Effects of SPTA1 Gene Variants on the Hematological Phenotype of Mexican Patients with Hereditary Spherocytosis

Abstract

Introduction:

Hereditary spherocytosis (HS) is a common hereditary hemolytic anemia characterized by chronic hemolysis, increased indirect serum bilirubin, the presence of reticulocytes and spherocytes in blood smears, and great heterogeneity at the clinical, biochemical, and molecular levels. The molecular pathology of HS includes genetic variants at five genes: ANK1, EPB42, SLC4A1, SPTA1, and SPTB. Alpha spectrin (SPTA1) deficiency is the second leading cause of HS in Mexican patients.

Aim:

To assess the effects of five SPTA1 variants on the hematological phenotype of Mexican patients with HS.

Materials and Methods:

This study included a retrospective cohort of 227 biologically unrelated patients with HS. Variants c.4339-99C>T and c.6531-12C>T in SPTA1 were identified by the amplification-refractory mutation system polymerase chain reaction (ARMS-PCR), and variants c.5572C>T, c.5992C>G, and c.6794T>C were identified by quantitive Real Time-Polymerase Chain Reaction (qRT-PCR) allelic discrimination. Risk tests were performed for each variant with respect to HS clinical severity.

Results:

The SPTA1 c.5992C>G variant showed association with moderately severe HS (p = 0.006, odds ratio = 5.67, confidence interval_95% = 1.6-19.9); the risk increased when the variant was in compound heterozygosity with α^LELY and c.6794T>C. Lower hematological levels were observed in simple α^Lely (c.5572C>T and c.6531-12C>T), and c.5992C>G heterozygotes (red blood cell [RBC] p = 0.028 and 0.010; hemoglobin [Hb] p = 0.030 and 0.002; packed cell volume [PCV] p = 0.034 and 0.002 respectively), and in c.5992C>G+c.6794T>C compound heterozygotes (RBC p = 0.043; Hb p = 0.033; PCV p = 0.043). Additional genetic traits were observed: 15% had HS+Gilbert syndrome and 13% HS+thalassemia.

Conclusion:

Although most of the studied variants are considered benign, we observed significant associations with phenotypic severity. Therefore, we recommend the inclusion of these variants in molecular screening for HS.

Introduction

Hereditary spherocytosis (HS) is a clinically, biochemically, and molecularly heterogeneous pathology characterized by chronic hemolysis, increased indirect serum bilirubin, and the presence of reticulocytes and spherocytes in blood smears (Andolfo et al., 2016). HS has been observed in all populations; being more frequent in Northern European populations (Perrotta et al., 2008). Its estimated prevalence is 1 in 2000-5000 families (Andolfo et al., 2016). Autosomal dominant and autosomal recessive inheritance patterns have been described in 75% and 25% of HS cases, respectively (Mohandas 2008). Clinical severity ranges from mild with normal hemoglobin (Hb) levels at 12.0-15.0 g/dL, moderate with Hb at 8-12.0 g/dL, moderately severe with Hb at 6-8 g/dL, and severe with Hb below 6 g/dL (Perrotta et al., 2008).

The diagnosis is based on several hematological tests, such as blood smear, red blood cell (RBC) and reticulocyte counts, osmotic fragility testing (by acidified glycerol lysis time, cryohemolysis, and, recently, by flow cytometry or ektacytometry), and erythrocyte membrane protein electrophoresis (Bolton-Maggs et al., 2012; Ciepiela, 2018). Pathologies such as Gilbert syndrome (GS) and thalassemia (Thal) could modify the clinical and hematological features of HS (Aggarwal et al., 2020).

The molecular bases of HS include variants in five genes: ANK1 (Ankyrin-1 8p11.21); EPB42 (Protein 4.2 15q15.2); SLC4A1 (Band 3 or anion exchange protein 17q21.31); SPTA1 (Spectrin alpha chain 1q23.1); and SPTB (Spectrin beta chain 14q23.3) (He et al., 2018; Iolascon et al., 2019).

