A genomic perspective of benign hematological disorders in the era of next-generation sequencing

A genomic perspective of benign hematological disorders in the era of next-generation sequencing
Review the core principles of Next-Generation Sequencing (NGS) technologies relevant to hematology
Differentiate between the applications of targeted panels, WES, WGS in benign hematology
Analyze the challenges and limitations of implementing NGS in clinical practice
Evaluate the impact of NGS on personalized management and genetic counseling
The role of NGS in diagnosing benign hematological disorders through case studies
NGS provides rapid, comprehensive diagnosis with a high diagnostic yield. It discovers novel mutations and is crucial for differentiating between inherited and acquired conditions, which directly impacts clinical management. It facilitates genetic counseling and family planning. In essence, NGS has ended the diagnostic odyssey for many of our patients, providing clarity and guiding personalized care

1. A genomic perspective of benign hematological disorders in the era of next-generation sequencing Dr. Amir Abdelazim , MD Biochemical and Molecular Pathology Kuwait cancer control center NGS Laboratory -2025

2. Review the core principles of Next-Generation Sequencing (NGS) technologies relevant to hematology Differentiate between the applications of targeted panels, WES, WGS in benign hematology Analyze the challenges and limitations of implementing NGS in clinical practice Learning Objectives Evaluate the impact of NGS on personalized management and genetic counseling The role of NGS in diagnosing benign hematological disorders through case studies

3. NGS Pathway Coverage depth (30-100x for germline, 500-1000x for somatic) Uniformity of coverage, GC bias, mapping quality

4. Clinical Utility of NGS Diagnostic Capabilities Early Diagnosis Rapid identification of genetic causes Detection of novel or rare mutations Differential diagnosis in complex cases Carrier Detection & Family Screening Identification of at-risk family members Genetic counseling for family planning Cascade testing in related individuals Clinical Applications Risk Stratification Predicting disease progression Assessment of malignancy risk Surveillance planning for complications Guiding Therapy Personalized treatment selection Informing transplantation decisions Avoiding ineffective or harmful therapies NGS Approaches Targeted Panels Focus on specific genes Whole Exome Panel All protein-coding regions Whole Genome Complete genomic DNA Key Quality Standards The College of American Pathologists (CAP) and Association for Molecular Pathology (AMP) guidelines establish minimum thresholds for NGS assay validation, including sensitivity ≥95%, specificity ≥98%, and reproducibility of ≥95%.

5. Targeted resequencing panels(t-NGS) Whole Exome Sequencing (WES) Whole Genome Sequencing (WGS) Clinical Utility of NGS

6. High-throughpu t sequencing enables simultaneous analysis of multiple genes. Detects a wide range of mutations , including SNVs, indels, fusions, and CNVs. Faster turnaround time by eliminating sequential testing. Personalized therapy selection based on actionable mutations. False positives/negatives – sequencing errors can affect variant calling. Distinguishing germline vs. somatic mutations requires additional filtering & Additional sample Data interpretation complexity – bioinformatics pipelines are evolving. Cost and accessibility – despite cost reductions, affordability varies globally. Advantages Challenges Advantages and challenges of clinical NGS

7. only in cases where acquired causes excluded Patient provide consent for the test To confirm conditions when there is diagnostic uncertainty Before undertaking splenectomy or other irreversible procedures such as bone marrow transplantation, where the genetic variant should be excluded from a potential stem cell sibling donor Question 1 :When is NGS for inherited D. necessary and/or of additional value in the diagnosis?

8. Early if there are known familial mutations If the clinically examination real uncertain diagnosis If the investigation is equivocal If special test consist with diagnosis for confirmation by NGS Question 2 : At which point in the diagnostic pathway should NGS be used?

9. If known familial mutation If target NGS panel for If target NGS was negative or Genes list not included If Whole exome was negative Question 3:What are the important considerations in choosing the most appropriate NGS method? Target method if available as Sanger Sequencing Target NGS hematology panel WES Re-analysis of WES Confirm CNV included Whole genome if available

10. Variants to which a pathogenic role can be attributed with certainty Variants with a well-known pathogenic role not related to clinical suspicion. Variants with unknown clinical and functional role intronic/splice (noncanonical) · 5' and 3' variants , Known benign or polymorphism Question 4: What criteria should be used for reporting NGS variants identified? Pathogenic Incidental VUS Not reported

11. Question 5:How should variants identified be stored/shared between labs? . Labs should make a reasonable effort to share variants with other labs analysing the same genes . Labs should ensure they are using commonly used transcripts which have been shown to be expressed in erythroblasts Question 6:What criteria are essential for a lab who offer clinical-NGS? Consent for DNA analysis should be sought. Phenotypic information must be provided (for phenotypic- genetoypic ) It should be clear which genes have been tested, the coverage of these genes, and whether or not CNVs are detectable by this method Ideally a false negative rate should be provided The whole panel must be fully validated before use

12. 04 Genotype-driven diagnosis using NGS provides molecular precision that overcomes the limitations of phenotype-based approaches, especially for complex or atypical presentations Key Message results in: no diagnosis overlapping 03 Inappropriate or inadequate treatment approaches Psychological burden for patients and families Missed opportunities for targeted therapy or genetic counseling 02 Studies show 10-40% of patients receive incorrect or no diagnosis when relying solely on phenotype 01 Phenotype-driven diagnosis may lead to diagnostic odysseys Similar symptoms across different disorders (anemia, jaundice, thrombocytopenia) Similar morphologic findings in peripheral blood smears Ambiguous bone marrow morphology Incomplete phenotypic expression Phenotype vs Genotype Driven Diagnosis

