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  1. NGS Panel – Genetic Testing for Spherocytosis

Spherocytosis

July 03, 2017

Disease synonyms

Hereditary spherocytosis, Congenital spherocytic hemolytic anemia, Congenital spherocytosis, HS, Spherocytic anemia

Inheritance pattern

Autosomal recessive, autosomal dominant


Clinical features

Hereditary spherocytosis is a rare inherited disorder characterized by haemolytic anaemia resulting from red blood cell membrane protein anomalies. Hereditary spherocytosis is the most common inherited anemia in individuals of European ancestry, with a prevalence of 1-5 in 10,000 or higher when the very mild forms, which are frequently underdiagnosed, are included 1.

There are four forms of hereditary spherocytosis, which are distinguished by the severity of signs and symptoms 2, 3:

  • Mild form (20-30% of affected)
  • Moderate form (60-70%)
  • Moderate/severe form (10%)
  • Severe form (3-5%)

Jaundice is the first clinical manifestation in newborns, with severe anemia developing the first few days after birth. Splenomegaly is a frequently observed feature. Age of onset and severity vary considerably. The majority (60-70%) of patients with hereditary spherocytosis have moderate anemia, resulting from incomplete compensation of hyperhemolysis. In the absence of jaundice, splenomegaly, or complications the disease may remain undetected.

Clinical diagnosis of hereditary spherocytosis is suspected in individuals with any of the following findings:

  • Jaundice (usually intermittent and due to unconjugated hyperbilirubinemia, resulting from exacerbated hemolysis)
  • Chronic, non-immune hemolytic anemia
  • High mean corpuscular hemoglobin concentration (MCHC)
  • Presence of spherocytes in the peripheral blood smear
  • Splenomegaly
  • Cholelithiasis in the second or third decade of life

Laboratory findings include the following:

  • Anemia
  • Reticulocytosis
  • High mean corpuscular hemoglobin concentration (MCHC)
  • Presence of spherocytes and occasionally few ovalocytes and elliptocytes in the peripheral blood smear
  • Significantly decreased or absent haptoglobin
  • Mildly increased osmotic fragility
  • Increased osmolarity index (Omin=osmolality at which 50% of red blood cells hemolyze)

Hereditary spherocytosis is inherited in an autosomal dominant manner in 75% of cases through mutations in the ANK1, SPTB, SLC4A1, SPTA1, and EPB42 genes (accounting for 60%, 10%, 15%, 10%, and 5% cases, respectively)3. Mutations in the ANK1 gene are responsible for approximately half of all cases of hereditary spherocytosis4. The other genes associated with hereditary spherocytosis each account for a smaller percentage of cases of this condition (see Table).

EPB42-associated hereditary spherocytosis is responsible for 40-50% of hereditary spherocytosis in Japan, where the carrier frequency of p.Ala142Thr among healthy persons is as high as 3%5, 6. The p.Ala142Thr variant has been observed in a homozygous state in affected individuals. While this variant has a carrier frequency of approximately 3% in persons of Japanese ancestry, it was also found in an affected individual from central Italy who had no Japanese ancestry7. Although the frequency in the Japanese population is likely explained by a founder effect, the apparently random occurrence of this variant in another population may be explained by the fact that the G>A transition occurs within a CpG site on the antisense DNA strand8. In other populations, EPB42-HS accounts for 5% or less of HS5.


