	HETRO (48%)	Likely Pathogenic	SAMD9 related bone marrow failure
C50	17/M	Pancytopenia with hypocellular bone marrow; Dysmorphism	MECOM	c.2765A>G	p.His922Arg	Missense	HETRO (48%)	Likely Pathogenic	MECOM related bone marrow failure
C52	28/M	Isolated thrombocytopenia; Chronic and Cyclical	MPL	c.1120A>G	p.Thr374Ala	Missense	HETRO (48%)	Likely Pathogenic	MPL related cyclical thrombocytopenia
C53	25/M	Myelodysplastic syndrome on bone marrow; Monosomy 7 and 20q del on FISH	SAMD9+RUNX1(SOMATIC)	c.3812T>C; c.521G>A	p.Phe1271Ser+ p.Arg174Gln	Missense and Missense	HETRO (40%)+ HETRO (14%)	Likely Pathogenic	SAMD9 related bone marrow failure with myelodysplastic syndrome development

Footnote:- variations; IHETRO: Heterozygous; HOMO: Homozygous; Comp HETRO: Compound Heterozygous; HEMI: Hemizygous; FISH: Fluorescent in situ hybridization INDEL: Insertion-Deletion

Table 3: Genetic data of pathogenic/likely pathogenic variation positive cases (IAA group; n=8)
Case ID	Age at presentation/Gender	Haematological and Clinical Phenotype	Gene variations on Targeted NGS Panel/WES Result	Codon	Protein change	Nature	Heterozygous (HETRO)/ Homozygous (HOMO) (VAF)	ACMG Classification	Final Diagnosis*
C54	10.5/F	SAA; Partial IST response 8 months	SAMD9	c.3644C>T	p.Pro1215Leu	Missense	HETRO (42%)	Likely Pathogenic	SAMD9 related bone marrow failure
C55	12/M	SAA; No IST response at 7 months; Partial Danazol response	DKC1	c.264-3CA>AT	p?	Splice site	HEMI (99%)	Pathogenic	Dyskeratosis Congenita
C57	12/F	SAA; Partial IST response at 9 months	SAMD9	c.4666G>A	p.Ala1556Thr	Missense	HETRO (51%)	Likely Pathogenic	SAMD9 related bone marrow failure
C58	11/M	SAA; No IST response at 8 months	MECOM	c.276A>T	p.Lys92Asn	Missense	HETR0 (51%)	Likely Pathogenic	MECOM related bone marrow failure
C59	10/F	SAA; Pre-IST screen	FANCG + FANCA	c.366G>C; c.2236G>T	p.Trp122Cys; p.Ala746Ser	Missense; Missense	Comp HETRO (53%+50%)	Likely Pathogenic	Fanconi Anemia
C60	4.5/M	SAA; Pre-IST screen	DKC1	c.1192C>G	p.Leu398Val	Missense	HEMI (100%)	Pathogenic	Dyskeratosis Congenita
C61	23/M	SAA; Pre-IST screen	FANCG +FANCA	c.1074delG; c.3551G>C	p.Arg359Glyfs Ter44; p.Arg1184Pro	INDEL; Missense	Comp HETRO (53%+50%)	Likely Pathogenic	Fanconi Anemia
C63	47/M	SAA; Pre-IST screen	FANCC+FANCD2	c.1555G>C; c.1540C>T	p.Asp519His; p.His514Tyr	Missense; Missense	HETRO (51%+51%)	Likely Pathogenic	Fanconi Anemia

Footnote: SAA: Severe aplastic anemia; IAA: Idiopathic aplastic anemiaIST: Immunosuppressive therapy; HETRO: Heterozygous; HOMO: Homozygous; Comp HETRO: Compound Heterozygous; HEMI: Hemizygous; INDEL: Insertion-Deletion

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Genetic data of Variants of unknown significance (VUS) cases (n=8)

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Targeted next generation sequencing panel based screening and diagnosis of bone marrow failure cohort reveals diverse genomic landscape of pathogenic germline variants

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