13. Inherited bone marrow failure syndromes  benign hematological disorders 06 01 02 03 04 05 MAIN Hereditary hemolytic anemias CONGENITAL METHEMOGLOBINEMIAS Iron-related disorders:   HEREDITARY ERYTHROCYTOSIS Coagulation and bleeding disorders 6 Step HEXAGONAL Shape Infographic Inherited thrombocytopenias THROMBOPHILIA Applications of NGS in benign hematological disorders

14. THALASSEMIA HEMOGLOBINOPATHIES

15. THALASSEMIA AND HEMOGLOBINOPATHIES The molecular genotypes of these patients were tested using the Sanger sequencing, multiplex-ARMS , GAP-PCR and MLPA methods. NGS demonstrates superiority in detecting rare variants, resolving complex hematological cases But there are limitations prevent it from replacing traditional methods as a stand-alone tool.  T-NGS panels often exclude alpha globin genes ( HBA1  and  HBA2 ) due to several challenges: High sequence homology: The extensive similarity between  HBA1  and  HBA2  sequences complicates analysis, increasing the likelihood of false-positive results. Copy number variations (CNVs): Pathogenic variants in globin genes frequently involve CNVs , such as large deletions (e.g., α3.7 deletion) or gene multiplications, which are challenging for NGS to detect accurately. Deep intronic mutations: These mutations may not be captured during exome sequencing, leaving gaps in diagnostic capability. If NGS used : Aligns sequences to reference genome, flags variants, and cross-references with databases HbVar ). Large deletions in alpha genes require complementary methods (MLPA)/or MicroArray -if NGS lacks robust CNV detection. Rare/novel variants need functional studies to confirm pathogenicity. For most cases of thalassemia and hemoglobinopathies, clinical and laboratory fndings provide strong diagnostic clues, often making extensive genetic testing unnecessary. Annually, 300,000 to 500,000 children are born with severe hemoglobinopathies, indicating a significant global health challenge. Detects more than 500 pathogenic variants of thalassemia in a single test. (Traditional methods usually detect less than 30 variants)· Detects novel / rare pathogenic variants

16. This current study updated the HBB gene variations with newly identified variants of HBB gene Case Study : Beta-Thalassemia Major - Novel HBB Variant Patient Presentation: 8-year-old female with severe anemia (Hb 6.5 g/dL) . Microcytic hypochromic anemia (MCV 65 fL ) . Hepatosplenomegaly, growth retardation . Transfusion-dependent since age 1 . Family history: other affected sibling, parents asymptomatic NGS Findings NGS identified a novel variant in the HBB gene:c.118C>T (p.Gln40*) - Nonsense mutation Clinical Impact Confirmed beta-thalassemia major diagnosis Early management: Transfusion protocol optimized. Precise genetic counseling for family planning

17. CONGENITAL METHEMOGLOBINEMIAS Molecular testing allows for precise identification of these genetic abnormalities, differentiating between: autosomal recessive cytochrome b5 reductase deficiency (  CYB5R3  gene ) dominant HbM disease. (globin genes ( HBB  or  HBA1/2 ) ) mutations in the  CYB5R3  gene can result in : Type 1 methemoglobinemia, limited to red blood cells, or Type 2, which involves multiple tissues and presents with severe neurological manifestations. In cases of HbM disease, single-point mutations in the globin genes result in amino acid substitutions that stabilize the ferric (Fe 3+ ) state of heme. Molecular analysis helps avoid misdiagnosis with acquired forms of methemoglobinemia and provides a foundation for developing targeted therapeutic interventions.

18. Hereditary Hemolytic Anemias

19. Clinical Spectrum of Hereditary Hemolytic Anemias Membrane defects: Hereditary spherocytosis, elliptocytosis, pyropoikilocytosis Clinical: Splenomegaly, jaundice, increased MCHC, spherocytes on smear Genes:  SPTB, SPTA1, ANK1, SLC4A1, EPB42 Enzyme deficiencies: Glycolytic and pentose phosphate pathway disorders Pyruvate Kinase (PK) deficiency: Most common glycolytic defect, hemolytic crisis, transfusion-dependent in severe cases G6PD deficiency: X-linked, oxidant-induced hemolysis, >230 variants identified Genes:  PKLR, G6PD, GPI, TPI1, ALDOA Hemoglobinopathies : Structural or synthesis defects affecting globin genes Sickle cell disease, unstable hemoglobin variants, thalassemias Genes:  HBB, HBA1/HBA2, and other globin genes Complex phenotypes : Disorders with combined or unusual presentations Congenital dyserythropoietic anemias, combined membrane-enzyme defects Cases with cryptic or multigenic inheritance patterns NGS Impact on HHA Diagnosis NGS has revealed extensive genetic heterogeneity in HHA, providing definitive diagnosis in 40-70% of previously unexplained cases and identifying novel pathogenic mechanisms and phenotype-genotype correlations. Diagnosis by routine morphology and biochemical analysis may be difficult, particularly in transfusion-dependent

