cells Article Concomitant Hereditary Spherocytosis and Pyruvate Kinase Deficiency in a Spanish Family with Chronic Hemolytic Anemia: Contribution of Laser Ektacytometry to Clinical Diagnosis Joan-Lluis Vives Corrons 1, * , Elena Krishnevskaya 1 , Laura Montllor 1 , Valentina Leguizamon 1 and Marta Garcia Bernal 2 1 Red Cell Pathology and Hematopoietic Disorders, Institute for Leukaemia Research Josep Carreras, 08916 Badalona, Spain; ekrishnevskaya@carrerasresearch.org (E.K.); lauramontllor@gmail.com (L.M.); valen.velilla.vl@gmail.com (V.L.) 2 Pediatric Hematology Department, University Hospital Mútua Terrassa Terrassa, 08221 Barcelona, Spain; mgarcia@mutuaterrassa.es * Correspondence: jlvives@clinic.cat Abstract: Background: Hereditary spherocytosis (HS) and pyruvate kinase deficiency (PKD) are the most common causes of hereditary chronic hemolytic anemia. Here, we describe clinical and genetic characteristics of a Spanish family with concomitant β-spectrin (SPTB) c.647G>A variant and pyruvate kinase (PKLR) c.1706G>A variant. Methods: A family of 11 members was studied.  Hematological investigation, hemolysis tests, and specific red cell studies were performed in all  family members, according to conventional procedures. An ektacytometric study was performed Citation: Vives Corrons, J.-L.; using the osmoscan module of the Lorca ektacytometer (MaxSis. RR Mechatronics). The presence of Krishnevskaya, E.; Montllor, L.; the SPTB and PKLR variants was confirmed by t-NGS. Results: The t-NGS genetic characterization of Leguizamon, V.; Garcia Bernal, M. the 11 family members showed the presence of a heterozygous mutation for the β-spectrin (SPTB; Concomitant Hereditary Spherocytosis and Pyruvate Kinase c.647G>A) in seven members with HS, three of them co-inherited the PKLR variant c.1706G>A. In Deficiency in a Spanish Family with the remaining four members, no gene mutation was found. Ektacytometry allowed a clear diagnostic Chronic Hemolytic Anemia: orientation of HS, independently from the PKLR variant. Conclusions: This family study allows Contribution of Laser Ektacytometry concluding that the SPTB mutation, (c.647G>A) previously described as likely pathogenic (LP), to Clinical Diagnosis. Cells 2022, 11, should be classified as pathogenic (P), according to the recommendations for pathogenicity of the 1133. https://doi.org/10.3390/ American College of Medical Genetics and the Association for Molecular Pathology. In addition, after cells11071133 6 years of clinical follow-up of the patients with HS, it can be inferred that the chronic hemolytic Academic Editor: Marcin Majka anemia may be attributable to the SPTB mutation only, without influence of the concomitant PKLR. Moreover, only the family members with the SPTB mutation exhibited an ektacytometric profile Received: 28 January 2022 characteristic of HS. Accepted: 23 March 2022 Published: 28 March 2022 Keywords: anemia; hemolysis; RBC membrane; pyruvate-kinase deficiency; beta-spectrin; hereditary Publisher’s Note: MDPI stays neutral spherocytosis with regard to jurisdictional claims in published maps and institutional affil- iations. 1. Introduction Hereditary spherocytosis (HS) is the most common cause of hereditary hemolytic ane- Copyright: © 2022 by the authors. mia (HHA), affecting approximately 1 in 1000 to 2000 individuals of European ancestry [1], Licensee MDPI, Basel, Switzerland. and with a relatively high incidence in Spain [2]. It is transmitted as an autosomal dominant This article is an open access article character in 75% of cases, and it has become clear that it is a highly heterogeneous disorder, distributed under the terms and in terms of clinical expression and inheritance [3]. In clinical practice, HS diagnosis is based conditions of the Creative Commons on a workup which includes clinical, laboratory