Introduction: 
Hereditary spherocytosis (HS) is the most frequent cause of non-immune haemolytic anaemia (HA) among individuals of Northern European ancestry; its estimated prevalence is 1:2000. HS results from various molecular defects leading to decreased production or abnormality of one or more red blood cell (RBC) membrane proteins i.e., ankyrin, protein 4.2, band 3 and spectrin. The majority of HS cases (75%) are inherited in an autosomal dominant manner. Especially because of the spherical shape, RBCs are trapped in the spleen and have a shortened life span. Among other factors, according to the importance of the deficiency or protein abnormality it conducts to a clinical expression that may range from asymptomatic to severe chronic HA. Most often a phenotypic diagnosis can be made but next-generation gene sequencing (NGS) has a place in difficult cases i.e. unsolved cases of HA, transfusion prior to diagnosis and discrepancy between clinical phenotype and diagnosis. We report here the case of a 18-year-old patient with a suspected HS, prominent splenomegaly and unknown personal or family history.

Case report: 
The patient complained of fatigue for several months and grade 2 dyspnea. In the past, he repeatedly presented with biliary colic pain. He apparently had no personal or family medical history. Two years ago, he nevertheless had jaundice secondary to haemolytic anaemia. The evolution was spontaneously favourable and no further investigation was undertaken. He has a twin brother and a 20-year-old sister, both in good health. The clinical examination found a prominent splenomegaly. Haemoglobinopathy and most frequent RBC enzyme deficiencies were excluded. Given the high clinical suspicion of HS, his parents and siblings (although asymptomatic) were screened for HS. The main biological data are reported in the table below.

Test (unit; reference value)	Propositus	Mother	Father	Sister	Fraternal twin
Hb  (g/dL; 13-16 M – 12-15 F) 	9.1	13.5	15.3	12.9	15.8
MCV (µm³; 80.8-99.2)	85.9	80.3	89.6	84.2	77
MCHC (g/dL; 32-36)	34.7	35.5	34.6	33.5	35.8
Reticulocytes (103/mm3; 30-111)	350	192	74	54.8	84.2
LDH (x upper ref. value)		1.1	< 1.0	<1.0	<1.0
Unc. Bilirubin mg/dl (< 0.7; mg/dL)	1.6	0.62	0.15	0.38	0.53
Haptoglobin mg/dl (mg/dL; 30-200)	< 10	< 10	66	101	36
Blood smear	Rare microspherocytes		Normal		
Direct Coombs	Negative	-	-	-	-
EMA (%; < 19)	21	29	< 2.0		
CH (%; <10)	19	30	9.3		
Ektacytometry	Compatible with HS	Compatible with HS	Normal		
SDS-PAGE	Band 3 deficiency		Normal		
Molecular analysis	Mutation c.224G>A/wt	Mutation c.224G>A/wt	wt/wt	Ongoing	Ongoing
(MCV= mean corpuscular volume; MCHC=mean corpuscular haemoglobin concentration; EMA= eosine-5’-maleimide; CH= cryohemolysis)

Molecular genetic analyses were performed: NGS targeted sequencing panel, containing panel 40 genes associated to haemolytic anemia, and confirmation by Sanger identified a nonsense mutation on the cytoplasmic domain of Band3. The mutation is novel and drastic; it causes a stop codon at 75 residues. The mutation is transmitted by the mother. NGS targeted sequencing analysis did not demonstrate any other abnormalities that could explain the phenotypic variability observed in the propositus and his mother.
Given the profound anaemia associated to the voluminous spleen (main diameter of 25 cm by CT-scan evaluation) and given the presence of symptomatic gallstones, the patient underwent a splenectomy and a cholecystectomy. After the procedure, he recovered from fatigue and the haemoglobin level returned to normal with no signs of residual haemolysis.

Conclusion:
This case reports a HS related to a new nonsense mutation in SLC4A1. The transmission is autosomal dominant but the penetrance is heterogeneous within the same family.

Contribution of NGS in the diagnosis of hereditary spherocytosis: case report