Neutropenia in the age of genetic testing: advances and challenges

Abstract

Identification of genetic causes of neutropenia informs precision medicine approaches to medical management and treatment. Accurate diagnosis of genetic neutropenia disorders informs treatment options, enables risk stratification, cancer surveillance, and attention to associated medical complications. The rapidly expanding genetic testing options for the evaluation of neutropenia have led to exciting advances but also new challenges. This review provides a practical guide to germline genetic testing for neutropenia.

Introduction

Neutropenia, typically defined as an absolute neutrophil count <1500/μL, is a common cause of referral to both adult and pediatric hematologists. Although most cases of neutropenia are transient and extrinsic to the bone marrow, the diagnosis of an underlying genetic neutropenia disorder profoundly alters clinical management. Many of the genetic neutropenia disorders are associated with leukemia predisposition, or additional medical co-morbidities (Table 1). ^1,2 This review focuses on practical considerations for germline genetic testing of neutropenia, including which patients to test, which tests to send, and interpretation of results. The reader is referred to other excellent recent publications for a comprehensive review of genetic neutropenia disorders^3,4,5 or a review of the clinical evaluation and management of neutropenia disorders. ^6,7,8

Table 1:

Genetic Neutropenia Disorders

	Disease	Gene(s)	Disease pathogenesis	Inheritance	Other clinical findings	Hematologic phenotype	Myeloid malignancy	Other cancers	REF
Predominantly neutropenia	SCN1	ELANE	Neutrophil elastase (class of serine proteases). ELANE mutations lead to unfolded protein response, ER stress, and apoptosis	AD		Severe neutropenia. Promyelocyte death and differentiation arrest, aumiddlehagy of myeloid cells. Variable response to G-CSF	Yes		46
	Cyclic Neutropenia					Neutropenia is cyclic (21 day cycles. Monocytes cycle in reverse to neutrophils	No
	SCN2	GFI1	Zinc finger transcriptional repressor, mutations cause change in transcription	AD		Severe neutropenia, maturation arrest in myeloid cells, and abnormal monocytes and lymphocytes	Possibly		47,48
	SCN3 (Kostmann)	HAX1	HAX1 protects against cell death, stabilizes mitochondrial membrane potential and prevents apoptosis through X-linked inhibitor of apoptosis	AR	Patients with mutations in isoforms A and B have neurologic disease. Splenomegaly	Severe neutropenia, maturation arrest at promyelocyte or myelocyte stage	Yes		35,49–51
	SCN4	G6PC3	G6PC3 is a homolog of glucose-6- phosphatase, mutations lead to abnormal glucose metabolism	AR	Cardiovascular anomalies, urogenital anomalies, bone abnormalities, immune dysregulation, deafness, poor growth, facial dysmorphologies, hyperelastic skin, endocrinopathies, inflammatory	Severe neutropenia, intermittent thrombocymiddleenia, T cell lymphopenia	Possibly		38,52–54
	SCN5	VPS45	VPS45 is critical for endosomal trafficking and mutations in VPS45 cause decreased motility and maturation, and increase in apoptosis	AR	Nephromegaly, hepatosplenomegaly	Neutropenia and neutrophil dysfunction, myelofibrosis, defective platelet aggregation. Not responsive to G-CSF			55–57
	SCN6	JAGN1	JAGN1 is important in the secretory pathway and mutations cause abonrmal glycosylation of glycoproteins including of	AR	Skeletal and dental defects	Neutropenia with decreased neutrophil granules, differentiation arrest at promyelocyte-myelocyte stage. Typically poor response to G-CSF	Possibly		25,58
	SCN7	CSF3R	CSF3R encodes the GCSF receptor	AR		Peripheral neutropenia, full myeloid maturation seen in bone marrow. Not responsive to G-CSF, may respond to GM-CSF			59,60
	Cohen Syndrome	VPS13B	Abnormal vesicle trafficking	AR	Microcephaly, retinal dystrophy, psychomotor retardation, pulmonary alveolar proteinosis, deafness	Intermittent neutropenia			61
	Poilkiloderma with neutropenia	USB1	Abnormal chemokinesis, abnormal RNA splicing	AR	Poilkiloderma, photosensitivity	Neutropenia			62, 63
	TCIRG1-neutropenia	TCIRG1	Mutations affect pH of oragnelles	AD	Hemangiomas	Neutropenia			64
	X linked Neutropenia	WAS	Activating mutations in WAS cause defects in mitosis and cytokinesis	XL		Neutropenia, monocymiddleenia			65
Oculocutaneous Albinism and bleeding diathasis	Chediak-Higashi syndrome	LYST	LYST mutations affect lysosomes, cytotoxic graules	AR	Oculocutaneous albinism, neurodegenerative disease. Impaired NK cell and cytotoxic T cell function	Neutropenia with peroxidase-positive giant inclusions in white blood cells, HLH, bleeding diathesis with decreased platelet dense-bodies		Skin cancer with albinism	66, 67