investigations, family studies, and the Attribution (CC BY) license (https:// exclusion of other causes of hemolytic anemia [4,5]. Laboratory investigations require, at creativecommons.org/licenses/by/ least, three essential conditions: (a) the presence of circulating spherical red cells (sphero- 4.0/). cytes), (b) a positive eosin-maleimide binding test (EM-binding test), and (c) a characteristic Cells 2022, 11, 1133. https://doi.org/10.3390/cells11071133 https://www.mdpi.com/journal/cells Cells 2022, 11, 1133 2 of 8 osmotic gradient ektacytometry (OGE) profile. In HS the primary molecular abnormality may affect one or more red cell membrane proteins that, in the order of frequency, as found in Spain, are β-spectrin, Band 3, Ankyrin, α-spectrin, and Protein 4.2 [5]. These proteins are encoded by SPTB, SLC4A1 ANK, SPTA1, and EPB42 genes, respectively. Basically, the inheritance pattern of HS is autosomal dominant and its clinical phenotype is mild to moderate or severe hemolytic anemia. The association of HS with other RBC congenital defects is rare, and it has been described with sickle-cell anemia [6], β-thalassemia [7], glucose-6-phosphate dehydrogenase [8], and pyruvate kinase deficiency [9–13]. Congenital pyruvate kinase deficiency (PKD: EC. 2.7.1.40) is the most common enzyme abnormality in the erythrocyte glycolytic pathway, with an estimated prevalence of 1:20,000 in the global Caucasian population [14,15]. Clinical manifestations of PK deficiency are, in general, only present in subjects homozygous or double-heterozygous for two different allele mutations; whereas, in heterozygous carriers, the enzyme deficiency is usually asymptomatic. In some cases, however, the patients exhibit overt neonatal jaundice or hemolytic crises, triggered by different stress situations such as pregnancy, infections, or concomitant metabolic diseases [16,17]. In Spain, PK deficiency is a very rare disease, and practically all descriptions refer to sporadic cases [18–20]. We describe, here, a Spanish family of 11 members, where seven members presented with a HS due to the c.647G>A mutation in the β-spectrin gene (SPTB). In three out of these seven members, a concomitant PKLR c.1706G>A mutation was found. The SPTB variant has been previously described by van Vuren et al. [21] and classified as LP-4, according to the recommendations for pathogenicity of the American College of Medical Genetics and the Association for Molecular Pathology [22]. Here, after the proband’s familial segregation study, we confirm that, on the basis of its phenotype, this variant can be re-classified as P-5. It should be mentioned that genetic analysis is not yet recommended for HS diagnosis, since hundreds of mutations exist; however, the finding of this, same SPTB mutation in a non-related patient of our cohort of patients with chronic hemolytic anemia due to HS [5] provides further support to its P-5 pathogenicity. Finally, since, independently from the coexistence of the heterozygous PK mutation, all the family members with HS have been followed for 6 years since the diagnosis and have exhibited similar clinical phenotype and OGE pattern, it can be assumed that the PKLR deficient variant does not modulate the clinical expression of HS, and that ektacytometry is as a valuable clinical tool for the diagnosis of HS. 2. Materials and Methods Blood samples were collected in EDTA-K3 anticoagulant after obtaining informed con- sent and approval from the Institutional Ethical Committee. All the diagnostic procedures and investigations were performed in accordance with the Helsinki Declaration of 1975. Studies were carried out via a stepwise process, including basic and hemolysis laboratory tests according to Vives Corrons and Aguilar Bascompte [23], RBC enzyme activities were measured by Beutler’s procedure [24], and