In clinical practice, HS results in frequent medical evaluation. Because HS is considered a relatively benign disease, biochemical and molecular research in Mexico has paid little attention to this pathology. In Mexican patients, biochemical bases for HS analyzed by membrane protein electrophoresis showed that the main protein deficiency was the Band 3 anion exchange protein, followed by the spectrin alpha chain (Sánchez-López et al., 2003). The molecular approach performed in Mexican patients revealed benign polymorphic variants in ANK1 and SLC4A1 (Camacho-Torres et al., 2006) and a rare pathogenic variant in SLC4A1 (Sánchez-López et al., 2010).

Mutations in SPTA1 have been reported in recessive HS (Chonat, 2019; Gallagher et al., 2019). However, to our knowledge, no clinical or hematological variability in HS has been associated with single nucleotide variants (SNVs). In the present study, we analyzed five SPTA1 SNVs in Mexican HS patients: (1)

c.4339-99C>T (rs200830867) named α^LEPRA located at nucleotide −99 from acceptor site of intron 30 (Gallagher et al., 2019);

(2)

c.5572C>G (rs3737515); a transversion in exon 40, considered a benign variant (Wilmotte et al., 1993);

(3)

the c.5992C>G (rs77877855) variant, located in exon 43 and produces a missense variant (p.Ala1998Pro) (Alías et al., 2010); based on in silico analyses by Sorting Intolerant From Tolerant (SIFT) and PolyPhen scores obtained from the Ensembl database, this variant is considered deleterious and possibly damaging (Howe et al., 2021);

(4)

c.6531-12C>T (rs28525570) in intron 45, 12 nucleotides upstream of the acceptor site (Wilmotte et al., 1993) this transition produces an alternative splicing site that synthetizes 25% of mRNA lacking exon 46, which destabilizes the erythrocyte cytoskeleton (Wilmotte et al., 1993, 1999);

(5)

c.6794T>C (rs952094) in exon 49, p.Ile2265Tre (Howe et al., 2021) this variant is considered benign by SIFT and PolyPhen scores.

Variants c.5572C>G and c.6531-12C>T are in complete linkage disequilibrium and are known as α^LELY. α^LEPRA and α^LELY variants were included because both have been associated to HS and hereditary elliptocytosis, respectively (Maillet et al., 1996), while c. 5992C>G and c.6794T>C because they are missense variants found in exons encoding domains involved in the formation of the lateral dimer of Alpha and Beta spectrin, which together with other cytoskeletal proteins are essential for erythrocyte membrane stability (Begg et al., 2000). Therefore, the aim of this work was to assess the effects of five SPTA1 variants c.4339-99C>T, c.5572C>T, c.5992C>G, c.6531-12C>T, and c.6794T>C on the hematological phenotype of Mexican patients with HS.

Materials and Methods

Subjects and ethics statement

This study included a retrospective cohort of 227 anonymous biologically unrelated patients with HS diagnosed by a positive osmotic fragility test, acidified glycerol lysis test (AGLT) or cryohemolysis, >5% of spherocytes in blood smears, and a background of hemolytic crisis, chronic anemia, or relatives with HS. The study was approved by the local Research and Ethics Committees (Approval No. R-2019-1305-045). All subjects voluntarily agreed to participate and signed informed consent forms. Underaged patients (over 7 years of age) assented to participate in the study, and informed consent was obtained from their parents or tutors.

All patients were referred for suspecting heredity hemolytic anemia by Hematology and Genetics Services from Hospital de Especialidades, and Hospital de Pediatría from Instituto Mexicano del Seguro Social, and Hospitales Civiles de Guadalajara from Universidad de Guadalajara. In all patients, a diagnosis of autoimmune hemolytic anemia was excluded by a negative direct Coombs test. Peripheral blood was collected in tubes containing ethylenediaminetetraacetic acid for hematological, biochemical, and molecular testing. Hematological parameters were determined by cytometry (ADVIA 2120i; Siemens Healthcare, Erlangen Germany), including RBC count, Hb, packed cell volume (PCV), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration, and red cell distribution width.

