Novel histotypes of sporadic Creutzfeldt–Jakob disease linked to 129MV genotype

The MV1 and MV2 subtypes of sporadic Creutzfeldt–Jakob disease (sCJD) are linked to the heterozygous methionine (M)/valine (V) polymorphism at codon 129 of the prion protein (PrP) gene. MV2 is phenotypically heterogeneous, whereas MV1, due to its low prevalence, is one of the least well characterized subtypes. In this study, we investigated the biochemical properties of PrPSc and phenotypic expression of cases diagnosed as sCJD MV1 and MV2. We describe four MV2 histotypes: 2C, with cortical (C) coarse pathology; 2K, with kuru (K) plaque deposits; 2C-K, with co-existing C and K histotypic features; and the novel histotype 2C-PL that mimics 2C in the cerebral cortex and cerebellum, but exhibits plaque-like (PL) PrP deposits in subcortical regions (e.g., basal nuclei, thalamus and midbrain). Histotype prevalence is highest for 2C-K (55%), intermediate for 2C (31%), and lowest for 2C-PL and 2K (7%). Nearly every MV2 case expressed both PrPSc types, with T2 being the predominant type (“MV2-1”). MV1 cases typically show a rapid disease course (≤ 4 months), and feature the 1C histotype, phenotypically identical to sCJDMM1. Co-existing PrPSc types, with T1 significantly exceeding T2 (“MV1-2”), are detected in patients diagnosed as MV1 with longer disease courses. We observed four histotypes among MV1-2 cases, including two novel histotypes: 1V, reminiscent of sCJDVV1; 1C-2C, resembling sCJDMM1-2 with predominant MM1 histotypic component; and novel histotypes 1C-2PL and 1C-2K, overall mimicking 1C in the cerebral cortex, but harboring T2 and plaque-like PrP deposits in subcortical regions (1C-2PL), and T2 and kuru plaques in the cerebellum (1C-2K). Lesion profiles of 1C, 1V, and 1C-2C are similar, but differ from 1C-2PL and 1C-2K, as the latter two groups show prominent hippocampal and nigral degeneration. We believe that the novel “C-PL” histotypes are distinct entities rather than intermediate forms between “C” and “C-K” groups, and that 1C-2PL and 1C-2K histotypes may be characterized by different T1 variants of the same size. Supplementary Information The online version contains supplementary material available at 10.1186/s40478-023-01631-9.

Recently, a novel mechanism of phenotypic determination has been described in sCJDMV2 [30].According to this model, the amount of PrP C -129M/-129V that is converted into PrP Sc has a strong effect on the resulting phenotype, which encompasses three histotypes: i) MV2C, which mimics MM2 sCJD, and is characterized by cortical ("C") spongiform degeneration (SD) with large vacuoles and perivacuolar/coarse PrP deposition; ii) MV2K, identifiable by the presence of kuru (K) plaques and lack of cortical MM2 pathology; and iii) MV2C-K, which includes a spectrum of 2K and 2C mixed histopathological features [1,30].These three histotypes are the result of the preferential conversion of PrP C -129M into 2C, and of PrP C -129V into 2K.Moreover, regional variability in the relative abundance of PrP C -129M and -129V results in the distinct severity and topographic distribution of the lesions in 2K and 2C histotypes of 2C-K [19,30].Unlike the 2C histotype, which is associated with PrP Sc T2, the western blot profile of 2K shows a ~ 20-19 kDa doublet (T1 20 -T2) with T2 > T1 20 in the neocortex, and T1 20 > T2 in the hippocampus and subcortical regions [1,7,40].However, whether (i) a prominent T1 21 is consistently detected in 2C-K cases, and (ii) intermediate histotypes exist between 2C and 2C-K has not been explored extensively, likely due to 2C and 2C-K with a predominant C component being rare histotypes [1].Because of its rare occurrence, little is also known about the MV1 subtype.A recent report has contributed to further dissect the histopathology of this subtype, and a small subset of five "sCJDVM1" cases was shown to have clinical, histopathological and molecular features resembling the VV1 subtype [23].All VM1 cases had ≥ 10 months disease duration, which is significantly longer than that of classical MV1 [21,35].Given the high phenotypic heterogeneity of sCJDMV2, and the recent report on VM1, we wondered whether further unrecognized histotypes may exist.To answer this question, we retrospectively examined 110 sCJD cases linked to codon 129MV genotype (sCJDMV) to assess the disease phenotype and molecular features of PrP Sc .The large sCJDMV population included 79 MV2 and 31 MV1 cases that were referred to the National Prion Disease Pathology Surveillance Center (NPDPSC) in Cleveland, USA.Sporadic CJDMV2 was classified into 2K, 2C, or 2C-K according to the histopathological features mentioned above.In addition to these well-known histotypes, we identified a novel histotype that resembles 2C in the cerebral cortex and cerebellum, but forms plaque-like PrP deposits in subcortical regions, and more severe lesions in the midbrain.We named this histotype "2C-plaque-like" or "2C-PL".In cases diagnosed as MV1, the classical histotype with fine SD and diffuse PrP (identified as 1C), is the most common and is typically observed in subjects with short disease duration (≤ 4 months).However, a small proportion of "MV1" cases with slower disease progression harbored small amounts of focally distributed resPrP Sc T2.We identified two novel histotypes in this case cohort.The first one, analogous to 2C-PL, mimics the 1C histotype except for the presence of subcortical plaque-like PrP deposits (1C-2PL histotype).The second, and perhaps the most interesting, is reminiscent of 1C in the cerebral cortex, and of 2K in the cerebellum (1C-2K histotype).Overall, the lesion profiles of 1C-2K and 1C-2PL were similar, and they both differed from those of the other histotypes in the degree of subcortical pathology.Finally, a single case with 31-month disease duration resembled the recently reported VM1 (1V histotype) [23].Our study defines the histotype prevalence, resPrP Sc type distribution, and clinical features in a large cohort of sCJDMV cases, allowing identification and classification of these novel histotypes by neuropathologists.