eosin-5-maleimide (EMA) binding test, as de- scribed by King et al. [25] and revisited by King and Zanella [26]. RBC deformability and other rheological parameters were analyzed by osmotic gradient ektacytometry (OGE), using the osmoscan module of the Laser-assisted Optical Rotational Deformability Cell Analyser LoRRca (MaxSis. RR Mechatronics), as previously described [4,5] The parameters used for the OGE evaluation were (1) EImax (maximum elongation index) or the maximal value of RBC deformability; (2) Omin (osmotic value where EI is minimal), which represents the conventional RBC osmotic fragility (OF); (3) Ohyper (osmotic value corresponding to 50% of the EImax ), which reflects the cellular hydration status; and (4) AUC (Area under the curve), which reflects RBC deformability in the whole gradient osmolality profile. Genetic identification of SPTB and PKLR mutations was performed by SGene capture, followed by t-NGS, as previously described [4,5]. Cells 2022, 11, x FOR PEER REVIEW 3 of 9 Cells 2022, 11, 1133 3 of 8 3. Results The family tree is represented in Figure 1. The proband (ID 1) is a 4 year-old boy, 3. Results who was admitted for moderate anemia (Hb: 104 g/L), high MCHC (346 g/L), and The family tree is represented in Figure 1. The proband (ID 1) is a 4 year-old boy, who marked reticulocytosis (237 × 109/L). Few spherocytes were observed in peripheral blood was admitted for moderate anemia (Hb: 104 g/L), high MCHC (346 g/L), and marked and physical examination9 showed a splenomegaly of 3 cm under the left costal margin. reticulocytosis (237 × 10 /L). Few spherocytes were observed in peripheral blood and The hemolysis physical was confirmed examination showed aby an increasedof serum splenomegaly non-conjugated 3 cm under bilirubin the left costal margin.(2.50 The mg/dl; normal range 0.5–0.8), lactic dehydrogenase (LDH: 767 hemolysis was confirmed by an increased serum non-conjugated bilirubin (2.50 mg/dL; U/L; normal range: 230–460), normal rangeand 0.5–0.8), a low serumlactic haptoglobin dehydrogenase (<20(LDH: mg/dL;767normal range:range: U/L; normal 30–200230–460), mg/dL).and Thea direct antiglobulin test (DAT) was negative, high precision low serum haptoglobin (<20 mg/dL; normal range: 30–200 mg/dL). The direct antiglob- liquid chromatography (HPLC) ulin testdisclosed (DAT) was hemoglobin negative, abnormalities, high precisionand the chromatography liquid RBC enzyme activities (HPLC) were found disclosed to be within the normal range. There was presence of spherocytosis, hemoglobin abnormalities, and the RBC enzyme activities were found to be within the nor- together with an increased RBC osmotic fragility (ROF) on both fresh and incubated mal range. There was presence of spherocytosis, together with an increased RBC osmotic blood, the positive acidified glycerol fragility (ROF) lysis fresh on both test (AGLT: <300 s;blood, and incubated normalthe value: positive >1800 s), and acidified the reduced glycerol lysis test binding (AGLT: <300 s; normal value: >1800 s), and the reduced binding of eosin 5<0 11). of eosin 5′ maleimide (EMA-binding test) to −28.43 (Normal Value Addi- maleimide tionally, a mutation (EMA-binding test) of to SPTB −28.43c.647G>A (Normal(p.Arg216Gln) was found by at-NGS Value < 11). Additionally, analysis, mutation al- of SPTB lowing c.647G>A the (p.Arg216Gln) confirmation ofwas HS.found The OGE profileanalysis, by t-NGS was characteristic allowing the of confirmation HS, as observed in of HS. all seven family members with the SPTB c.647G>A mutation (Figure The OGE profile was characteristic of HS, as observed in all seven family members with 2). The proband’s brother the SPTB (IDc.647G>A 2) and hismutation mother (ID3) (Figurewere 2). healthy. Proband’s The proband’s father(ID brother (ID2)4), a 37 and hisyear-old mother man, (ID3) were