Total, direct, and indirect serum bilirubin levels were determined in jaundiced subjects. An osmotic fragility test was performed by AGLT and/or a cryohemolysis test, and erythrocyte morphology was evaluated in blood smears. Hb analyses included alkaline Hb electrophoresis, and fetal hemoglobin was determined by Singer's alkaline denaturation and hemoglobin A₂ (HbA₂) quantification by diethyl-aminoethyl cellulose microchromatography. No patient presented hemolytic crisis at the time of the sample collection, or had they been transfused during the past 120 days.

Molecular testing

Using the amplification-refractory mutation system polymerase chain reaction (ARMS-PCR), SPTA1 variants c.4339-99C>T and c.6531-12C>T were identified, and c.5572C>T, c.5992C>G, and c.6794T>C were identified by quantitive Real Time-Polymerase Chain Reaction (qRT-PCR) allelic discrimination.

ARMS-PCR was performed using the following primers: in c.4339-99C>T, forward 5′-GATTCACCCTCTACATAGG-3′ and reverse 5′-CCTGTCTAGTACACGTTGGAG-3′ (359 bp), a specific forward primer 5′-GACCCTGAGTATATCTTCTGTATT-3′ (227 bp) was used for the variant allele; in the c.6531-12C>T variant, the genotypes were identified in separate reactions, using the same reverse primer 5′-CTTGCCAGACTGATGAGAAGG-3′ and different specific forward primers—5′-TGTGATTCCTGTTCCGCACT-3′ for variant alleles (350 bp) and 5′-TGTGATTCCTGTTCCGCACC-3′ for wild-type alleles (350 bp).

Both genotyping assays included an internal amplification control using primers, forward 5′-TCGGTAGTTCTGTCCTTGCC-3′ and reverse 5′-CTGAGATTACAGGCCTGAGC-3′ for exon 13 of SLC4A1. All PCR products were visualized in 2% agarose gel stained with SybrSafe^®.

Variants c.5572C>T, c.5992C>G, and c.6794T>C were genotyped using the TaqMan SNP Genotyping Assay (Thermo Fisher Scientific), according to the manufacturer's recommendations. C_27490193_10, C_99640804_10, and C_11334046_20 were used for each variant, respectively.

The identification of variant c.*238-6785_*238-6786insTATATATA or (TA)_nA of UGT1A1 (rs8175347) was performed by fragment analysis as previously described (Beutler et al., 1998). Patients with suspected Thal were analyzed according to previously established molecular screening (Rizo-de la Torre et al., 2021).

Data analysis

Allelic and genotypic frequencies, as well as Hardy-Weinberg equilibriums, were calculated using the Microsoft Excel^® program. Haplotype assignation and linkage disequilibrium were determined using Haploview software. Descriptive and inferential statistical analyses were performed using IBM^® SPSS^® Statistics 25, and a p-value <0.05 was considered statistically significant. Homogeneity (Levene) and data distribution (Kolmogorov-Smirnov) tests were applied to all observations.

Normally distributed descriptive values were compared by the t-test and one-way analysis of variance (ANOVA), while non-normal distributed values were analyzed by the Mann-Whitney U test and the Kruskal-Wallis test with the Bonferroni correction. Odds ratio tests were performed for each of the variants to determine HS clinical severity. The GRCh38.p13 (GCF_000001405.39) NC_000001.11 (158610498.158686715) sequence from GenBank was used as reference for SPTA1 gene.