	Griscelli syndrome type 2	RAB27A	RAB27A mutations lead to abnormal priming of cytotoxic membranes	AR	Oculocutaneous albinism, neurologic disease. Impaired NK cell function	Absence of giant granules in leukocytes, HLH in GS2		Skin cancer with albinism	67
	Hermansky-Pudlak Syndrome type 2	AP3B1	AP3B1 mutations lead to abnormaltiies in lysosomal processing	AR	Oculocutaneous albinism, horizontal nystagmus, epicanthal folds, retrognathia, posteriorly rotated ears	Neutropenia, bleeding diathasis with platelet aggregation studies showing abnormal response to collagen and ADP. Decreased platelet dense-bodies		Hodgkin’s lymphoma, skin cancer	67
	p14 deficiency	LAMTOR2/ROBLD3	Mutations lead to abnormalities in endosomal trafficking	AR	Oculocutaneous albinism, failure to thrive, nystagmus	Transient leukopenia, absence of platelet dense-granules		Skin cancer with albinism	67
Metabolic or mitochondrial disorders	AK2 deficiency	AK2	Defects in mitochondrial homeostasis lead to increased apoptosis	AR	Reticular dysgenesis, hearing loss	Agranulocytosis, absence of T and NK lymphocytes			68
	3-Methyglutaconic aciduria type VII	CLPB	Mutations may affect mitochondrial chaperone function	AR	Neurologic defects, cataracts, aciduria, facial dysmorphisms, endocrinopathies, cardiomyopathy	Neutropenia	Yes		69,70
	Barth Syndrome	TAZ	Mutations lead to mitochondrial dysfunction	x-linked	Ca rdiomyopathy, growth failure, myopathy	May have cyclic oscillations in neutrophil counts, monocytosis			71
	Glycogen storage disease Ib	SLC37A4	Abnormal glucose metabolism	AR	Hepatomegaly, hypoglycemia, lactic acidosis	Neutropenia and neutrophil dysfunction			72, 73
	Pearson Syndrome	mitochondrial DNA deletions	Mitochondrial dysfunction	maternal inheritance	Exocrine pancreatic insufficiency, lactic acidosis, liver failure	Marrow failure, sideoblastic anemia, vacuoles seen in erythroid precursors			74, 75
	STK4 deficiency	STK4	Mitochondrial membrane dysfunction	AR	Congenital heart defects, lymphoproliferative disorder	Intermittent neutropenia, T and B cell lymphopenia			76
	Transcobalamin II deficiency	TCN2	Cells cannot internalize vitamin B12 in transcobalamin deficiency	AR	Poor growth, aciduria, neurologic abnormalities	Megaloblastic anemia, pancymiddleenia			77
Immune System	Cartilage Hair Hypoplasia	RMRP	Abnormal ribosome assembly and cell cycle control	AR	Immunodeficiency, dwarfism, hair abnormalities, intestinal dysplasia	Marrow failure		NHL, skin cancer	78, 79
	CD40LG deficiency	CD40LG	Defect in signaling through CD40ligand	x-linked	Hyper IgM immunodeficiency	Episodic, cyclic, or chronic neutropenia			80
	WHIM	CXCR4	CXCR4 gain of function mutations cause hemamiddleoietic stem cell homing to marrow and retention of neutrophils by defective release and trafficking of neutrophil granulocytes	AD	Warts, B and T cells are also affected, leading to hypogammaglobulinemia and infections	Neutropenia with lymphopenia and monocymiddleenia, myelokathexis			45, 81
	PAMI	PSTPIP1	PSTPIP1 is a cytoskeleton-associated protein, mutations increase PSTPIP1-pyrin binding interaction which leads to autoinflammatory cascade	AD	Hepatosplenomegaly, failure to thrive, chronic inflammation, arthritis/arthralgias, elevated zinc levels	Neutropenia in most cases			82
	GATA2 deficiency, Emberger Syndrome, Mono-Mac Syndrome	GATA2	Abnormal hemamiddleoietic stem cell differentiation	AD	DCML deficiency (dendritic cell, monocyte, B and NK lymphocyte deficiency), Emberger Syndrome (lymphedema), Mono-MAC syndrome (recurrent infections especially	Neutropenia, monocymiddleenia, marrow failure	Yes		83–85
Marrow failure disorders	Ataxia-Pancymiddleenia	SAMD9L	Abnormal cell proliferation	AD	Cerebellar ataxia	Marrow failure	Yes		14, 15
	Dyskeratosis Congenita	DKC1, TERC, TERT, NOP10, NHP2, TINF2, WRAP53/TCAB1, CTC1, RTEL1, ACD/TPP1 PARN NAF1 STN1	Disorders of telomere maintenance	XLR, AR, AD	Nail dystrophy, skin pigmentation abnormalities, oral leukoplakia, pulmonary fibrosis, liver disease, vascular abnormalities	Marrow failure	Yes	SCC, skin cancer, NHL	87, 88
	Fanconi Anemia	FANCA, FANCB, FANCC, FANCD1/BRCA2, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCJ/BRIP1, FANCL, FAMCM, FANCN/PALB2, BANFO/RAD51C, FANCP/SLX4, FANCq/ERCC4, FANCR/RAD51, FANCS/BRCA1, FANCT/UBE2T, FANCU/XRCC2, FANCV/REV7, FANCW	DNA repair defect	XLR, AR, AD	short stature, hyper or hypopigmentation, facial dysmorphology, radial anomalies, cardiac anomalies, GI anomalies, CNS anomalies	Marrow failure	Yes	Head and neck SCC, skin cancer, HCC	89, 90
	Mirage Syndrome	SAMD9	Abnormal cell proliferation	AD	Infections, IUGR, adrenal hypoplasia, genital anomalies enteropathy (MIRAGE)	Marrow failure	Yes		91, 92
	Shwachman-Diamond Syndrome	SBDS	Mutations cause abnormal ribosome biogenesis	AR	Exocrine pancreatic dysfunction, skeletal defects, poor growth	Marrow failure	Yes		93–95
	SDS-like disorders	SRP54, DNAJC21, EFL1	Abnormal ribosome function	AR	Variable	Marrow failure			96–101