healthy. Proband’s father (ID 4), a 37 year-old man, had displayeduntil had displayed moderate anemia and high reticulocyte count since birth sple- moderate nectomy, anemia and performed at the agecount high reticulocyte of 18since years, which birth untilwas followed byperformed splenectomy, a normalization of at the age hemoglobin concentration of 18 years, which and the was followed byreticulocyte a normalizationcount.of hemoglobin concentration and the reticulocyte count. Figure 1. Family tree Scheme 647. G> A (p.Arg216Gln), in beta-spectrin gene; PKLR (+/−): het- Figure 1. Family tree Scheme 647. G> A (p.Arg216Gln), in beta-spectrin gene; PKLR (+/−): hetero- erozygous mutation, c.1706G>A (p.Arg569Gln), in pyruvate kinase gene; Hb: hemoglobin (g/dL); zygous mutation, c.1706G> A (p.Arg569Gln), in pyruvate kinase gene; Hb: hemoglobin (g/dL); Retics: reticulocytes (×109 /L); OGE: osmotic gradient ektacytometry, (+) profile characteristic of HS, Retics: reticulocytes (×109/L);.OGE: osmotic gradient ektacytometry, (+) profile characteristic of HS, (−)normal (−) normalprofile; profile;EMA: EMA:EMA-binding EMA-bindingtest test(+) (+)positive (−)normal; positive;; (−) normal;Spleen Spleen(age (ageofofsplenectomy); splenectomy); (*):values (*): valuesoutside outsidethe thereference referencerange. range. Theproband’s The proband’sfather father(ID4) (ID4)had hadaa41-year-old 41-year-oldsister sister(ID5), (ID5),with withaasimilar similarclinical clinicaland and hematological picture, characterized by slight anemia and reticulocytosis since birth hematological picture, characterized by slight anemia and reticulocytosis since birth that that disappeared after splenectomy performed when she was 22 years old. ROF, on both NaCl disappeared after splenectomy performed when she was 22 years old. ROF, on both NaCl fresh and incubated blood, a positive acidified glycerol lysis test, a positive EMA-binding fresh and incubated blood, a positive acidified glycerol lysis test, a positive EMA-binding test, and an OGE profile were characteristic of HS (Table 1). High performance liquid test, and an OGE profile were characteristic of HS (Table 1). High performance liquid chromatography (HPLC) rejected the co-inheritance of an hemoglobinopathy, but the chromatography (HPLC) rejected the co-inheritance of an hemoglobinopathy, but the measurement of RBC enzyme activities showed a decrease of PK activity (7.5 IU/g Hb, measurement of RBC enzyme activities showed a decrease of PK activity (7.5 IU/g Hb, Normal range: 8.4–15.2) and of the PK/HK ratio (6.3, normal range: 7.2–15.6), suggesting Normal range: 8.4–15.2) and of the PK/HK ratio (6.3, normal range: 7.2–15.6), suggesting the existence of a heterozygous PK deficiency (PKD). The PK activity, also measured in ID 5 children, was found to be within normal range. ID10) exhibited a clinical history of neonatal jaundice, positive EMA-binding tests, and typical HS OGE profiles. ID8 suffered, at 11 months of age, an aplastic crisis with severe anemia, due to a B19 parvovirus infection that recovered after a blood transfusion. ID 9 at 11 years-old did not present a clinical history of neonatal anemia or jaundice and main- Cells 2022, 11, 1133 tained a slightly decreased hemoglobin concentration of about 115 g/L throughout his 4 of 8 life. Cells 2022, 11, x FOR PEER REVIEW 5 of 9 Figure 2. Osmotic Figure Osmotic Gradient GradientEktacytometry Ektacytometry(OGE) (OGE)curves. (a)(a) curves. Family members Family with members HS HS with (7 out (7 of out11); of 11); (b) family (b) family members members without without HS HS (4 (4 of out out of 11). 