ISSN: 2469-2778 HTIJ

Hematology & Transfusion International Journal
Case Report
Volume 2 Issue 6 - 2016
Two novel mutations in the Ankyrin-1 gene associated with Hereditary Spherocytosis
Del Orbe-Barreto R1*, Arrizabalaga B1,2, Erquiaga S2, De la Hoz-Rastrollo AB1, Martin-Martitegui X2, García-Orad A3, Molina J4, Bento C5 and García-Ruiz JC1,2
1BioCruces Health Research Institute, Spain
2Hematology Department, University Hospital Cruces, Spain
3Department of Genetics, Physical Anthropology and Animal Physiology, University of the Basque Country (UPV / EHU), Spain
4Pediatrics Department, Complejo Hospitalario de Navarra, Spain
5Hematology Department, Centro Hospitalar e Universitário de Coimbra, Portugal
Received: October 18, 2016| Published: November 10, 2016
*Corresponding author: Rafael Andrés Del Orbe Barreto, BioCruces Health Research Institute. Plaza de Cruces s/n. 48903, Barakaldo, Spain, Europe, Tel: +34946006000; +34946006089; Email:
Citation: Del Orbe-Barreto R, Arrizabalaga B, Erquiaga S, De la Hoz-Rastrollo AB, Martin-Martitegui X, et al. (2016) Two novel mutations in the Ankyrin-1 gene associated with Hereditary Spherocytosis. Hematol Transfus Int J 2(6): 00054. DOI: 10.15406/htij.2016.02.00054


Mutations in the ankyrin-1 gene (ANK1) underlie half of the cases of Hereditary Spherocytosis (HS), and of these, two thirds are due to mutations inherited in a dominant pattern, while the others are due to sporadic mutations. Here we report two novel ANK1 mutations responsible for HS (c.1800+1G>A and c.1196_1196delC) and propose possible mechanisms of pathogenicity for each mutation described.

Keywords: Hemolytic anemia; Red cells; Membrane disorders; Molecular biology; Pathogenicity; Genotyping


ANK1: Ankyrin-1 Gene; HS: Hereditary Spherocytosis; CBS: Center of Biological Sequence Analysis; BC: BioCruces; NGS: Next Generation Sequencing


Hereditary spherocytosis (HS) is the most common inherited red cell membrane disorder with one case out of 2000-3000 individuals and probably even higher prevalence due to under diagnosis of minor or moderate forms of HS [1]. In this heterogeneous disorder abnormalities of red blood cell structural proteins lead to loss of erythrocyte membrane surface area, resulting in spherical-shaped, hyperdense, poorly deformable red blood cells with a shortened life span [2]. Mutations in the Ankirin-1 gene (ANK1) accounts 50% of the HS cases, which two thirds are inherited in a dominant pattern and the remaining cases are due to sporadic mutations; To date, more than seventy mutations have been described [1,3,4], which demonstrate the high level of allelic heterogeneity in ANK1 mutations so that makes necessary the search of new variants in this gene to establish the biological mechanisms of pathogenicity. Here we report two novel mutations in ANK1 found in patients diagnosed with HS.

Patients and Methods

Clinical and laboratory data of cases are summarized in Table 1.

Case 1: ANK1:c.1800+1 G> A

Case 2: ANK1: c.1196_1196 delC

Age at diagnosis

1 month

2 months




Hemoglobin (g/dL)



Reticulocyte count (%)

310.000/mm3 (11,8%)

522.500/mm3 (12,9%)

Mean corpuscular volume (fL)



Mean corpuscular hemoglobin concentration (g/dL)



Serum lactate dehydrogenase (U/L)



Total Bilirubin mg/dL



Eosin-5’-maleimide test






Osmotic frailty




De novo


Table 1: Clinical and biochemical variables of individuals with overweight-obesity.

SD: Standard Deviation; BMI: Body Mass Index; WC: Waist Circumference; AC: Abdominal Circumference; HC: Hip Circumference; RER: Respiratory Exchange Ratio; HR: Hear Rate.

Genetyc Analysis

The Euskadi Research Ethics Committee approved the study protocol in accordance with the principles of the Declaration of Helsinki (PI2014160). Informed consent was obtained from all of the adult subjects or from the parents of the children for genetic testing. We performed a targeted sequencing of genes that encode membrane proteins on a Next Generation Sequencing (NGS) platform as previously described [5] and identified two new mutations in ANK1 (NM_000037) in heterozygous state related with pathogenesis of HS. These newly identified variants were no found in the Human Gene Mutation Database [6], ClinVar [7], 1000 Genomes Project dataset [8], Exome Aggregation Consortium (ExAC, [9]) and the UCSC SNPs database and after a comprehensive review of current literature.

Case Description

Case 1 was a male baby who presented with jaundice, severe anemia and extravascular hemolysis. His parents, no-consanguineous had no medical history of HS. Sequencing revealed a change of a Guanine to an Adenine in intron 16-17 at position c.1800+1 in ANK1 (c.1800+1G>A). The consulted splicing prediction software (Center of Biological Sequence Analysis (CBS); [10]) indicates that this mutation can affect the mRNA transcription with a high coefficient of confidence (0.95), resulting in a different transcript, 315 base pairs longer than the canonical sequence, and this corresponds to the addition of 105 amino acids to the protein, which would imply changing its structure; nevertheless, it would be necessary to conduct mRNA analysis to confirm this. The inheritance pattern revealed that it is a “de novo” mutation, since genotyping of the parents not showing the variation. Previously, seven other mutations that affect splicing in ANK1 have been identified as causative of HS, leading to exon skipping or intron inclusion [11,12].

Case 2 was a child diagnosed with hemolytic anemia few weeks after birth. His mother and uncle were diagnosed with HS during her childhood and splenectomized, but there was no data from molecular studies. Genotyping done in the child and his mother showed a deletion of cytosine at position 1196 in ANK1. This frameshift mutation determines a displacement of the reading frame causing the occurrence of a premature stop codon obtaining a peptide of only 406 amino acids instead of 1880 (c.1196_1196delC; p.Ala399Glyfs*7). The new truncated protein would cause disturbance of the structure of the erythrocyte membrane, due to lacking of both spectrin-binding domain and C-terminal regulatory domain [13]. Actually, nineteen small-deletions and five small-insertions mutations in ANK1 have been reported as pathogenic [4].


In summary, we identified two novel ANK1 mutations responsible for HS and demonstrated their genotype/phenotype correlation and we propose a possible mechanisms of pathogenicity for each described mutation. Nowadays, the increasing use of NGS technologies as a genetic diagnostic tool in congenital hemolytic anemia will lead us to know many novel mutations implicated in this disease which will conduct to a better understanding of the biology of HS.


The author was supported by a grant from BBK- BioCruces (BBK-BC/POST-MIR/2015/001).


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