Table 1. Overview of genes associated with Spherocytosis

Gene OMIM (Gene) Associated diseases (OMIM) Inheritance CentoMD® exclusive variant numbers (++)
ABCB7 300135 Anemia, Sideroblastic, and Spinocerebellar Ataxia XLR 1
ACTN1 102575 Bleeding disorder, platelet-type, 15 AD 0
ADAMTS13 604134 thrombotic thrombocytopenic purpura AR 45
AK2 103020 Reticular dysgenesis AR 0
ALAS2 301300 X-linked sideroblastic anemia; Protoporphyria, erythropoietic, X-linked XL, XLR 4
AMN 605799 AR 10
ANK1 612641 Spherocytosis, type 1 AD, AR 9
ANKRD26 610855 thrombocytopenia type 2 AD 6
AP3B1 603401 Hermansky-Pudlak syndrome type 2 AR 18
ATM 607585 familial breast-ovarian cancer type 2; ataxia-telangiectasia AD, AR 82
ATRX 300032 Alpha-Thalassemia Myelodysplasia Syndrome; Alpha-Thalassemia/Mental Retardation Syndrome, X-Linked; Mental retardation-hypotonic facies syndrome, X-linked XLD, XLR 16
BLM 604610 Bloom syndrome AR 9
BLOC1S3 609762 Hermansky-Pudlak syndrome 8 AR 3
BRCA1 113705 familial breast-ovarian cancer type 1; pancreatic cancer type 4; Fanconi anemia, complementation group S AD, AR 262
BRCA2 600185 familial breast-ovarian cancer type 2; Medulloblastoma; Prostate Cancer; Wilms tumor, type 1; Fanconi anemia complementation group D1; pancreatic cancer type 2 AD, AR 240
BRIP1 605882 familial breast-ovarian cancer type 2; Fanconi anemia of complementation group J AD 16
CASP10 601762 Autoimmune lymphoproliferative syndrome, type II; Lymphoma, non-Hodgkin; Gastric Cancer AD 4
CBL 165360 Leukemia, juvenile myelomonocytic; Noonan syndrome-like disorder with or without juvenile myelomonocytic leukemia AD 9
CBLIF 609342 Intrinsic factor deficiency AR 3
CD36 173510 Platelet glycoprotein IV deficiency; resistance to malaria AR 5
CD40LG 300386 Immunodeficiency With Hyper-Igm, Type 1 XLR 4
CDAN1 607465 congenital dyserythropoietic anemia 1 AR 12
CDIN1 615626 congenital dyserythropoietic anemia 1b AR 1
CENPJ 609279 primary microcephaly 6; Seckel syndrome 4 AR 13
CEP152 613529 Seckel syndrome 5; primary microcephaly 9 AR 10
CLCN7 602727 Osteopetrosis, autosomal dominant 2; Osteopetrosis, autosomal recessive 4 AD, AR 25
CLPB 616254 3-methylglutaconic aciduria type VII with cataracts, neurologic involvement and neutropenia AR 2
CSF3R 138971 Neutropenia, severe congenital, 7, autosomal recessive AR 1
CTC1 613129 Cerebroretinal microangiopathy with calcifications and cysts AR 3
CUBN 602997 megaloblastic anemia 1 AR 46
CXCR4 162643 WHIM syndrome AD 4
CYB5R3 613213 methemoglobinemia type I AR 8
CYCS 123970 Thrombocytopenia 4 AD 1
DHFR 126060 AR 0
DIAPH1 602121 Deafness, autosomal dominant 1; Seizures, cortical blindness, microcephaly syndrome AD, AR 5
DKC1 300126 X-linked dyskeratosis congenita XLR 3
DTNBP1 607145 Hermansky-Pudlak syndrome 7 AR 6
ELANE 130130 Neutropenia, cyclic; Neutropenia, severe congenital 1, autosomal dominant AD 3
EPB42 177070 Spherocytosis, type 5 0
ERCC4 133520 xeroderma pigmentosum complementation group F; XFE prpgeroid syndroem; Fanconi anemia of complementation group Q AR 4
FANCA 607139 Fanconi anemia complementation group A AR 61
FANCB 300515 Fanconi anemia of complementation group B XLR 6
FANCC 613899 Fanconi anemia of complementation group C AR 9
FANCD2 613984 Fanconi anemia of complementation group D2 AR 24