11). Table 1. Clinical, hematological, and genetic data of the family members. Reference ID1 ID2 ID3 ID4 ID5 ID6 ID7 ID8 ID9 ID10 ID11 Values Age 4 years 7 years 41 years 37 years 41 years 41 years 12 years 7 years 11 years 4 months 54 years Neonatal Yes No No No No No Yes No No Yes No jaundice Cells 2022, 11, 1133 5 of 8 Table 1. Clinical, hematological, and genetic data of the family members. Reference ID1 ID2 ID3 ID4 ID5 ID6 ID7 ID8 ID9 ID10 ID11 Values Age 4 years 7 years 41 years 37 years 41 years 41 years 12 years 7 years 11 years 4 months 54 years Neonatal jaundice Yes No No No No No Yes No No Yes No Splenomegaly Yes No No Yes No No Yes Yes No No No 2 1 Transfusion (No. of units) No No No No No No (6 and No No No (1 year) 8 years) Hemoglobin (g/dL) * 94 130 127 156 140 145 133 108 115 114 142 121–167 MCV (fL) * 76.1 81.6 89.9 85.8 86.9 93.3 83.5 87.1 81.5 83.3 93.4 80–94 MCHC (g/L) * 351 345 323 366 333 343 335 348 368 347 330 310–350 Reticulocytes (109 /L) * 236.7 65.8 46.8 144.1 72.4 50 108.4 544 191.6 175.2 59.7 24–84 Unconjugated bilirubin 2.6 0.1 0.2 0.61 0.60 0.56 2.04 0.98 0.6 5.94 0.54 0.2–0.8 (mg/dL) Haptoglobin (mg/dL) <20 65 72 40 78 42 20 20 42 <20 52 30–200 Lactate Dehydrogenase 767 250 380 520 266 318 280 582 310 393 183 230–460 (IU/L) Direct antiglobulin test (−) (−) (−) (−) (−) (−) (−) (−) (−) (−) (−) (−) Splenectomy (years) No No No Yes (16) Yes (18) No Yes (12) No No No No No RBC OSMOTIC FRAGILITYY AND EMA–BINDING TEST OF in NaCl on fresh blood Increased Decreased Normal Normal normal normal Increased increased increased increased normal normal OF in NaCl on incubated Decreased Decreased Normal Decreased increased normal increased increased increased increased normal normal blood Acidified Glycerol Lysis 1800 1800 1800 1800 <1800 >1800 >120 <120 <120 >1800 <120 >1800 (s) EMA-binding test –28.43 –12.15 –8.4 –26.2 –15.62 –4.1 –26.23 –18.4 –27.45 –24.09 –2.2 >–11 OSMOTIC GRADIENT EKTACYTOMETRY (OGE) EImax 0.168 0.147 0.151 0.164 0.585 0.619 0.565 0.579 0.586 0.583 0.619 0.60–0.63 Omin 158 131 139 157 154 150 162 177 162 164 143 127–159 Ohiper 446 439 473 425 461 455 462 476 436 470 468 447–480 AUC 145.8 165.4 177.5 134.6 155.8 162.2 146.6 144.7 137.3 147.9 172.1 159–175 ACTIVITY PYRUVATE KINASE (PK) PK (IU/gHb) 11.5 12.31 14.9 8.59 7.95 9.17 9.8 9.61 9.61 14.6 8.73 8.4–15.2 GENETIC DIAGNOSIS BY t-NGS, >500x PKLR c.1706G>A heterozygous heterozygous heterozygous heterozygous (p.Arg569Gln) SPTB c.647G>A heterozygous heterozygous heterozygous heterozygous heterozygous heterozygous heterozygous (p.Arg216Gln) *: After splenectomy; MCV: mean cellular volume; MCHC: mean cellular hemoglobin concentration; OF: osmotic fragility; EImax, Omin, Ohyper, AUG: osmotic gradient ektacytometry parameters (see text description). ID5 married two times: the first with 41-year-old healthy man (ID6), and the second with a 54-year-old man (ID11). From the first marriage, she had three sons (ID7, ID8, and ID9) and from the second marriage, she had a daughter (ID10). Two of her sons (ID7 and ID10) exhibited a clinical history of neonatal jaundice, positive EMA-binding tests, and typical HS OGE profiles. ID8 suffered, at 11 months of age, an aplastic crisis with severe anemia, due to a B19 parvovirus infection that recovered after a blood transfusion. ID 9 at 11 years-old did not present a clinical history of neonatal anemia or jaundice and maintained a slightly decreased hemoglobin concentration of about 115 g/L throughout his life. In order to discard an eventual co-inheritance of PK deficiency with HS in this family, a t-NGS study was undertaken in the propositus (D1), his father (ID4), his aunt (ID5), and in all the aunt’s children (ID7, ID8, ID9, and ID10). The t-NGS study demonstrated the existence of the β-Spectrin (SPTB) gene mutation (c.647G>A; p.Arg216Gln) in seven out of 11 family members and of a PKLR gene mutation (c.1706>a; p.Arg569Gln) in three of the seven members with the SPTB mutation, confirming the co-inheritance of HS and PKD in ID5, ID8, and ID9 (Figure 1). PKLR c.1706>a (p.Arg569Gln) mutation has been previously described by Fermo et al. in a 40 year-old women splenectomized for a severe hemolytic anemia [27]. Clinical, hematologic, biochemical, and molecular data of all family members at the time of diagnosis are summarized in Table 1. 