20. 54-year -old male has chronic hemolytic anemia since childhood . Splenomegaly (16 cm)- Cholecystectomy for gallstones at 16 years old- Non-cirrhotic liver steatosis with liver iron overload, treated with oral iron chelator (HFE: heterozygous H63D)- Moderate anemia since childhood:. Hb 9-9.5 g/dL, MCV 105 fL , PLT 312 x10^9/L, WBC 7.8 x10^3/ mcL (normal differential count). LDH 1.6 xULN , Unc Bil 3.7 mg/dL, Hp <20 mg/dL, retics 190 x10^9/L, DAT neg, PNH neg newborn male , Elective labor induction in a local Hospital at 34 GW for anhydramnios Pale skin, irregular breathing, Hb 6.6 g/dL, MCV 124 fL , Retics 186H, Unconjug.bilirubin13.5H , Osmotic fragility tests Normal APGAR score: 5 (1'), 5 (5') Transfused with 1 packed RBC unit > Transferred to Neonatal ICU: · 3 pRBC units · s.c. rhEPO (5 administrations), folic acid, cyanocobalamin Patient: 25-year-old male History:Chronic hemolysis, splenomegaly, negative DAT Lab Values:Hb : 10.4 g/dL, Reticulocytes: 237×10⁹/L, MCHC: 346 g/L, Os Hereditary Spherocytosis for : Splenectomy motic fragility test: Positive, No history of anemia, transfusions · Non consanguinity of parents Parental consanguinity Mother: Hb HPLC neg Father :Hb HPLC neg No history of anemia, transfusions · Non consanguinity of parents NGS revealed homozygous pathogenic variant in PKLR gene PKLR c.1706G>A (p.Arg569Gln) , confirming PKD diagnosis rather than HS, despite similar clinical presentation and positive osmotic fragility NGS revealed compound heterozygous pathogenic variant in PKLR gene NGS study Negative One year after onset: Re-test By NGS panel include CNV r eveal Large deletion of 1149 base pairs that cause exon 11 skipping of the  PKLR  gene c.1437-518_1618+440del1140 Confirmed by MicroArray test Pyruvate kinase deficiency FDA has approved  Pyrukynd ( mitapivat ) tablets    to treat hemolytic anemia (a disorder in which red blood cells are destroyed faster than they can be made) in adults with pyruvate kinase (PK) deficiency. Avoided unnecessary splenectomy (limited benefit in PKD vs HS) Confirmed autosomal recessive inheritance for genetic counseling Enabled targeted management of PKD-specific complications Facilitated family testing and counseling Triple cases PKD 1 2 3 NGS result Impact of NGS Family history

21. 1 For diagnosis Pyruvate kinase deficiency, in situations where PK enzyme levels may be inconclusive or potentially confounded NGS is an effective 2 Most pathogenic variants identified were small sequence variants, but large deletions in PKLR are not uncommon and should be part of the NGS panel. PKLR pathogenic varaint 3 over 40% of the pts tested were >30 yrs of age, some of whom were without a previous diagnosis, emphasizing the complexity of a PK deficiency diagnosis in pts with lifelong anemia. complexity of a PK deficiency Pyruvate kinase deficiency

22. Congenital dyserythropoietic anemia type II (CDAN2) A R presents : jaundice and normocytic anemia (mild to severe) in neonates but in some cases symptoms may be so mild that diagnosis can be delayed until adulthood. Splenomegaly and hepatomegaly Less commonly, posterior mediastinal or paravertebral masses (that consist of extramedullary hemopoietic tissue) are present. In rare cases, hydrops fetalis (see this term) has been associated with CDA II. Long-term complications include secondary hemochromatosis that, if left untreated, can lead to organ damage. Confirmed by Sanger sequencing. Both parents are heterozygous carrier. 3 mont hs neonate born 2018 Presented with : Normocytic anemia/ erythroblasts Increased serum unconjugated bilirubin Increased reticulocytes No family history signs and symptoms mild and overlap with those of other disorders NGS analysis detected a compound heterozygous variant in SEC23B gene . Both variants are previously reported as pathogenic variants in patients suffered from congenital dyserythropoietic anemia type II (CDAN2 The first one is a nonsense variant c.1648C>T, p.(Arg550*) in exon 14 and the second one is a missense variant c.1489C>T, p.(Arg497Cys) in Exon 13. CDA type I:   CDAN1  or  CDIN1  gene CDA type II:  SEC23B  gene CDA type III:  KIF23  or  RACGAP1  gene CDA type IV: KLF1  gene Case study

23. used the targeted gene panel approach of NGS in identifying disease causing mutations in unexplained haemolytic anaemias/dyserythropoietic anaemia . Variants in  Piezo1  gene causing rare haemolytic anaemia such as hereditary xerocytosis (HX) could be identified in five patients of unexplained haemolytic anaemia