FANCE 613976 Fanconi anemia of complementation group E AR 7
FANCF 613897 Fanconi anemia of complementation group F 4
FANCG 602956 Fanconi anemia of complementation group G 13
FANCI 611360 Fanconi anemia of complementation group I AR 33
FANCL 608111 Fanconi anemia of complementation group L AR 15
FANCM 609644 Spermatogenic failure 28 AR 16
FAS 134637 Autoimmune lymphoproliferative syndrome AD 4
FASLG 134638 Lung Cancer; Autoimmune lymphoproliferative syndrome AD 1
G6PC3 611045 Neutropenia, severe congenital 4, autosomal recessive AR 3
G6PD 305900 glucose-6-phosphate dehydrogenase deficiency; resistance to malaria XLD 10
GATA1 305371 X-linked congenital dyserythropoietic anemia with thrombocytopenia; Anemia, X-linked, with/without neutropenia and/or platelet abnormalities; beta-thalassemia - X-linked thrombocytopenia XLR 4
GFI1 600871 Neutropenia, severe congenital 2, autosomal dominant AD 0
GFI1B 604383 Bleeding disorder, platelet-type, 17 AD, AR 0
GLRX5 609588 Anemia, sideroblastic, 3, pyridoxine-refractory AR 3
GNE 603824 Sialuria; Nonaka myopathy AD, AR 6
GP1BA 606672 Bernard-Soulier syndrome, type A2 (dominant); platelet type von Willebrand disease; Bernard-Soulier syndrome, type C AD, AR 12
GP1BB 138720 Bernard-Soulier syndrome, type C AR 2
GP9 173515 Bernard-Soulier syndrome, type C AR 2
GPI 172400 nonspherocytic hemolytic anemia due to glucose phosphate isomerase deficiency AR 0
GSS 601002 Glutathione synthetase deficiency AR 1
HAX1 605998 autosomal recessive severe congenital neutropenia type 3 AR 1
HBA1 141800 Heinz Body Anemias; alpha-Thalassemia; Hemoglobin H Disease AD 8
HBA2 141850 Heinz Body Anemias; alpha-Thalassemia; Hemoglobin H Disease AD 2
HBB 141900 Heinz Body Anemias; Delta-beta thalassemia; dominantly inherited inclusion body beta-thalassemia; sickle cell anemia; resistance to malaria; beta-thalassemia AD, AR 4
HFE 613609 Alzheimer Disease; hepatoerythropoietic porphyria; variegate porphyria; hemochromatosis type 1; susceptibility to microvascular complications of diabetes type 7; Transferrin serum level QTL2 AD, AR 11
HOXA11 142958 Radioulnar synostosis with amegakaryocytic thrombocytopenia 1 AD 2
HPS1 604982 Hermansky-Pudlak syndrome type 1 AR 14
HPS3 606118 Hermansky-Pudlak syndrome type 3 AR 12
HPS4 606682 Hermansky-Pudlak syndrome type 4 AR 6
HPS5 607521 Hermansky-Pudlak syndrome type 5 AR 7
HPS6 607522 Hermansky-Pudlak syndrome type 6 AR 4
HSPA9 600548 Sideroblastic anemia type 4 AD, AR 1
IL2RG 308380 Severe X-linked combined immunodeficiency; moderate X-linked combined immunodeficiency XLR 7
ITGA2B 607759 Bleeding disorder, platelet-type, 16, autosomal dominant; Glanzmann thrombasthenia AD, AR 9
ITGB3 173470 Bleeding disorder, platelet-type, 16, autosomal dominant; Glanzmann thrombasthenia; Myocardial infarction, decreased susceptibility to AD, AR 5
ITK 186973 Lymphoproliferative syndrome 1 AR 0
JAGN1 616012 Neutropenia, severe congenital, 6, autosomal recessive AR 2
KLF1 600599 Blood group--lutheran inhibitor; Fetal hemoglobin quantitative trait locus 6; Anemia, congenital dyserythropoietic, type iv AD 2