4. Discussion Hereditary spherocytosis (HS) is a highly heterogeneous rare anemia in terms of clinical expression, inheritance, and underlying genetic defects. Its estimated prevalence in Europe is about 1:2000 [28], and its incidence in Spain is relatively high [2,29]. In more than 80% of cases, HS has an autosomal dominant inheritance pattern, and its clinical manifestation is a long life chronic hemolytic anemia with variable intensity, from a severe, transfusion-dependent anemia, to an almost silent phenotype, depending on the membrane protein(s) involved, the type of mutation, and other phenotypic modifiers [30,31]. The hallmark of HS is a loss of red cell membrane and a consequent change of the biconcave shape, to a more spherical and dense cell, called spherocyte, that, due to its markedly decreased deformability, is removed from the circulation by the spleen [32]. Cells 2022, 11, 1133 6 of 8 Hereditary pyruvate kinase deficiency (PKD: EC. 2.7.1.40) is the most common anaer- obic glycolysis defect and is a rare, autosomal recessive disease, affecting about 1:20,000 worldwide Caucasian individuals. PKD essentially differs from HS by a relatively more severe clinical phenotype and the absence of circulating spherocytes. PKD hampers cellular energy supply, because glycolysis is the only source of red blood cell ATP production, and the severity of anemia differs considerably, depending on the type of mutation and phenotypic modifiers. Moreover, in some cases, the peripheral blood smear may show few echinocytes and/or tear-drop cells, but RBC morphology is altered to a far lesser extent than in HS [33]. The clinical manifestations of PKD are present in homozygous or double- heterozygous conditions, whereas the heterozygous condition is usually asymptomatic, with the exception of some rare cases with overt neonatal hemolytic anemia and jaundice or with hemolytic crisis triggered by situations of stress, such as pregnancy, infections, or concomitant metabolic diseases [16]. Co-inheritance of HS and PKD is very rare, and, to date, only eight cases have been reported [6–13]. The first case was published in 1970 by Brook and Tanaka [9], and after our first report of this association in Spain [10], we concluded that heterozygous PKD did not modulate the HS clinical expression. Vercellati et al. [11] arrived at the same conclusion after their study of a patient with co-inheritance of HS and a PKLR deficient variant. More recently, however, van Zwieten et al. [12] considered that in a family with HS, the co-inheritance of a partial PKD may aggravate the phenotypic expression of band 3 deficiency, due to the deleterious effect of the ATP decrease. Finally, Andres et al. [13] described a novel association of HS and partial PKD, apparently caused by the loss of membrane-bound PK as the consequence of the impaired structural integrity of the RBC membrane, due to band 3 deficiency. In this paper, we describe a Spanish family of 11 members, where seven exhibited a dominant HS associated with a moderate chronic hemolytic anemia. The β-Spectrin (SPTB) c.647G>A mutation found in this family has been previously described by van Vuren et al. in a cohort of 95 patients with HS [21] and by our group in another study involving a cohort of 45 patients with several hereditary membranopathies (5). As shown in the family tree (Figure 1) the proband (ID1), his father (ID4), and his aunt (ID5) showed a similar clinical and hematological picture, characterized by a chronic hemolytic anemia with increased reticulocyte count and increased MCHC. The