24. I nherited B one M arrow F ailure S yndromes

25. Suspected case of IBMFS Pancytopenia with hypocellular marrow Stress Cytogenetics Abnormal Normal/Equivocal Mutation testing for Fanconi anemia Mutation testing for mosaic Fanconi anemia / Dyskeratosis congenita / evolved Shwachmann Diamond syndrome or Amegakaryocytic thrombocytopenia Single cytopenia Anemia Mutation testing for Diamond Blackfan anemia Neutropenia Exocrine pancreatic dysfunction/cystic fibrosis Thrombocytopenia Bilateral absent radil with normal thumbs Consider Severe congenital neutropenia Mutation testing for Shwachmann Diamond syndrome Mutation testing for Amegakaryocytic thrombocytopenia Mutation testing for TAR- Thrombocytopenia absent radii All patients need bone marrow biopsy, cytogenetic studies for clonal abnormalities, chromosome breakage for Fanconi anemia by diepoxybutane [DEB] test, flowcytometry for CD55/59 for paroxysmal nocturnal hemoglobinuria (PNH), and telomere length study for dyskeratosis congenita (DC). Initial testing involves stress cytogenetics using agents such as diepoxybutane or mitomycin C. However, tests have limitations. For example, somatic mosaicism may lead to false-negative results, and other diseases, such as Nijmegen breakage syndrome, may produce false-positive findings in chromosome breakage. I nherited B one M arrow F ailure S yndromes

26. Patient: 8-year-old female with Progressive cytopenias , no classic physical stigmata of FA, Lab Values : Pancytopenia, hypocellular bone marrow, Chromosomal breakage test inconclusive Diagnosis: Idiopathic Aplastic Anemia under Standard immunosuppression 15-year-old with Pancytopenia Persistent pancytopenia for 6+ months conditioning Subtle physical anomalies (short stature, café-au-lait spots). Initially diagnosed as idiopathic aplastic anemia Non-responsive to immunosuppressive therapy Chromosomal breakage test inconclusive HSCT being considered but donor selection uncertain 8ys-male - Bicytopenia ( PLt and RBC), hypocellular bone marrow, below 5th percentile in all parameters. Born 2012 teste 2020 Third child born to consanguineous parents , developmental delay NGS identified biallelic pathogenic variants in the FANCA gene, diagnostic of Fanconi Anemia, despite absence of classic physical features that often accompany this syndrome. NGS Revealed Biallelic pathogenic variants in FANCG gene Compound heterozygous mutations: c.1066C>T (p.GIn356*) and c.307+1G>C Diagnosis revised: Fanconi Anemia NGS analysis detected new homozygous likely pathogenic frameshift insertion variant in FANCA gene, exon 20, chr16:89845222; c.1812_1813insA, p.Glu605fs*8. Drastically altered management strategy Avoidance of alkylator-based immunosuppression which could be harmful Triple cases FA NGS result Impact of NGS Implemented cancer surveillance protocol a high predisposition to cancer. NGS diagnosis will help us to plan appropriate treatment and also to select suitable donor for hematopoietic stem cell transplantation (HSCT) and to plan for genetic counseling.  Aplastic Anemia Initial Diagnosis NGS Panel Testing FANCA, FANCB, FANCC,FANCG ... Fanconi Anemia Revised Diagnosis Fanconi Anemia 1 2 3 FANCA  are the most frequent among FA patients worldwide which account for 60- 65%. BMFS should be considered in children presenting with isolated aplastic anemia (AA), myelodysplastic syndrome (MDS), or leukemia

27. 1 is a useful tool in the molecular diagnosis of IBMFSs and a reasonable option as the first tier genetic test in these disorders due to Overlapping phenotypes, variable penetrance, and atypical presentations NGS IBMFS gene panel 2 mainly because they are characterized by a wide range of syndromes and, at the same time, have a high degree of phenotypic overlap.  IBMFSs remain underdiagnosed 3 Since IBMFS syndromes are inherited, diagnosing an underlying genetic cause allows for family member testing to identify carriers or asymptomatic individuals who may also require monitoring or treatment. Genetic Counseling Inherited Bone Marrow Failure Syndromes 4 A related donor carrying the same genetic defect could compromise the success of the transplant. Donor Selection for HSCT

28. THROMBOCYTOPENIA

29. Clinical Features . 42-year-old female with mild thrombocytopenia . Platelet count: 80,000/ uL (persistently low) . Sensorineural hearing loss since age 30 . Family history: Mother with mild bleeding tendency Initially diagnosed with immune thrombocytopenia (ITP) . Unresponsive to standard ITP treatment . Blood smear showed giant platelets Clinical Impact of NGS Diagnosis Avoided unnecessary immunosuppressive therapy Initiated surveillance for r enal disease Family screening identified 2 affected relatives Appropriate pre-procedural management One month male Bilateral absence of radius, Thrombocytopenia Platelet count: 20,000/ uL , Monocytosis , Atrial septal defect No family history Thrombocytopenia-absent radius syndrome TAR AR With juvenile myelomonocytic leukemia AD NGS helps classify thrombocytopenia into syndromic or non-syndromic forms WES can uncover novel mutations and non-coding region variants that might be missed by targeted panels. NGS  clarifies genetic causes, enabling differentiation from acquired thrombocytopenia and informing treatment decisions such as platelet transfusion or transplantation. INHERITED THROMBOCYTOPENIA NGS result Impact of NGS 1 2 NGS result: Homozygous mutation in RBM8A c.-21G>A (((Non-coding ))) Heterozygous mutation in NF1 MPL gene no mutation NGS FindingsMYH9 Gene Structure . Heterozygous pathogenic variant in MYH9 gene . c.5797C>T (p.Arg1933*) - nonsense mutation · Diagnostic of MYH9-related disorder (MYH9-RD)

30. CONGENITAL NEUTROPENIA There are about 24 genes which are responsible for this syndrome.  such as  ELA STASE, N EUTROPHIL- E XPRESSED ELANE most common, G6PC3, HAX1 , and  CXCR4 . Mutations in some genes such as  GATA2  and  CSF3R  can be either germline or somatic. The germline status can be confirmed by analyzing DNA extracted from skin fibroblasts, nails, or hair follicles.

