Page 106 - 2020_02-Haematologica-web

R. Huisjes et al.
ing, reflecting band 3 protein loss, was seen in all patients. Patients with SPTA1 mutations tended to have greater EMA staining (Figure 1A), although the number of patients was too low to draw firm conclusions. The max- imal deformability of RBC, reflected by a decrease in maximal elongation index (EImax) as determined by the Osmoscan, was decreased in all HS patients compared to that in healthy controls. This decrease was more pro- nounced in patients with ANK1 and SPTB mutations than in patients with mutations in SPTA or SCL4A1 (Figure 1B). On the other hand, cells from patients with SLC4A1 mutations tended to be more dehydrated as their Ohyper values (hypertonic osmolarity at 50% of EImax) were lower than those in both control blood samples and patients with SPTA1 mutations (Figure 1C). The latter patients also showed the least pronounced loss of surface area-to- volume ratio, reflected by a normal Omin (hypotonic osmolarity where EI is minimal) (Figure 1D) on the Osmoscan and normal results in the osmotic fragility test
(50% lysis point) (Figure 1E). Membrane stability was compromised in all other HS patients.
As expected, RBC turnover was increased in all patients: reticulocyte counts were high (Table 1) and the band 4.1a:b ratio, a marker of aging, was lower in all patients than in healthy controls (Table 1).
The heterogeneity of the RBC, reflected by the red blood cell distribution width (RDW), was greater in patients with HS than in healthy controls (Figure 1F) as was the MCHC (Figure 1G). Intracellular K+ content was reduced in all patients but tended to be higher in HS patients with SPTA1 mutations (Figure 1H).
An increase in heterogeneity in cell projected areas (mean projected area distribution width, MPA DW) (Figure 2A) and a decrease in absolute mean values in pro- jected area (MPA) (Figure 2B) were seen in all HS patients compared to those in controls. Patients with SLC4A1 and SPTA1 mutations had cellular projected areas more simi- lar to those of the healthy control group (Figure 2A, B).
Table 1. Hemocytometry, chemistry parameters, cell-age markers and genotypes of patients with mild or moderate/severe hereditary spherocytosis (HS) and splenectomized HS patients included in this study.
N. Sex Age Genotype (years)
Hb RBC (g/dL) (1012
Hct MCV Ret (%) (fL) (%)
MCHC RDW EMA Omin EImax Ohyper OFT Band CD71 (g/L) (%CV) (%) (mOsmo (A.U.) (mOsmo (g/L 4.1/4.2 (%)
/L)
4.1
4.7 4.9 4.0 3.7 4.1 4.1 3.5
l/L) l/L) NaCl) ratio (A.U.)
1 ♂ 75
2 ♂ 46
3 ♂ 40
4 ♀ 55
5 ♀ 18
6 ♂ 58
7 ♂ 40
8 ♂ 54
9 ♂ 4
10 ♀ 3
11 ♂ 5
12 ♀ 26
13 ♂ 1
14 ♂ 3
15 ♂ 4
16 ♀ 42
17 ♂ 31
18 ♀ 46
19 ♀ 84
20 ♂ 71
21 ♂ 40
ANK1 c.344T>C p.Leu115Pro
SLC4A1 c.1030C>T p.Arg344*
SLC4A1 c.1421C>A p.Ala474Asp
SLC4A1 c.2057+1G>A (splicing)
SLC4A1 c.2057+1G>A (splicing)
SLC4A1 c.2348T>A p.Ile783Asn
SPTA1 c.678G>A p.Glu227fs + αLELY
SPTA1 c.[4339-99C>T; c.4347G>T] p.[(?; Lys1449Asn)]; c.4339-99C>T p.(?)
ANK 1 c.341C>T p.Pro114Leu ANK1 c.1943delC p.Ala648fs ANK1 c.2394_2397delCAGT p.Ser799fs ANK1 c.2559-2A>G (splicing) SPTB c.154delC p.Arg52fs
SPTB c.2470C>T p.Gln824*
SPTB c.5937+1G>A p.(?)