Results

Of the 227 patients, 112 (49.3%) were male; the age range was 1-76 years. The physical examination of 210 patients showed that 19% had splenomegaly, 6% hepatomegaly, and 3% hepatosplenomegaly. According to their clinical background, only 3% had been splenectomized. A total of 146 (64.3%) were identified as having only HS. Eighty-one (35.7%) patients had the following additional genetic traits: 34 (15.0%) had GS, 29 (12.8%) had Thal (identified by presenting erythrocytosis, microcytosis, MCV <80 fL, and normal or increased HbA₂). The remaining 18 (7.9%) patients had complex hematological phenotypes (iron deficiency, sickle cell trait, and/or G6PD deficiency), and they were excluded from the hematological analysis.

Variant analysis

The allelic and genotypic frequencies are shown in Table 1. At least one SPTA1 variant was identified in 47.1% (107/227) of patients, 25.5% (58/227) had more than one variant and 23.8% (54/227) were homozygous wild-type genotype. All minor alleles were identified. The most common was c.6794C at a frequency of 0.46, while c.4339-99T was observed at a frequency of only 0.01. All variants were in Hardy-Weinberg equilibrium (p > 0.05). Linkage disequilibrium tests were performed for the five SNVs; only variants c.5572C>G and c.6531-12C>T of SPTA1 were observed in complete linkage disequilibrium (p = 0.000; D′ = 1; Z = 74.7; r = 1). They were referred to as a single variant known as α^LELY and observed at a frequency of 0.15.

Table 1.

Allele and Genotype Frequencies of the Studied Variants and Hardy-Weinberg Equilibrium

Genotype	c.4339-99C>T		c.5572C>G		c.5992C>G		c.6531-12C>T		c.6794T>C
Genotype	n	f	n	f	n	f	n	f	n	f
wt/wt	225	0.99	167	0.74	211	0.93	167	0.74	62	0.27
wt/mut	2	0.01	52	0.23	16	0.07	52	0.23	123	0.54
mut/mut			8	0.03			8	0.03	42	0.19

Allele	n	p	n	p	n	p	n	p	n	p
wt	452	0.99	386	0.85	438	0.97	386	0.85	247	0.54
mut	2	0.01	68	0.15	16	0.03	68	0.15	207	0.46
HWE (p)	0.95		0.13		0.58		0.13		0.16

HWE, Hardy-Weinberg Equilibrium; Mut, mutated; Wt, wild-type.

HS+GS, and HS+Thal

Additional hematological pathologies were observed in 81 patients: 34 patients had HS+GS, among which 33 were homozygous (AT)₇A/(AT)₇A (g.175492TA [8]), and only one was heterozygous (AT)₇A/(AT)₈A. Twenty-nine patients had suspected HS+Thal. In 15 patients α-Thal was confirmed, 7 of which were -^3.7α/αα (g.34164_37967del3804), 6 were α^−59C>Tα /αα (HBA2:c.-59C>T), 1 was—^MEX5/αα (g.(?_47181)_(113714_143666)del), and 1 was—^Fil/-α^3.7 (g.201640_232315del306751/g.34164_37967del3804). Ten patients had minor β-Thal, three patients were β^Cd39C>T/β (HBB:c.118C>T), three patients were β^IVS1:1G>A/β (HBB:c.92 + 1G>A), two patients had δβ-Thal Spanish type (g.60375_153285del92911), one patient was β^FSCd77/78-C/β (HBB:c.234_c.235delC), and one β^{FS Cd 11-T}/β (HBB:c.36delT).

In the remaining four patients, we could not identify the mutation causing Thal but we cannot rule it out because they may present another variant not included in the screening.

Hematological parameters

According to Perrotta's classification of the clinical staging of patients with HS, in our study, 42.7% (97/227) had mild HS, 44.9% (102/227) had moderate HS, 11.0% (25/227) had moderately severe HS, and only 1.3% (3/227) had severe HS.