Abbreviations: HLH (hemophagocytic lymphohistiocytosis), NHL (Non-Hodgkin’s lymphoma), BCC (basal cell carcinoma), SCC (squamous cell carcinoma), HCC (hepatocellular carcinoma), REF (references)

Accurate and early diagnosis of genetic neutropenias informs clinical care. Correct diagnosis allows for informed treatment decisions, initiation of surveillance strategies for patients at increased risk of leukemia, and for informed donor selection in the case of hematopoietic stem cell transplantation (HSCT) to avoid selecting an affected but clinically cryptic family member. Timely diagnosis of a genetic neutropenia disorder prior to the development of MDS or AML is essential since the long time-frame for genetic testing may preclude diagnosis for such patients who urgently require a hematopoietic stem cell transplant (HSCT). Families also benefit from genetic counseling for family planning, consideration of preimplantation genetic diagnosis to both test for the genetic neutropenia disorder and to match HLA type, and to discuss testing of additional family members. The identification of the genetic causes of congenital neutropenia advances our understanding of neutrophil biology and function which in turn provides the basis for new targeted therapies.⁹

Genetic testing: Whom to test?

Although many patients with a genetic neutropenia disorder present in childhood, some patients who are otherwise asymptomatic or have mild or moderate neutropenia with fluctuating counts present to medical care in adulthood with neutropenia, cytopenias, or a myeloid malignancy. Indeed, in a recent study of MDS, 4% of patients who required HSCT were found to have compound heterozygous mutations in SBDS, of whom only 2 out of the 7 carried a clinical diagnosis of Shwachman Diamond syndrome (SDS). Notably, these patients were significantly younger than the average MDS patient in the cohort, highlighting their genetic predisposition to myeloid malignancy.¹⁰