SPTA1 c.2755G>T p.Glu919* + αLELY
ANK1 c.341C>T p.Pro114Leu ANK1 c.344T>C p.Leu115Pro SLC4A1 c.2057+1G>A (splicing) SPTB c.2136_2137delinsTT SPTB c.3449G>A p.Trp1150*
136
153 140 136 131 132 127 121
116 117 120 102 86 78 84 113
152
143 160 164 163
n.a.
n.a. n.a. 36.5 35.7 36.5 35.9 34.7
Mild HS11
n.a. 7.2
n.a. 3.2 n.a. 4.0 91.1 7.8 96.9 9.4 88.4 8.9 87.8 5.8 98.3 3.9
n.a. 16.2
n.a. 15.2 n.a. 12.3 372 14.0 367 13.1 363 15.7 355 15.0 348 15.3
85 162
71 163 93 162 73 158 77 166 68 169 94 175 94 160
72 162 59 164 67 185 84 170 66 168 71 180 74 173 89 178
76 179
74 158 67 164 77 173 73 183
0.555 457 7.0
0.563 417 6.6 0.557 427 5.7 0.554 405 6.4 0.569 410 6.1 0.548 429 6.8 0.600 453 5.6 0.566 458 5.7
0.509 398 6.6 0.503 424 6.6 0.472 462 7.8 0.537 446 6.4 0.532 456 5.7 0.534 475 6.7 0.541 459 7.0 0.567 471 5.9
0.533 437 7.4
0.590 423 7.4 0.565 417 7.1 0.509 445 7.3 0.512 440 7.8
0.70 0.8
0.87 1.4 0.99 1.2 0.82 2.7 0.84 1.4 n.a. 3.5 0.84 0.9 n.a. 0.9
0.72 2.2 n.a. 0.2 n.a. 7.3 0.60 5.3 n.a. 4.8 n.a. 2.9 n.a. 5.5 n.a. 2.1
1.08 0.1
1.18 0.2 1.07 0.2 0.99 0.3 1.22 0.3
Moderate/severe HS11
3.8 30.0 4.02 36.3 4.1 32.2 3.3 29.4 3.38 30.2 2.9 23.7 3.09 27.7 3.5 31.7
80.1 9.1 90.4 11.9 78.6 18.4 89,5 18.3 89.5 11.9 81.9 10.6 89.7 16.7 91.8 8.2
386 14.5 321 21.1 372 25.4 346 24.1 284 24.7 328 23.8 303 23.4 355 16.0
Splenectomized HS11
4.9 84.7 41.9
4.6 n.a. n.a. 4.8 121.8 45.1 4.84 100.9 48.8 4.94 92.0 45.5
8.4 362 11.8
2.8 n.a. 12.4 12.6 354 11.0 2.2 337 13.2 12.2 359 11.9
Clinical severity in non-splenectomized HS patients was assigned according to Bolton-Maggs et al.11 on the basis of (i) hemoglobin concentration and (ii) reticulocyte count.Mild HS was defined as hemoglobin levels between 110–150 g/L, moderate HS as hemoglobin levels between 80–120 g/L and severe HS as hemoglobin levels lower than 80 g/L. HS patients with hemoglobin levels between 110 and 120 g/L were categorized as having mild or moderate disease on the basis of their reticulocyte levels (i.e., lower or higher than 6% reticulocytes). Novel mutations are displayed in bold font and the pathogenicity of novel missense variants was predicted with SIFT, PolyPhen-2, and MutationTaster (results not shown). Notation of αLELY represents SPTA1 c.[5572C>G; 6531- 12C>T] p.[(Leu1858Val);(?)]. N: number; Hb: hemoglobin; RBC: red blood cells; Hct: hematocrit; MCV; mean corpuscular volume; Rct: reticulocytes; MCHC: mean corpuscular hemoglobin con- centration; RDW: red cell distribution width; EMA: eosin-5′-maleimide; %CV: percent coefficient of variation; HS: hereditary spherocytosis; n.a.: not available.
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haematologica | 2020; 105(2)