The hematological and biochemical parameter analyses (Table 2) showed that patients with only HS and those with HS+GS had higher Hb values (mean 11.6 and 11.8 g/dL, respectively). The HS+Thal patients had erythrocytosis (mean RBC 4.9 × 10⁶/μL), anemia (mean Hb 10.7 g/dL), microcytosis (mean MCV 67.2 fL), and hypochromia (mean MCH 21.7 pg). Moreover, the HS+GS patients displayed hyperbilirubinemia (mean total and indirect serum bilirubin of 4.69 and 3.51 mg/dL, respectively).

Table 2.

Mean and Standard Deviation of Hematological Parameters Classified by Groups

Group	n (%)	Sex (M/F)	Age (years)^a	RBC ( × 10⁶/μL)	Hb (g/dL)	PCV (%)
HS	146	70/76	9 (1-69)	4.0 ± 1.0	11.6 ± 2.9	34.3 ± 8.5
Mild	70 (47.9)	39/31	19 (1-69)	4.8 ± 0.6	14.0 ± 1.7	41.2 ± 5.5
Mod	56 (38.4)	21/35	15 (1-67)	3.6 ± 0.6	10.2 ± 1.1	30.4 ± 3.4
M-S	18 (12.3)	9/9	18 (1-69)	2.5 ± 0.5	7.0 ± 0.6	22.0 ± 4.0
Sev	2 (1.4)	M	2	1.8	5.3	15.6
		F	2	1.9	5.8	16.5
HS+GS	34	19/15	15 (1-75)	3.9 ± 1.1	11.8 ± 3.1	35.2 ± 9.2
HS+Thal	29	15/14	9 (1-46)	4.9 ± 0.7	10.7 ± 1.9	33.1 ± 5.1

Age is represented in median and range.

GS, Gilbert syndrome; Hb, hemoglobin; HS, hereditary spherocytosis; Mod, moderate; M-S, moderately severe; PCV, packed cell volume; RBC, red blood cells; Sev, severe; Thal, thalassemia.

SPTA1 variants and HS hematological phenotype

According to dominant models of SPTA1 variants, median comparisons of hematological indexes showed that RBC, Hb, and PCV were significantly lower in minor allele carriers of α^LELY and c.5992C>G and in compound heterozygotes for c.5992C>G+c.6794T>C. Carriers of the three minor alleles, α^LELY+c.5992C>G+c.6794T>C, were significant only at lower Hb levels (Table 3).

Table 3.

Model Comparison of Hematological Values by Mann-Whitney U Test

SPTA1 variant	Model	n	RBC ( × 10⁶/μL)	Hb (g/dL)	PCV (%)
α^LELY	C/C+C/C	113	4.3	12.2	35.3
	Dominant model	33	3.6	10.5	32.2
	P		0.028	0.030	0.034
c.5992C>G	C/C	133	4.1	12.2	35.3
	Dominant model	13	3.6	9.6	26.5
	P		0.010	0.002	0.002
c.5992C>G+c.6794T>C	C/C+T/T	140	4.1	11.8	34.7
	Dominant model	6	3.0	8.8	26.7
	P		0.043	0.033	0.043
α^LELY+c.5992C>G+c.6794T>C	C/C+C/C+C/C+T/T	142	4.1	11.8	34.6
	Dominant model	4	3.2	7.8	24.7
	P		NS	0.038	NS

NS, nonsignificant.

When comparing HS patients with SPTA1 variants by clinical group (mild, moderate, moderately severe, and severe), we observed a significant association between the presence of c.5992C>G and the moderately severe phenotype of HS (p = 0.006, odds ratio [OR] = 5.673, confidence interval [CI]_95% = 1.616 − 19.912). An association with the same phenotype was found in SPTA1 variants in compound heterozygosity (Table 4). No statistical association was observed in the HS+GS and the HS+Thal patients.

Table 4.