The reader is referred to excellent reviews on the general diagnostic evaluation of neutropenia.^11,6,7 Genetic testing should be considered as part of the work-up for patients who are suspected of having a primary neutropenia disorder after acquired causes are excluded. Persistent symptomatic or severe neutropenia (ANC <500 cells/μL) without an apparent cause, or neutropenia along physical anomalies or medical comorbidities suggestive of a genetic neutropenia disorder, a family history suggestive of a neutropenia syndrome, or a personal or family history of MDS or acute myeloid leukemia (AML) should prompt consideration of an inherited syndrome.¹² In addition, unexplained red cell macrocytosis or an elevated hemoglobin F percentage may be signs of an underlying marrow failure condition and prompt further evaluation of a possible germline condition. Physical findings such as cardiac, urogenital, or skeletal abnormalities; organomegaly; or abnormal skin pigmentation may provide clues to the diagnosis of genetic neutropenias (Table 1). The presence of oral ulcers, severe dental caries, periodontal inflammation, perianal abscesses, or cellulitis may indicate severe or chronic neutropenia. However, many patients do not present with classic phenotypic features, given the broad phenotypic spectrum of these disorders and range of onset of symptoms, even among members of the same family.^{13,14,15,16,17,18,19,20} Those who have subtle findings or lack physical findings may be more likely to present in adulthood, compared to those with a more severe phenotype who are typically diagnosed during infancy or childhood.

Similarly, although a detailed family history can provide important clues, a negative family history does not necessarily rule out an underlying germline condition. Family history will be negative with de novo mutations or parental mosaicism (systemic or gonadal), and may be negative in the setting of an autosomal recessive disorder. Many genes have variable penetrance and disease phenotype can be highly variable even within the same family. For example, ELANE mutations can lead to SCN or cyclic neutropenia in different individuals in the same kindred.²¹ However, a family history of neutropenia or other cytopenias, frequent infections, early death, malignancy at a young age (particularly MDS or acute myeloid leukemia [AML]), or excessive toxicity from chemotherapy should prompt consideration of genetic testing.

Genetic testing: Which tests to send?

Patients and families should receive counseling regarding the indications, risks, and limitations of genetic testing; the expected time frame for receiving results; and the potential impact on clinical decision-making. A joint discussion involving both a hematologist and a genetic counselor experienced with genetic neutropenia disorders is recommended. Insurance coverage for genetic testing remains a challenge despite its clinical importance. The prolonged time frame of weeks to months for genetic testing may be challenging for clinically urgent cases such as patients heading to transplant or who have progressed to MDS/AML. For this reason, early genetic evaluation prior to the development of clinical complications is recommended.

The sample source for genetic testing may affect results. Although a blood sample is typically drawn for genetic testing, hematopoietic cells may acquire somatic mutations. Therefore, a non-hematopoietic tissue may be tested to distinguish germline versus somatic mutations. Buccal swab or saliva samples may be contaminated by hematopoietic cells. Therefore, skin fibroblasts are typically used for confirmation, although it takes weeks for the skin fibroblasts to grow in culture before their DNA can be sequenced. Hair follicles or fingernails have been used in research laboratories.

It is critical to ensure that the genetic test is optimized for germline testing. Certain genes overlap between those present in germline neutropenia disorders and those that are somatically mutated in MDS, leukemia, or lymphoma, for example RUNX1, GATA2, and CSF3R. Tests differ in their design and analysis depending on whether germline versus somatic mutations in a given gene are sought, so mutations in a given gene may be missed if the incorrect test is ordered.²² Somatic testing panels for hematologic malignancies and aplastic anemia typically target specific regions of the gene and therefore may result in false negatives if they are sent in lieu of panels designed for germline hematologic disorders which may require sequencing of the entire gene. Each testing panel also uses a different variant allele fraction threshold to determine whether to report an identified mutation and this differs between somatic and germline, as described in a genomic analysis of both germline and somatic variants in MDS and leukemia.²²

Genetic Testing Platforms

Table 2 (Adapted from ²³) lists testing considerations of the various testing platforms. The advantages and disadvantages of each approach must be considered when selecting a testing methodology.