KRAS 190070 Arteriovenous malformations of the brain; Bladder Cancer; familial breast-ovarian cancer type 2; Gastric Cancer, Hereditary Diffuse; Schimmelpenning-Feuerstein-Mims Syndrome; Lung Cancer; Pancreatic Cancer; acute myeloid leukemia; Noonan syndrome 3; Autoimmune lymphoproliferative syndrome type IV; Cardiofaciocutaneous syndrome 2 AD 9
LIG4 601837 LIG4 syndrome AR 3
LPIN2 605519 Majeed syndrome 3
LYST 606897 Chediak-Higashi syndrome AR 30
MLH1 120436 Muir-Torre syndrome; mismatch repair cancer syndrome; hereditary nonpolyposis colorectal cancer-2 AD, AR 19
MPL 159530 somatic myelofibrosis with myeloid metaplasia; thrombocythemia type 2; congenital amegakaryocytic thrombocytopenia AD, AR 8
MRE11 600814 Ataxia-telangiectasia-like disorder type 1 AR 16
MSH2 609309 Lynch syndrome; Muir-Torre syndrome; mismatch repair cancer syndrome AD, AR 30
MSH6 600678 mismatch repair cancer syndrome; endometrial cancer; hereditary nonpolyposis colorectal cancer-5 AD, AR 21
MTR 156570 homocystinuria-megaloblastic anemia, cblG complementation type; folate-sensitive neural tube defects AR 29
MTRR 602568 Homocystinuria-Megaloblastic Anemia, Cble Complementation Type; folate-sensitive neural tube defects AR 16
MYH9 160775 Fechtner syndrome; Macrothrombocytopenia and progressive sensorineural deafness; May-Hegglin anomaly; Sebastian syndrome; deafness type 17 AD 8
NBN 602667 Nijmegen breakage syndrome; Aplastic Anemia; Acute lymphoblastic leukemia AR 11
NF1 613113 neurofibromatosis type 1; Neurofibromatosis-Noonan syndrome; Leukemia, juvenile myelomonocytic AD 140
NHP2 606470 Dyskeratosis Congenita, Autosomal Recessive, 2 AR 0
NOP10 606471 Dyskeratosis Congenita, Autosomal Recessive, 1 AR 0
NRAS 164790 colorectal cancer; Melanocytic nevus syndrome, congenital, somatic; Nevus, Epidermal; Schimmelpenning-Feuerstein-Mims Syndrome; Thyroid Carcinoma, Follicular; Neurocutaneous melanosis, somatic; Noonan syndrome 6; Autoimmune lymphoproliferative syndrome type IV AD 5
PALB2 610355 familial breast-ovarian cancer type 2; Fanconi anemia of complementation group N; Pancreatic cancer, susceptibility to, 3 AD 14
PARN 604212 Dyskeratosis congenita, autosomal recessive 6; telomere-related pulmonary fibrosis and/or bone marrow failure type 4 AD, AR 3
PC 608786 pyruvate carboxylase deficiency AR 13
PDHA1 300502 Pyruvate dehydrogenase E1-alpha deficiency XLD 14
PDHX 608769 Lacticacidemia due to PDX1 deficiency AR 12
PKLR 609712 pyruvate kinase deficiency AD, AR 35
PMS2 600259 mismatch repair cancer syndrome; hereditary nonpolyposis colorectal cancer-4 AR 31
PRF1 170280 familial hemophagocytic lymphohistiocytosis 2; Lymphoma, non-Hodgkin; Aplastic Anemia AR 17
PTPN11 176876 LEOPARD syndrome 1; Noonan syndrome 1; Leukemia, juvenile myelomonocytic AD 13
PUS1 608109 myopathy, lactic acidosis and sideroblastic anemia type 1 AR 7
RAC2 602049 Neutrophil immunodeficiency syndrome 0
RAD51C 602774 Fanconi anemia of complementation group O; Breast-ovarian cancer, familial, susceptibility to, 3 AR 5
RBBP8 604124 Jawad syndrome / Microcephaly with mental retardation and digital anomalies; Seckel syndrome 2 AR 8
RBM8A 605313 Thrombocytopenia-absent radius syndrome AR 3
RIT1 609591 Noonan syndrome 8 AD 2
RPL11 604175 Diamond-Blackfan anemia 7 AD 7
RPL15 604174 Diamond-Blackfan anemia 12 AD 4
RPL26 603704 Diamond-Blackfan anemia 11 AD 0