proband’s mother (ID3) and his brother (ID2) were clinically and hematologically normal. ID4 and ID5 were splenectomized at 18 and 22 years, respectively, and this was followed by a fully compensated hemolysis, without anemia. ID5 married two times with healthy men (ID6 and ID11) and from the first marriage she had three sons (ID7, ID8, and ID9) with chronic hemolytic anemia, more severe in ID7 than in ID8 and ID9 until splenectomy was performed at the age of 12 years (Figure 1). From the second marriage she had a four- month-old daughter (ID 10) with neonatal jaundice, a positive EMA binding test and typical HS OGE profile. The t-NGS study performed in the 11 family members demonstrated the existence of the SPTB mutation in seven members (Figure 1, marked in red), and three of them exhibited a heterozygous PKLR gene mutation (c.1706>a; p.Arg569Gln), confirming the co-inheritance of HS and PKD in patients ID5, ID8, and ID9 (Figure 1, marked in blue). The question here arises whether the presence of the PKLR c.1706G>A mutation may modulate the clinical expression of HS due to the SPTB c.647G>A mutation. In our previous study of a patient with coinheritance of HS and PK ‘Mallorca’ [10], we concluded that the defective PKLR gene did not influence the HS clinical phenotype. We provide here a further support to this assumption, as can be seen in the large number of family members included in our study where the patients with concomitant SPTB and PKLR mutations (ID5, ID8, and ID9) seem to exhibit the same phenotypic expression as the patients with the SPTB mutation alone. (Figure 1). The slight differences in clinical expression of SPTB mutation between the different members of this family may be explained by the known intra-family variability of clinical phenotype in HS [34] or additional factors (even acquired) that may modify clinical phenotype in these patients [35]. Cells 2022, 11, 1133 7 of 8 5. Conclusions Our findings provide three main messages: (a) The SPTB mutation (c.647G>A), with a moderate clinical phenotype has to be re-classified as P-5; (b) The tight correlation between the SPTB mutations and the OGE profile provides a further demonstration of the clinical utility of RBC deformability measurement in the diagnosis of HS; and (c) Key glycolytic enzymes activity measurement in patients with chronic hemolytic anemia due to an underlying RBC membrane defect may improve our knowledge of the epidemiologic and genetic characteristics of eventual co-inherited RBC defects, as well as their frequency in the European population. Author Contributions: M.G.B. gathered the clinical data and provided the patient samples; J.-L.V.C. and M.G.B. designed the family study; E.K. performed the diagnostic tests for HS, analyzed the patient’s data and designed the family tree; L.M. performed PKD molecular and genetic studies; V.L. is a pre doctoral fellow in practices for a Master degree. J.-L.V.C. and E.K. wrote the paper. All authors have read and agreed to the published version of the manuscript. Funding: This research received partial financial support from the European Community’s Seventh Framework Program (FP7/2007-2013) (grant number 602121). Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board (or Ethics Committee) of the Institute for Leukaemia Research Josep Carreras. Informed Consent Statement: All subjects gave their informed consent for inclusion before they participated in the study (Project identification code: PI-21-308). Data Availability Statement: Data sharing policies concern the minimal dataset that supports the central findings of a published study. In accordance with journal guidelines, MDPI Research Data Policies generated data should be publicly available and cited in accordance with journal guidelines. 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