Table 2:

Genetic testing platforms

Method	Use	Limitations
Single gene analysis	•  Clinical suspicion for a specific genetic mutation is high •  Specific gene with a characteristic clinical phenotype •  Screening family members if proband has known genetic mutation	•  Only returns results for a single gene with some platforms optimized only to return results for a particular mutation or a specific region of the gene •  Some platforms may miss gene deletions and duplications
Targeted Multigene panel	•  Consider when there is a strong suspicion of a congenital neutropenia •  Several genes can produce the clinical phenotype	•  Some genetic panels may miss gene deletions and duplications •  Must ensure targeted panel covers genes of interest and gene regions of interest
Whole exome sequencing	•  Performed on a clinical or research basis •  May be sent after an initial genetic evaluation returns negative but there is a strong suspicion of a germline mutation	•  Variable coverage and may have poor gene coverage in areas of interest •  Will miss pathogenic noncoding regulatory regions (promoters and enhancers)
Whole genome sequencing	•  Typically done on a research basis if whole exome sequencing is negative •  Strong suspicion that a mutation may be in a region missed by whole exome sequencing	•  Analysis is challenging

In certain cases, Sanger sequencing for mutations in a single gene or a sequential series of genes for a proband can be a cost-effective strategy. For example, if a patient has classic cyclic neutropenia, sequencing the ELANE gene is reasonable. Another effective use of Sanger sequencing of a single gene is to screen additional family members for a specific mutation identified in the proband. However, even if a patient has a relatively classic phenotype, mutations could be in one of multiple candidate genes. For example, patients with oculocutaneous albinism and neutropenia could potentially harbor mutations in LYST, AP3B1, RAB27A, or LAMTOR2. In addition, Sanger sequencing may miss deletions and copy number variants which can be detected on some of the other testing platforms.

Therefore in many cases, multiplexed targeted analysis with next-generation sequencing panels for multiple neutropenia genes are generally cost effective and efficient especially considering the wide variability and overlap in clinical phenotypes.²⁴ However, the utility of any targeted gene panel is dependent on 1) the specific set of genes included, 2) the coverage of coding regions and pathogenic noncoding regions, and 3) the post-sequencing analysis.

For those patients for whom a genetic cause is not identified by the above methods, whole exome sequencing (WES) or whole genome sequencing (WGS) can be powerful tools. In WES, the exomes are sequenced using next generation sequencing techniques which can identify mutations in both protein-coding and non-coding-RNA genes. Identification of new, previously unrecognized genetic mutations via WES has increased our understanding of the pathogenesis of many disease types including recently SCN6 due to JAGN1 mutations.²⁵ However, it is necessary to note that some regions of the genome are poorly covered by WES (including promoter and enhancer regions), non-coding RNAs may be filtered or missed depending on the platform, and mitochondrial genome mutations and deletions are filtered out due to the high copy number mtDNA sequences. Conserved pseudogenes such as in with SBDS, and segmental duplications can complicate analysis.

WES results can be challenging to analyze and require expertise in genomics as well as knowledge of neutropenia genetics. Analysis pipelines are not standardized. In order to improve the coverage of specific regions or genes of interest, targeted capture sequencing approaches added to WES can improve sensitivity, as can supplementation with other types of methodologies such as array comparative genomic hybridization (array CGH) or multiplexed ligation-dependent probe amplification to detect copy number variants.^23,22

Currently, whole genome sequencing (WGS), which is largely performed in research laboratories, can evaluate the non-exonic content missed by WES or non-coding variants such as deep intronic mutations and mutations in promoter and enhancer regions. WGS may also be more sensitive for copy number variants. However, the depth of coverage is reduced with increasing breadth of coverage, and bioinformatic analysis and clinical interpretation of such large datasets can be challenging. Currently, cost and time required for genomic analysis are often prohibitive, particularly for urgent clinical cases. As coverage depth and post-sequencing analytic tools improve, WES and WGS technologies are very likely to be utilized more frequently in the clinical setting.

Genetic testing: Interpretation of results

In some instances, the results are easy to interpret if there is a clearly deleterious mutation or mutations which have previously been reported to be pathogenic. However, if there are compound heterozygous mutations in a recessive disorder, then it is important to demonstrate that the mutations are in trans and not in cis, in which case only a single allele would be affected. One straightforward way of determining this information is to test both parents, as each parent would be expected to carry one mutation if the mutations are indeed in trans. If it is not possible to test the parents, it can sometimes be technically possible to determine whether mutations are in cis or in trans by subcloning and Sanger sequencing if the variants are located in close proximity.