31. Symptoms: 3 months female Neutropenia early postnatal period, recurrent infections .  dysmorphic facial features, echocardiography revealed the presence of patent foramen, oval mitral valve regurgitation, and tricuspid regurgitation. Flow cytometry and immunoglobulin level assessments yielded normal results.   G6PC3: c.479C>T; Ser160Leu Dursun syndrome AR   9‐year‐old boy with failure to thrive and dysmorphic features a history of recurrent admissions due to frequent infections since early neonatal days.  abdominal pain, joint pain, chronic diarrhea, and oral ulcers, tricuspid, mitral regurgitation splenomegaly, and the patient was at Tanner stage 2 with anal tags. CBC results showed hypochromic microcytic anemia with neutropenia Family history : a sister diagnosed with inflammatory bowel disease and experiencing persistent neutropenia until her unfortunate passing at the age of 7 years. Additionally, an infant boy died at the age of 8 months, although the parents were unable to provide a specific cause of death. G6PC3 c.481C>T ; Arg161Ter Dursun syndrome Congenital neutropenia disorders, while rare, can easily go unnoticed in patients with neutropenia, especially those of younger age.  Persistent neutropenia should always be taken seriously .  Severe and recurrent infections must always initiate the search for diagnosis as may result in irretrievable end-organ damage or even death of the patient. Timely referral is the key to the successful diagnosis and management of patients with SCN SEVERE CONGENITAL NEUTROPENIA NGS result Impact of NGS 1 2

32. IRON-RELATED DISORDERS

33. IRON-RELATED DISORDERS Iron-refractory iron deficiency anemia (IRIDA) : IRIDA is caused by mutations in the  TMPRSS6  gene, which encodes matriptase-2, a regulator of hepcidin, leading to inappropriate hepcidin overproduction, reduced iron absorption, and poor response to oral iron therapy ABCB7 PUS1 ALAS2 SLC11A2 ATP4A SLC19A2 ATP7B SLC25A38 CP SLC40A1 FECH SLC46A1 FTH1 STEAP3 FTL TF GLRX5 TFR2 HAMP TFRC HFE TMPRSS6 HJV TRNT1 LARS2 YARS2 NDUFB11 Hereditary hemochromatosis (HH): (Hepcidin deficiency) HH is a condition characterized by iron overload due to dysregulated intestinal absorption of iron. Based on the gene defect, HH was formerly classified into 4 types HFE is the most common gene associated with HH, particularly the C282Y and H63D mutations. Some cases show a digenic inheritance deriving from the combination of pathogenic variants in 2 different genes involved in iron metabolism (e.g., single p.Cys282Tyr + heterozygous variants in hemojuvelin [HJV], HAMP, or transferrin receptor 2 [TFR2]) Some cases do not display variants in any of the 5 classical HC genes (i.e., HFE, HAMP, HJV, TFR2, and SLC40A1). Recently, a small case series have shown variants in the BMP6 gene to be associated with HH. BMP6 encodes one of the major activators of hepcidin expression in response to iron. The role of such variants is still controversial; nonetheless, they broaden the spectrum of genetic defects potentially responsible for HH. The new classification of HH recommended by the BIOIRON society Congenital hypotransferrinemia : This rare disorder is caused by mutations in the  TF  gene , leading to insufficient transferrin and impaired iron transport to cells, causing severe anemia with iron overload in non-hematopoietic tissues. [ 9 ] Ferroportin disease : Mutations in the  SLC40A1  gene impair iron export from macrophages and enterocytes, leading to iron sequestration and anemia. [ 10 ] Sideroblastic anemia : Sideroblastic anemia is characterized by defective heme synthesis, resulting in mitochondrial iron accumulation in erythroid precursors and microcytic hypochromic anemia. Mutations in genes such as  ALAS2 , SLC25A38 , and  GLRX5   are implicated. Aceruloplasminemia : Aceruloplasminemia is a rare autosomal recessive disorder caused by mutations in the   CP  gene , leading to absent ceruloplasmin activity, impaired iron metabolism, and iron accumulation in tissues. It presents with neurological symptoms, diabetes, and anemia due to iron overload. Isolated hyperferritinemia : Isolated hyperferritinemia refers to elevated serum ferritin levels without evidence of systemic iron overload or inflammation. It is commonly associated with hereditary hyperferritinemia-cataract syndrome due to mutations in the FTL  ferritin light chain  gene or can result from metabolic conditions like fatty liver or alcohol use. [ 10 ] Porphyria : Porphyrias are a group of disorders caused by defects in heme biosynthesis enzymes. NGS panels targeting genes such as HMBS, PPOX, and CPOX have improved the diagnosis of conditions such as acute intermittent porphyria and erythropoietic protoporphyria. Disorders of iron transport, utilization, and recycling 5 groups 1 2