Real-time quaking-induced conversion (RT-QuIC)
Before performing RT-QuIC, we measured the amount of insoluble PrP Sc (obtained at 100,000 × g) that was extracted from the thalamus of 1C-2PL and 1C-2K cases.Samples with larger PrP Sc amounts were diluted so that all cases had the same PrP Sc amount, which was determined by western blot analysis.RT-QuIC was carried out as previously described with some modifications [4].Bacterially expressed, truncated (amino acids 90-231) Syrian hamster recombinant PrP (recHaPrP) was used as substrate; kinetic reactions were average of three reaction wells of sample with 10 -4 , 10 -6 , or 10 -8 dilution.

Molecular genetics
DNA was extracted from frozen brain tissue.Genetic analysis was performed to identify possible mutations in the PrP gene (PRNP), and to determine the polymorphism at codon 129 of PRNP.Genetic analysis was performed as previously described [34,36].

Clinical evaluations
Medical records are requested when cases are submitted to the NPDPSC for neuropathological examination, the legal next of kin completes an autopsy consent form that includes information on recognized and possible acquired prion disease risk factors.Data was collected on demographics such as gender, race/ethnicity, age at disease onset, and disease duration.Medical records, including past medical and surgical histories, as well as other risk factors for the development of an iatrogenic prion disease, clinical symptoms, family history, and diagnostic test results were obtained and reviewed by a clinician, but were not available in all cases.

Image acquisition and statistical tests
All microphotographs were taken with Leica DFC 425 digital camera that is mounted on a Leica DM 2000 microscope.When needed, densitometric analysis of resPrP Sc unglycosylated fragments was carried out with LI-COR application software 3.0.Statistical significance was determined by (i) Student's t-test (two-tailed) for age at onset, disease duration, and total tau; (ii) Chi-square test for gender, race, MRI and positive CSF 14-3-3; (iii) Fisher's exact test for EEG with PSWCs (Table 1); (iv) One-way ANOVA for lesion profiles (Additional file 1: Table S3) and RT-QuIC.Statistical analyses as well as charts were generated using GraphPad Prism 9.5.0.
In the 32 MV2 cases with extensive western blot characterization, both resPrP Sc types co-exist in 31 cases (97%).Because T2 is the predominant resPrP Sc type, we refer to this group as "MV2-1".For convenience, the nomenclature "MV2-1" is used also for the only case with 2C histotype and "pure" T2 on western blot.Thus, 2C, 2C-K and 2K, as well as the novel 2C-PL histotype, are included in MV2-1 (Fig. 1).

Histopathology of sCJD MV1 and MV1-2 1C histotype
The lesion profile shows moderate damage (spongiform degeneration, and gliosis) in the cerebral cortex, striatum, anterior thalamus, with mild cerebellar degeneration (Fig. 2a, c, d).The hippocampus and substantia nigra are not affected (Fig. 2a and e).Vacuoles are predominantly of small in size, with occasionally medium-size vacuoles in two cases with long duration (≥ 10 months) (Additional file 1: Table S2).Cases with long survival also show significantly more cerebral cortical degeneration (Fig. 2a).PrP immunostaining (IHC) reveals the diffuse or "synaptic" pattern with scattered clusters of larger PrP granules throughout the brain (Fig. 2f-h; Additional file 1: Table S2) [30].In three cases, rare foci of coarse PrP (cPrP) are seen in the cerebral cortex (Fig. 2 i and j; Additional file 1: Table S2).

1V histotype
The lesion profile is similar to that of 1C (Fig. 2a); however, the presence of medium-size vacuoles and cortical ballooned neurons are distinctive features of this histotype (Additional file 2: Fig. S1a and b) [23].PrP staining is very fine in the cerebral cortex and subcortical regions (Additional file 2: Fig. S1c and d); the classic "brush stroke-like" pattern is found in the cerebellum (Additional file 2: Fig. S1e; Additional file 1: Table S2); the midbrain is spared (Additional file 2: Fig. S1f ).