RPL27 607526 AD 0
RPL35A 180468 Diamond-Blackfan anemia 5 AD 5
RPL5 603634 Diamond-Blackfan anemia 6 AD 13
RPS10 603632 Diamond-Blackfan anemia 9 AD 1
RPS17 180472 Diamond-Blackfan anemia 4 AD 4
RPS19 603474 Diamond-Blackfan anemia 1 AD 11
RPS24 602412 Diamond-blackfan anemia type 3 AD 10
RPS26 603701 Diamond-Blackfan anemia 10 AD 8
RPS27 603702 Diamond-Blackfan anemia 17 AD 0
RPS28 603685 Diamond-Blackfan anemia 15 with mandibulofacial dysostosis AD 1
RPS29 603633 Diamond-Blackfan anemia 13 AD 1
RPS7 603658 Diamond-Blackfan anemia 8 AD 7
RTEL1 608833 dyskeratosis congenita; that telomere-related pulmonary fibrosis and/or bone marrow failure type 3 AD, AR 8
RUNX1 151385 Platelet disorder, familial, with associated myeloid malignancy; acute myeloid leukemia AD 9
SAMD9 610456 normophosphatemic familial tumoral calcinosis; MIRAGE syndrome AD, AR 4
SBDS 607444 Shwachman-Bodian-Diamond syndrome; Aplastic Anemia AR 11
SEC23B 610512 congenital dyserythropoietic anemia 2; Cowden syndrome 7 AD, AR 11
SH2D1A 300490 Lymphoproliferative Syndrome, X-Linked, 1 XLR 3
SLC19A2 603941 Thiamine-responsive megaloblastic anemia syndrome AR 1
SLC19A3 606152 biotin-thiamine-responsive basal ganglia disease AR 13
SLC25A19 606521 Microcephaly, Amish type; Thiamine metabolism dysfunction syndrome 4 (progressive polyneuropathy type) AR 6
SLC25A38 610819 Anemia, sideroblastic, 2, pyridoxine-refractory AR 0
SLC4A1 109270 Autosomal dominant distal renal tubular acidosis; resistance to malaria; Renal tubular acidosis, distal, with hemolytic anemia; Spherocytosis, type 4 AD, AR 9
SLX4 613278 Fanconi anemia of complementation group P AR 7
SPTA1 182860 Elliptocytosis type 2; Pyropoikilocytosis; Spherocytosis, type 3 AD, AR 4
SPTB 182870 Spherocytosis, type 2; Elliptocytosis 3 AD 9
SRP72 602122 Bone marrow failure, familial AD 1
STIM1 605921 Myopathy, tubular aggregate, 1; Immunodeficiency 10 AD, AR 1
STX11 605014 Hemophagocytic lymphohistiocytosis, familial, 4 AR 2
STXBP2 601717 Hemophagocytic lymphohistiocytosis, familial, 5 9
TCN2 613441 Transcobalamin II deficiency AR 11
TERT 187270 acute myeloid leukemia; Dyskeratosis congenita 4; Bone marrow failure, telomere-related, 1 AD, AR 5
TINF2 604319 Revesz syndrome; Dyskeratosis congenita, autosomal dominant 3 AD 2
TP53 191170 familial breast-ovarian cancer type 2; colorectal cancer; Hepatocellular Carcinoma; Glioma susceptibility 1; Li-Fraumeni syndrome 1; Osteogenic Sarcoma; Pancreatic Cancer AD 13
TPK1 606370 Thiamine metabolism dysfunction syndrome 5 (episodic encephalopathy type) AR 5
TRNT1 612907 Sideroblastic anemia with B-cell immunodeficiency, periodic fevers, and developmental delay AR 0
UBE2T 610538 Fanconi anemia of complementation group T AR 0
UNC13D 608897 familial hemophagocytic lymphohistiocytosis 3 AR 16
VPS13B 607817 Cohen syndrome AR 28
VPS45 610035 Severe congenital neutropenia type 5 AR 1
WAS 300392 Neutropenia, severe congenital, X-linked; Wiskott-Aldrich syndrome; Thrombocytopenia 1 XLR 4
WRAP53 612661 Autosomal recessive dyskeratosis congenita type 3 AR 1
XIAP 300079 X-linked lymphoproliferative disease 2 XLR 6
XRCC2 600375 Fanconi anemia, complementation group U AR 8
YARS2 610957 Myopathy, lactic acidosis, and sideroblastic anemia 2 AR 0