34. Case study RESULTS AND INTERPRETATIONS: This patient is heterozygous in the FTL gene for a variant designated c .- 161C>T which t has been reported in an individual with hereditary hyperferritinemia-cataracts syndrome . This patient is also heterozygous in the HFE gene for a variant designated c.187C>G (p.His63Asp). This variant, when present in the compound heterozygous state with the c.845G>A (p.Cys282Tyr) variant, is associated with elevated serum ferritin, transferrin saturation, serum iron and decreased total and saturated iron binding capacity which is indicative of hereditary hemochromatosis ( Gurrin et al. 2009. PubMed ID: 19554541). However, individuals who are homozygous for the c.187C>G (p.His63Asp) variant display similar clinical parameters to unaffected individuals (Jackson et al. 2001. PubMed ID: 11529872; Pedersen and Milman. 2009.PubMed ID: 19159930). hereditary hyperferritinemia cataract syndrome Female 45ys presented with fatigue , pagophagia and brittle nail, bilateral cataracts , symptoms and CBC :Hb:8.3, RBC:3.6, MCV:73, MCH:22.2 were suggestive of iron deficiency but the was ferritin inexplicably high. only two conditions that can explain such a high ferritin level in the absence of any iron stores: Ferritin leak (e.g., cell necrosis) Hereditary Hyperferritinemia Cataract Syndrome

35. HEREDITARY ERYTHROCYTOSIS

36. HEREDITARY ERYTHROCYTOSIS polycythemia vera - JAK2 mutations. Molecular testing: Classification of hereditary erythrocytosis gene mutation: Type1: EPOR Type2:  VHL TYpe3:  EGLN1  ( PHD2 ) Type4:  EPAS1  ( HIF2A ) Type5:  EPO Type6:  HBB Type7:  HBA1, HBA2 Type8:  BPGM ERYTHROCYTOSIS Hereditary erythrocytosis Secondary erythrocytosis hypoxia or tumors  genes regulating oxygen sensing, erythropoiesis, or hemoglobin affinity High oxygen-affinity hemoglobin genes Oxygen-sensing genes No drug therapy targets the mutated hemoglobin. In cases with a higher risk of thrombosis, low-dose aspirin may be considered. Not typically associated with an increased risk of tumors. Targeted therapies are emerging. For example , belzutifan , a HIF-2α inhibitor, can be used for patients with  VHL  mutations to suppress HIF-driven EPO production and treat associated tumors. Mutations in genes like VHL and EPAS1 can increase the risk of tumors . Treatment protocols must include monitoring for associated tumors. personalized treatment Improves the long-term prognosis of patients with hereditary erythrocytosis.

37. COAGULATION AND BLEEDING DISORDERS

38. BLEEDING DISORDERS NGS panel offering a comprehensive approach to diagnosing complex or atypical presentations  atypical platelet disorders with milder bleeding phenotype haemophilia case, who were negative for  F8  intron 22 and intron 1 inversions, haemophilia B ( F9 ) mutations in  RASGRP2  and ITGA2B  gene large size of the F8 gene with its 28 exons, Sanger sequencing is labor-intensive and cost-prohibitive, making NGS an essential tool for pinpointing point mutations. Era of Gene therapy for hemophilia need precise molecular diagnosis

39. A 1-year-old boy was referred to a hematology clinic for a history of easy and unexplained bruising . On examination the patient was pale and had large bruise in the extremities. The patient had no known family history of a bleeding disorder Hb-10 gmsHematocrit-27%platelet count - 2.5 lakh/ cumm . Bleeding time-7 mins( n- 1to 3 mins) PT- 11 SEC (control-12 sec) APTT- 29 sec( control-34 sec) The NGS analysis identified a compound heterozygous mutation ITGA2B INTEGRIN, ALPHA-2B in exon 4 of the  gene. Glanzmann's thrombasthenia, AR relying solely on phenotypic and traditional tests can lead to misdiagnoses in 10–40% of cases, resulting in delayed or inadequate treatment.  Female 16 months presented with: Mucocutaneous bleeding , hematuria Prolonged bleeding time Impaired platelet aggregation Her family members, including mother, father, elder sister, and younger brother, had no history of bleeding . signs and symptoms mild and overlap with those of other disorders NGS analysis detected : A novel homozygous splice site variant (c.1412+2T>C) was detected in intron 12 of the RASGRP2 gene RAS GUANYL NUCLEOTIDE-RELEASING PROTEIN 2 .responsible for severe bleeding syndromes Bleeding disorder, platelet-type, 18 NGS result Impact of NGS 1 2 BLEEDING DISORDERS

40. THROMBOPHILIA The FVL mutation, caused by a single nucleotide polymorphism (c.1691G>A), results in resistance to activated protein C and is the most common inherited thrombophilia the prothrombin G20210A mutation increases plasma prothrombin levels, elevating the risk of thrombotic events Target-NGS panels can uncover rare or novel mutations , such as those in the  SERPINC1, PROC , and  PROS1  genes, which encode antithrombin, protein C, and protein S, respectively.  presence of the JAK2 V617F mutation