Risk of Moderately Severe Hereditary Spherocytosis According to Single or Compound Heterozygotes

SPTA1 variant	Sig.	OR	CI 95%
SPTA1 variant	Sig.	OR	Min	Max
α^LELY	0.076	2.570	0.905	7.302
c.5992C>G	0.006	5.673	1.616	19.912
c.6794T>C	0.149	2.590	0.710	9.440
α^LELY+c.6794T>C	0.040	3.008	1.049	8.625
c.5992C>G+c.6794T>C	0.007	6.914	1.660	28.791
α^LELY+c.5992C>G+c.6794T>C	0.047	7.750	1.199	8.889

CI, confidence interval; Max, máximum; Min, mínimum; OR, odds ratio; Sig, significance.

Discussion

This article is the first to report SPTA1 variants in Mexicans and their effects on hematological parameters in HS patients. The allelic frequencies of the five studied SNVs were similar to those reported worldwide in online databases gnomAD and Ensemble (Karczewski et al., 2020; Howe et al., 2021) (p > 0.05) (Table 1).

HS has been frequently observed in association with other genetic pathologies (Aggarwal et al., 2020). In the present study, GS and Thal were identified in 15.0% and 12.8% of the patient sample, respectively. The prevalence of GS in the Mexican population was previously reported to be 10.1% (Arámbula and Vaca, 2002), and thalassemic syndromes (α- and β-Thal) were shown to account for at least 10.0% of patients with microcytosis and/or hypochromia (Casas-Castañeda et al., 1998; Reyes-Núñez et al., 2006). Few studies have reported the coexistence of other hematological pathologies with HS. In a recent study, Aggarwal et al. (2020) found a prevalence of 41.1% for GS and 6.8% for α-Thal in a group of 73 index cases with HS. Chen et al. (2020) found that hematological features of Thal frequently overlapped those of HS.

The most frequent variant, c.6794T>C, was present in 72.7% of analyzed individuals with an allelic frequency of 0.46, similar to Mexican ancestry Los Angeles subjects (p = 0.88) (Howe et al., 2021). Heterozygous carriers showed no association with severe forms of HS; however, compound heterozygotes with c.5992C>G had significantly lower RBC, Hb, and PCV, and those with α^LELY+c.5992C>G had significantly lower Hb (p = 0.038) (Table 3).

The second most frequent variant was α^LELY, identified in 26% of patients. According to the online database gnomAD, this variant has been shown to have an allelic frequency of 0.15 and a worldwide frequency of 25% (Karczewski et al., 2020).

The α^LELY variant has been reported to be a modifier of elliptocytosis, and carrier patients have shown a severe elliptocytosis phenotype (Franck et al., 2018). According to the ClinVar database, conflicting interpretations of pathogenicity have been reported (Variation ID: 258954) (Delaunay et al., 2004; Landrum et al., 2018). In the present study, we observed a significant association with lower RBC, Hb, and PCV in simple or compound heterozygosity with c.5992C>G and c.6794T>C (Table 3). Although we did not identify the causal variant of HS, we observed that individuals who carried the α^LELY variant displayed more severe phenotypes. Similar findings were reported by Svidnicki et al. (2020), who identified null SPTA1 variants in four families with HS and discovered that compound heterozygotes with α^LELY had a slightly more severe phenotype.

SPTA1 variant c.5992C>G was identified in 16 patients, and a minor allele had an allelic frequency of 0.03. This variant showed a clear effect on the HS phenotype, and heterozygotes displayed significantly lower RBC, Hb, and PCV (p = 0.010, 0.002, and 0.002, respectively) (Table 3). A similar effect was observed in compound heterozygote individuals with c.6794T>C (Table 3). In contrast, carriers of c.5992C>G had a 5.7-times increased risk of presenting moderately severe HS (Table 4). Patients with α^LELY+c.5992C>G+c.6794T>C had a 7.8-times increased risk of presenting moderately severe HS (Table 4). This phenomenon could be attributed to a SPTA1 alpha helix alteration, because this variant involves an amino acid change (p.Ala1998Pro), and the introduction of a proline could disrupt the alpha helix, as previously suggested by Alías et al. (2010).