There is no cure for hereditary spherocytosis but many treatments, if applied in time, could result in significant benefit for the patient. In newborns, as for all types of hemolytic anemia, the treatment for hereditary spherocytosis involves management of jaundice to prevent hyperbilirubinemic encephalopathy. Further therapeutic approaches include red blood cell transfusions, folic acid supplementation, splenectomy, and many other approaches.

CENTOGENE offers a Spherocytosis panel that includes the following genes: ANK1, EPB42, SLC4A1, SPTA1, SPTB. We also offer several NGS panels associated with Spherocytosis-related phenotypes. Each gene in these panels can also be ordered individually as a single gene test.


Differential diagnosis

The differential diagnosis of Spherocytosis-related disorders – depending on the major symptoms in the initial case – includes the following diseases:

  • Hereditary elliptocytosis, stomatocytosis, or Southeast Asian ovalocytosis
  • Hemoglobin disorders
  • Erythrocyte enzymopathies, such as glucose-6-phospate dehydrogenase (G6PD) deficiency or pyruvate kinase (PK) deficiency
  • Congenital dyserythropoietic anemia.

Testing strategy

CENTOGENE offers advanced, fast and cost-effective strategy to test large NGS panels and diagnose complex phenotypes based on the PCR-free whole genome sequencing and NGS technology. This approach offers an unparalleled advantage by reducing amplification/capture biases and provides sequencing of entire gene at a more uniform coverage.

To confirm/establish the diagnosis, CENTOGENE offers the following testing strategy for spherocytosis using NGS Panel Genomic targeted towards this specific phenotype:

Step 1: Whole genome sequencing from a single filter card. The sequencing covers the entire genic region (coding region, exon/intron boundaries, intronic and promoter) for all the genes included in the Spherocytosis panel. Copy Number Variants analysis derived from NGS data is also included.

Step 2: If no mutation is identified after analysis of the Spherocytosis panel, based on the approval and consent, we further recommend to continue the bioinformatics analysis of the data obtained by whole genome sequencing to cover genes that are either implicated in an overlapping phenotype or could be involved in a similar pathway but not strongly clinically implicated based on the current information in literature.


Referral reasons

The following individuals are candidates for Spherocytosis panel testing:

  • Individuals with a family history of spherocytosis and presentation of the most common symptoms including jaundice and chronic, non-immune hemolytic anemia
  • Individuals without a positive family history, but with symptoms resembling spherocytosis
  • Individuals (infants) with a negative but suspected family history of spherocytosis, in order to perform proper genetic counseling.

Test utility

Sequencing, deletion/duplication of the panel genes should be performed in all individuals suspected of having spherocytosis and suspected phenotypes. In parallel, other genes reported to be related with this clinical phenotype should also be analyzed for the presence of mutations, due to the overlap in many clinical features caused by those particular genes.

Confirmation of a clinical diagnosis through genetic testing can allow for genetic counseling and may direct medical management. Genetic counseling can provide a patient and/or family with the natural history of the spherocytosis and related disorders, identify at-risk family members, provide disease risks as well as appropriate referral for patient support and/or resources.