41. PRIMARY IMMUNODEFICIENCY

42. PRIMARY IMMUNODEFICIENCY (PID) AND LYMPHOCYTE FUNCTION DISORDERS NGS has elucidated the genetic basis of PIDs and congenital disorders of lymphocyte function. Mutations in genes such as  BTK  (X-linked agammaglobulinemia),  IL2RG  (SCID), and  FOXP3  (IPEX syndrome) are now routinely identified, offering insights into pathophysiology and guiding targeted therapies. 6 October 2025 : The laureates’ discoveries launched the field of peripheral tolerance, spurring the development of medical treatments for cancer and autoimmune diseases . This may also lead to more successful transplantations. Several of these treatments are now undergoing clinical trials. Shimon Sakaguchi (1995):  immune system is more complex and discovered a previously unknown class of immune cells, which protect the body from autoimmune diseases. Mary Brunkow and Fred Ramsdell(2001): They had discovered that the mice have a mutation in a gene that they named  Foxp3 . They also showed that mutations in the human equivalent of this gene cause a serious autoimmune disease, IPEX.   Shimon Sakaguchi (2003) proved that the  Foxp3   gene governs the development of the cells he identified in 1995   Primary immune deficiency diseases (PID) comprise a genetically heterogeneous group of disorders that currently includes 354 distinct disorders with 344 different gene defects (2017 IUIS classification). Clinical diagnosis is often difficult due to overlapping symptoms and laboratory findings in different PIDs. (familial Hemophagocytic Lymphohistiocytosis :  PRF1, STX11, UNC13D , Hyper- IgE syndrome:  STAT3 , Severe Combined immunodeficiency: T-B+NK+ -  IL12RG  or T-B-NK+ -  RAG1/RAG2  or X linked agammaglobulinemia: BTK and Chronic Granulomatous Disease: CYBB)  mutations in genes which could be attributed to the pathogenesis of PID like  CD40LG, TBX1, DOCK2, TNFRSF11A, FTR ,

43. NGS tests In Kuwait

44. Clinical hematologist Investigation : CBC, BM , Immunophenotype, Electrophoresis, HPLC, Coagulation tests , Chromosomal break test, For acquired Somatic mutations as MPL , JAK2…. KCCC KMGC For Inherited hematology pane Whole exome sequencing Samples Pb or BM Refer the case for pre-counseling IF NGS indicated Patient consent needed family history needed Also inherited cancer panel Patient consent needed family history needed

45. GSTM1 RAF1 VPS13B ATM RAD50 ARTN FASN FARS2 TNKS1BP1 SUMF1 STK4 STAT1 ERCC2 IL7R HPS4 NPC2 MCFD2 F7 RAB40AL AHSP PUS1 TTC37 GCLM FANCM ANK1 CD46 CBS FANCE GLRX5 IL6 MMP9 CDKN1A CASP3 FANCG TBXAS1 MIF ALAS2 CCND1 RPL26 CSF3 PIF1 TFR2 LYST TBXA2R NBN CNTN1 GCLC HOXA11 HBM CYCS ALAS1 SERPINE1 AK2 GIF VENTX IRF1 APITD1 VKORC1 GPX1 CD59 HBQ1 PINX1 ANKRD26 SARS2 JAK2 ANO6 NOS3 SLC25A33 LCAT YARS2 TPI1 PPY ACP2 BTNL2 FECH CFB HBB IRF8 GP6 HBA1 EIF2AK2 CFH HK1 EPAS1 XPA BLM TNKS KITLG ABCB6 RTEL1 RAD51 STXBP2 THBS1 TSPAN12 HPS1 F2 FTL CBFA2T3 BGLAP BLOC1S3 ATR F13B SLX4 TFPI USP1 CLCN7 ABCA1 FANCB SEC23B F13A1 UNC13D ADAMTS10 CDAN1 SH2D1A STIM1 SERPINA1 PARP1 RABL2A HBA2 PF4 TERT PTPRC GP1BA GNAS SLC19A2 RAD51C FCGR2C RHCE PPARG MPL FGB THBD BST1 KCNA4 HMBS PEPD PGK1 THPO GBA NOP10 SLC22A3 CUBN MAN2B1 NFKB1 F5 SF3B1 EGLN1 LIPA SRP72 FERMT3 ACSL6 TNF HAX1 NQO1 P2RY12 MTAP C1orf86 TINF2 HBG1 CSF2 PLG PTEN FANCD2 F2RL2 FANCI COQ2 RAB27A FLT1 TET2 TEP1 TNFRSF11B SPTA1 PRF1 SERPIND1 SERPINC1 SF3B2 RPS14 PIGM CXCL12 RMRP ADAMTS13 TNKS2 PROS1 PDGFRB SLC25A19 F11 P2RX1 TELO2 KLKB1 ACSBG1 BMX UROS ERCC1 TMPRSS6 ELANE BST2 PLAU PLAUR SCGB1D1 F8A3 G6PD KRAS AP3B1 DTNBP1 ZNF160 ITK KNG1 PALB2 FANCC SLC40A1 ABCB7 GSTP1 BCL11A FANCF RPL35A SLC25A38 RPS26 FTH1 HMOX1 RPS17 MUC5B BRCA2 DHFR EPB41 FANCL IRF4 FGA ZNF224 FAS C21orf33 DKC1 GPI GNPDA1 WIPF1 CSF1 SERPINF2 F2RL1 FTMT CXCR4 BTF3P11 VWF PSTPIP1 LMBRD1 FASLG PROCR MTHFR RPS24 PROC NDUFS1 GP1BB LIG4 RPS19 F12 HPS6 CD3E GSTT1 BPGM SFTPA1 STX11 NT5C3A PROZ NF1 SLC35A1 CFHR5 HPS3 HOOK1 TERF2IP CFI IDH1 HRG ITGA2B GATA1 RHAG NFKBIA F3 PLAT RPL11 HBG2 RAC2 TERC SPTB BRIP1 POT1 MASTL MMRN1 SMPD1 SLC4A1 XK MECOM CD27 NPC1 EPB42 SERPING1 CSF2RB SLC46A1 PKLR NOG SYK DCLRE1B ACTN1 PGAP1 TERF1 TCIRG1 FGG RASGRP2 COL1A1 GLA TPMT CD63 SMAD7 ASXL1 DDX11 ALDH2 IL2RG ADA RPS10 AK1 NHP2 AMN RUNX1 ATRX F10 ITGA2 LRP5 ACVR1 ABCD1 AVPR2 PNP CISD2 SLC2A1 C3 POLG PRNP RAD54L GSS SFTPA2 COX4I2 FANCA GP9 F9 COX10 F2R IFNG FN1 RPL5 BMP4 ITGB3 GFI1B ABCD4 PDCD1 TCN2 MYH9 ALDOA NEU1 NDP C19orf70 XRCC1 PLA2G7 F8 HES1 CD36 TERF2 GP5 RBM8A LMAN1 ANXA4 FZD4 MT3 DIDO1 HBZ B4GALT1 WRAP53 SLC25A37 HBD MRPL36 GGCX SBDS OAT ANXA5 BTK DGKE WDR19 HPS5 RPS7 SMARCAL1 SLC11A2 C19orf40 PIGA TUBB1 KLF1 CSF3R CD3D B3GALT4 CCL14 Inherited Hematology panel 394 germline genes Patient consent needed family history needed