The other challenge that frequently arises is how to classify variants of unknown significance (VUS).²⁶ The broader the sequencing, the greater the chances of discovering a VUS. Variants found in multiple unrelated affected individuals and absent from control databases are more likely to be pathogenic although it is critical to note that some pathogenic mutations are present in control databases such as ExAC and gnomAD owing to variable penetrance, late clinical onset, or carrier states of recessive alleles. Assessing conservation across orthologues can be helpful in assessing the tolerability of various nucleotide or amino acid changes in a given location. In silico model-based systems assign prediction scores for pathogenicity, deleterious effect, and/or molecular functionality based on variables such as conservation, allele frequency, transcript information, protein scores, and reported variants. These models may provide helpful clues but must be interpreted with caution and different models can give contradictory results. ^27,28,29,30 Sequencing a trio (mother, father, and affected proband), and/or additional affected and unaffected family members can be helpful in filtering variants to identify a causative genetic mutation.

Ideally, these variants of unknown significance would be functionally validated and integrated with functional results and clinical phenotypes to more accurately interpret variants. This problem highlights a conundrum in translational research, as the interface between functional studies in research labs and clinical laboratory testing can be very challenging. Efforts are underway to develop centralized and regularly updated public databases collating identified variants together with expert annotation of functional significance and clinical pathogenicity.

Lastly, clinicians must determine how to proceed with negative results. For example, 30–40% of patients with SCN do not have mutations in ELANE and roughly 40% of patients have a negative result on targeted gene panels.^24,31,32 The clinician must assess which genes have not been queried and determine whether to send additional testing.

Genetic testing: Implications for management

The reader is referred to excellent reviews discussing the general management and treatment of neutropenia.^6,7 Notably for this discussion, identification of a specific genetic cause of a patient’s neutropenia may predict response to therapies such as G-CSF, and identify patients at risk of bone marrow failure and/or clonal evolution to MDS or AML which is information which otherwise would not have been available for the individual and his or her family. Knowledge of the underlying inherited disorder can lead to individually tailored surveillance strategies based on the risks and medical complications associated with specific genetic neutropenia disorders. Critically, identification of the specific affected gene can inform hematopoietic stem cell transplant decisions. Outcomes are superior and toxicities of conditioning regimens less if HSCT is performed prior to the development of MDS or leukemia. However, it is critical to genetically screen potential related donors to decrease the risk of selecting an HLA-matched donor who carries the same mutation with a reduced or absent phenotype,³³ as illustrated in a recent case series of patients with GATA2 mutations who developed donor-derived MDS and AML.³⁴

Genetic counseling is also paramount. Even in cases where the proband may be the only known family member with neutropenia, others in the family carrying the same allele may still have an elevated risk of developing a myeloid malignancy. Families may pursue preimplantation genetic diagnosis to screen for the causative mutation or select an HLA-matched embryo and must be counseled accordingly. While gene therapy is not yet available for patients with congenital neutropenia, this is likely to be a potential therapeutic option in the future.³⁵

Future directions

Increasingly powerful tools for genetic testing have spurred advances in the biology of hematopoiesis through the identification of previously unknown neutropenia genes.^36,37 However, there is a paucity of data regarding the phenotypic spectrum and natural history of genetic neutropenias,³⁸ and neutropenia registries and consortia continue to be instrumental for the study of congenital neutropenia syndromes.^39,40,41,13

Going forward, clinical utility of genetic testing and the ease of interpreting results will depend on the development of publicly accessible, regularly updated databases with expert annotation of genetic variants. Similarly, through exploration of neutropenia-related genetic pathways, we are entering a new era of targeted therapies. For example, neutrophil elastase inhibitors such as Sivelestat can overcome the differentiation block in SCN1 in pre-clinical studies and Plerixafor has moved into clinical trials for WHIM syndrome based on the understanding of the role of CXCR4 following the initial development of plerixafor as an inhibitor of CXCR4 for human immunodeficiency virus (Clinicaltrials.gov NCT02231879). ^42,43,44,45 These and other examples are discussed in a recent review.³⁶

Future studies are needed to develop optimal diagnostic tools, functional testing for these disorders, and treatment strategies including improved transplant regimens, supportive care, and targeted treatments. Additional research is required to develop strategies for surveillance for MDS and leukemia and inform the interpretation and management of specific somatic mutations and clonal evolution in these patients. Genetic testing and analysis coupled with clinical medicine and understanding of neutrophil biology will be paramount in providing the next generation of tailored therapy beyond G-CSF and transplant. In the meantime, helpful initiatives to drive forward this research and improve clinical care include 1) support for neutropenia registries and correlative studies and 2) the creation of a centralized database of annotated variants and list of laboratories which can perform functional validation of variants of unclear significance.

