1. NGS panel - Genetic testing for spherocytosis

Spherocytosis

July 03, 2017

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.


Overview of genes included in Spherocytosis panel offered by CENTOGENE

Gene
OMIM
chr.locus Protein Frequency of mutations Associated/allelic disorders
ANK1
(612641)
8p11.21 Ankyrin-1 60% 3
13/25 4
Spherocytosis type 1 (182900)
EPB42
(177070)
15q15.2 Erythrocyte membrane protein 4.2 5% 3 Spherocytosis type 5 (612690)
SLC4A1
(109270)
17q21.31 Anion transport protein 15% 3 Spherocytosis, type 4 (612653); Cryohydrocytosis (185020); Ovalocytosis, SA type (166900); Renal tubular acidosis, distal, (179800); Renal tubular acidosis, distal AR (611590)
SPTA1
(182860)
1q23.1 Spectrin alpha chain 1 10% 3 Spherocytosis, type 3 (270970); Elliptocytosis-2 (130600); Pyropoikilocytosis (266140)
SPTB
(182870)
14q23.3 Spectrin beta chain 10% 3
12/25 4
Spherocytosis, type 2 (616649); Anemia, neonatal hemolytic, fatal and near-fatal; Elliptocytosis-3

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.


More information on CENTOGENE´s Spherocytosis panel can be found in our genetic test catalogue.