46. Inherited Cancer panel AIP, AKT1, ALK, ANTXR1, APC, ASCC1, ATM, ATR, AXIN2, BAP1, BARD1, BLM, BMPR1A, BRCA1, BRCA2, BRIP1, BUB1, BUB1B, CACNA1D, CBL, CDC73, CDH1, CDK4, CDKN1B, CDKN2A, CHEK2, CTHRC1, CYLD, DDB2, DICER1, EGFR, ELAC2, EPCAM, ERCC2, ERCC3, ERCC4, ERCC5, EXT1, EXT2, FANCA, FANCB, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCL, FANCM, FH, FLCN, GPC3, HNF1A, HNF1B HOXB13, KDR, KIT, KLLN, LIG4, MAX, MEN1, MET, MITF, MLH1, MLH3, MRE11A, MSH2, MSH6, MSR1, MTAP, MUTYH, MYH8, NBN, NCOA4, NF1, NF2, NTRK1, PALB2, PALLD, PDGFRB, PHOX2B, PIK3CA, PMS2, POLD1, POLE, POLH, PPM1D, PRF1, PRKAR1A, PRSS1, PTCH1, PTCH2, PTEN, PTPN11, RAD50, RAD51C, RAD51D, RB1, RECQL4, RET, RHBDF2, RNASEL, RNF168, RSPO1, RUNX1, SETBP1, SBDS, SDHA, SDHAF2, SDHB, SDHC, SDHD, SH2D1A, SLX4, SMAD4, SMARCB1, SMARCE1, SPINK1, SPRED1, STK11, SUFU, TERT, TGFBR1, TMEM127, TP53, TSC1, TSC2, VHL, WAS, WRN, WT1, XPA, XPC, and XRCC2 134 germline genes Patient consent needed family history needed

47. Oncomine Myeloid Assay Somatic mutations

48. Genetic testing provides information for family planning and carrier screening for families affected by inherited disorders. Facilitating genetic counseling An accurate molecular diagnosis g uides treatment plans . For instance, an NGS diagnosis of Fanconi anemia would require a modified conditioning regimen for a hematopoietic stem cell transplant. clinical management NGS can help differentiate between an i nherited benign disorder and an acquired or malignant condition, which significantly impacts treatment and monitoring. For example, NGS can differentiate inherited thrombocytopenia from an acquired form or clarify the distinction between inherited IBMFS and acquired aplastic anemia. Differentiating diagnoses Whole-exome sequencing (WES), and whole-genome sequencing (WGS) can novel gene and reveal novel or rare genetic variants , including those in non-coding regions, that traditional methods might miss. novel mutations targeted gene panels can significantly increase the diagnostic rate . For instance, in patients with suspected inherited bone marrow failure syndromes (IBMFS), NGS-based testing has identified a pathogenic variant in up to 50% of cases High diagnostic yield Rapid and comprehensive diagnosis Many congenital hematological disorders share overlapping clinical features. NGS can expedite molecular diagnosis by simultaneously screening multiple candidate genes, particularly when routine laboratory tests are inconclusive. Studies show that relying only on phenotype and traditional testing can result in misdiagnosis in 10–40% of cases. Takeaway message

49. Machine learning algorithms to predict variant pathogenicity Integration of large genomic databases to improve accuracy Al-Assisted Variant Interpretation Liquid Biopsy & Cell-Free DNA Non-invasive genetic testing through blood samples . Early detection of disease progression Single-Cell Sequencing Enhanced resolution of cellular heterogeneity in hematologic disorders Future Perspectives

50. Dr.Amir