SPTA1 c.4339-99C>T variant, which is also known as α^LEPRA, was the least frequently observed SPTA1 variant (0.01). It has been shown to have a frequency of 0.008 in Europeans, while in Latin Americans it has been observed in 0.001 according to the online database gnomAD (Karczewski et al., 2020). This variant is considered pathogenic, and it has been described in families with erythrocyte membrane defects (Christensen et al., 2014; Niss et al., 2016; Ma et al., 2018). A heterozygous genotype of α^LEPRA was observed in only two patients, one with mild and the other with moderate HS; therefore, statistical analysis of hematological parameters was not performed in these patients.

According to the results of the data analysis, carriers of at least one minor allele of α^LELY or c.5992C>G were significantly associated with moderately severe forms of HS with lower RBC, Hb, and PCV hematological indices. As the number of minor alleles increased, the Hb values decreased, thus behaving as an additive model. Although these variants have not been considered a cause of HS, we observed a clear and notable influence on the hematological features of these patients (Table 3).

Variant c.5992C>G carrier patients have a 5.0-times increased risk of moderately severe HS. The presence of this variant, in addition to α^LELY and c.6794T>C, increased the risk up to 7.8 times; such associations have not been previously described in the literature (Table 4).

Additionally, our results showed that individuals with HS+GS showed higher indirect serum bilirubin levels than individuals with HS (3.51 vs. 2.12 mg/dL, respectively). Aggarwal et al. (2020) reported that homozygous (AT)₇A/(AT)₇A individuals (with GS) had higher levels of indirect serum bilirubin (1.43 mg/dL) than heterozygous (AT)₆A/(AT)₇A, and homozygous (AT)₆A/(AT)₆A, at 0.62 and 0.52 mg/dL, respectively. However, in our patients with HS+GS, indirect serum bilirubin was higher, which suggested a synergistic effect on the bilirubin levels of HS hemolysis and the presence of GS. Moreover, in cases of HS with some kind of Thal, there were lower values of MCV than expected for HS, as well as elevated RBC counts (Brancaleoni, 2016).

Although these individuals had HS, their clinical features and hematological phenotypes indicated GS and Thal rather than HS.

A methodological challenge for our laboratory is the search for pathological variants in genes involved in the molecular pathology of HS and their phenotypic effects on clinical, biochemical, and hematological variability. HS is a highly heterogenous disease not only in Mexican patients but in many populations worldwide.

Conclusions

In our population, we observed that individuals with HS frequently presented additional genetic alterations, such as GS and Thal, at 15% and 12.8%, respectively. All minor alleles of the five SPTA1 variants in our study were identified.

The most common was c.6794C at a frequency of 0.46, and α^LELY was observed at a frequency of 0.15. Carriers of α^LELY and c.5992C>G minor alleles and c.5992G with c.6794C compound heterozygotes were associated with decreased RBC, Hb, and PCV values. The c.5992C>G variant in the heterozygous state increased the risk of having moderately severe HS (p = 0.006, OR = 5.673, CI_95% = 1.616-19.912). This risk increased when the variant was in compound heterozygosity with α^LELY and c.6794T>C. Although most of the studied variants are considered benign, we observed significant associations with phenotype severity. Therefore, we recommend the inclusion of these variants in molecular screening for HS.

Footnotes

Acknowledgment

The authors thank CONACYT for granting scholarships to IMHT and LLEM.

Authors' Contributions

All authors fulfilled the criteria for authorship. Activities were performed as follows: I.M.H.-T.: methodology, analysis, draft preparation, and writing review and editing. L.L.E.-M.: analysis, draft preparation, and writing review and editing. L.C.R.-d.T.: validation, analysis, draft preparation, and writing review and editing. L.E.B.-S.: validation, draft preparation, and writing review and editing. B.I.-C.: validation, writing review and editing, reagents and equipment, and conceptualization. F.J.P.-D.: validation, writing review and editing, reagents and equipment, conceptualization, and project administration.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

No funding was received for this article.

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