SUMMARY TABLE:

Who might benefit from genetic testing for neutropenia?

The following features should prompt consideration of genetic testing: persistent symptomatic or severe neutropenia (ANC <500 cells/μL) without an apparent cause, a family history suggestive of a neutropenia syndrome, a personal or family history of MDS or acute myeloid leukemia (AML), excessive toxicity from chemotherapy, unexplained red cell macrocytosis, or an elevated hemoglobin F percentage. Physical findings which can raise the suspicion of a genetic neutropenia disorder include cardiac, urogenital, or skeletal abnormalities; organomegaly; abnormal skin pigmentation; short stature/poor growth. Common non-genetic causes of neutropenia to rule out include infections and medications.

When should testing be sent?

Early genetic evaluation prior to the development of clinical complications is recommended for those patients suspected of having a genetic neutropenia disorder to inform treatment decisions and medical management.

What is the role of genetic counseling?

Patients and families should receive counseling regarding the indications, risks, and limitations of genetic testing; the expected time frame for receiving results; and the potential impact on clinical decision-making. A joint discussion involving both a hematologist and a genetic counselor experienced with genetic neutropenia disorders is recommended.

What options are available for genetic testing?

1. Sanger sequencing of single gene can be cost-effective when suspicion for a specific gene is high, or when screening family members of a proband with a known mutation.

2. Multiplexed targeted analysis with next-generation sequencing panels for multiple neutropenia genes are generally cost effective and efficient in light of the wide variability and overlap in clinical phenotypes between different neutropenia disorders. However, the utility of any targeted gene panel is dependent on 1) the specific set of genes included (which are variable between panels), 2) the coverage of coding regions and pathogenic noncoding regions, and 3) the post-sequencing analysis.

3. In whole exome sequencing, next generation sequencing techniques can identify mutations in both protein-coding and non-coding-RNA genes, but coverage of the desired genes may be variable.

4. Whole genome sequencing (WGS) is currently largely performed in research laboratories and can evaluate the non-exonic content missed by WES or non-coding variants such as deep intronic mutations and mutations in promoter and enhancer regions. However, the depth of coverage is reduced with increasing breadth of coverage.

What are the differences between somatic and germline panels?

Certain genes may be mutated constitutionally in the germline to cause neutropenia and also mutated somatically in hematopoietic cells such as in MDS or hematologic malignancies. Somatic testing panels for hematologic malignancies typically target specific regions that are mutated somatically and therefore may result in false negatives if they are sent in lieu of panels designed for germline hematologic disorders which may require sequencing of the entire gene, evaluation of known pathogenic non-coding regions, and evaluation for partial or whole gene deletions. Each testing panel also uses a different variant allele fraction threshold to determine whether to report an identified mutation and this differs between somatic and germline genetic panels.

What are the sample requirements for testing?

Although a blood sample for DNA extraction is typically used for genetic testing, hematopoietic cells may acquire somatic mutations (see above). Therefore, testing of a non-hematopoietic tissue such as skin fibroblasts may sometimes be required to distinguish germline versus somatic mutations.

What do I do with variants of unknown significance (VUS)?

Variants are more likely to be pathogenic if absent from control databases, conserved across orthologues, and have higher pathogenicity scores using in-silico models. Sequencing a trio (mother, father, and affected proband), and/or additional affected and unaffected family members can be helpful to determine whether the variant tracks with the clinical phenotype, though variable penetrance may confound the analysis.. Variants can be functionally validated if a clinically relevant assay is available.

How might genetic testing affect clinical management?

Correct diagnosis allows for informed treatment decisions, initiation of surveillance strategies for patients at increased risk of leukemia, and for informed donor selection in the case of hematopoietic stem cell transplantation (HSCT) to avoid selecting an affected but clinically cryptic family member. Families also benefit from genetic counseling for family planning, consideration of preimplantation genetic diagnosis to both test for the genetic neutropenia disorder and to match HLA type, and allow counseling of any additional interested family members.

Consultation with an expert in genetic neutropenia disorders can be helpful to guide diagnostic genetic testing and interpretation of results.