1C-2PL histotype
Cortical severity is intermediate compared with 1C with short (≤ 4 months) and long disease duration; the substantia nigra and thalamus are more severely affected (Fig. 2a; Additional file 1: Table S3).A further distinctive feature is the presence of plaque-like PrP in subcortical regions, and, in one case, in the cerebellum (Additional file 1: Table S2; Fig. 3e).
PrP IHC reveals a granule-like PrP throughout the brain (Fig. 4c and d), and both "brush stroke-like" PrP and plaque staining in the cerebellum (Fig. 4e and f ).Plaque-like PrP deposits affecting the subcortical regions, and the cerebral cortex in one case (Additional file 1: Table S2).Rare foci of cPrP are noted in 2 cases (Additional file 1: Table S2).

2C-K histotype
The lesion profile of this large group (and of 2K, see below), differs significantly from the other histotypes, including: (1) a more severe pathology of the frontal cortex compared to that of the occipital cortex; (2) marked involvement of the hippocampus; and, (3) severe striatal pathological changes (Fig. 2b).The 2C-K histotype also differs from 1C-2K (Additional file 3: Fig. S2c).Spongiosis consists of a mixture of small and large vacuoles in different ratios, with clusters of large vacuoles occupying deeper cortical layers in some cases (Fig. 5c and d).Kuru plaques are easily identifiable in the cerebellar cortex, and occasionally within the cerebral cortex.PrP IHC shows coarse/perivacuolar (2C) and diffuse laminar (or pseudolaminar), perineuronal and plaque-like PrP (2K) patterns [30].
The percentage of cPrP in the cerebral cortex ranges from ~ 1% to 100% (Additional file 1: Table S4).If the 2C-K population is divided in two groups according to cPrP percentage being greater or smaller than 55% (mean cut-off ), disease duration is ~ 8 months shorter in subjects with low cPrP (18 vs. 10 months; P < 0.02) (Additional file 5: Fig. S4; Additional file 1: Table S4).

2K histotype
The lesion profile mimics that of 2C-K except for a more severe involvement of the substantia nigra (Fig. 2b; Additional file 1: Table S3).SD is laminar or pseudolaminar in the cerebral cortex; large vacuoles are absent (Fig. 5e and  f ).Kuru plaques are seen within the cerebellum, subcortical regions, and (to a lesser extent) the cerebral cortex.
No differences were noted between the two groups for CSF PrP RT-QuIC and percentage of brain MRIs typical for CJD.

Typing and brain distribution of resPrP Sc 1C histotype
Cases with short disease duration are associated with resPrP Sc T1 20 in all brain regions, even when blots are probed with 1E4 and tohoku-2 antibodies, which bind preferentially (1E4) or selectively (tohoku-2) to T2 (Fig. 7a; Additional file 6: Fig. S5; Additional file 1: Table S5) [7].PK-resistant PrP Sc T1 appears as T1 21−20  at buffer pH 6.9 or as T1 20 at buffer pH 8.0 (Additional file 7: Fig. S6a).In individuals with long disease course, western blot analysis on routine examination of 3 brain regions carried out at the NPDPSC (see materials and methods) reveals T1 20 .However, a minor T2 component is detected in one case with 27-month disease duration that was re-assessed by 1E4 in this study (data not shown).

1V histotype
T1 20 is detected in all brain regions.A minor T2 component is found only after probing with 1E4 (Additional file 8: Fig. S7a and b).

1C-2C histotype
T1 20 co-exists with a minor T2 component that is best represented in the cerebral cortex (Additional file 7: Fig. S6c and Additional file 9: Fig. S8c).As in MM1-2, gel mobility of T1 is influenced by the buffer pH (Additional file 7: Fig. S6c and d).

2C-PL histotype
The prevalence of "pure" T2 is high in the cerebral cortex (75%), and low (14%) in subcortical regions; "pure" T2 is never detected in the cerebellum (Additional file 1: Table S6).In two cases, T1 21 is well represented in the cerebral cortex and subcortical regions (Additional file 1: Table S5; Fig. 7e).Gel mobility of T1 variants and T2 is not affected by the buffer pH (Additional file 7: Fig. S6e).

2C-K histotype
This group is characterized typically by a co-occurrence of resPrP Sc T1 20 and T2.Prevalence of "pure" T2 is low in the cerebral cortex (27%) and absent in subcortical regions (Fig. 7f; Additional file 1: Table S6).In four cases, T1 21 is found in the cerebral cortex (29%) and subcortical regions (8%) (Additional file 1: Table S6).Gel mobility of T1 and T2 is not affected by the buffer pH (Additional file 7: Fig. S6f ).In the four cases with T1 21 , the 1C histotype is detected only in one (Additional file 1: Table S4).In this patient, diffuse and cPrP co-exist, with cPrP occupying only 15% of the cerebral cortex.In the other three cases, cPrP burden is greater than 80% (Additional file 1: Table S4).

RT-QuIC of 1C-2PL and 1C-2K histotypes
RT-QuIC was used to determine the seeding activity of thalamic PrP Sc .We used the thalamus because it (i) shows the "plaque-like PrP" pattern, (ii) expresses PrP Sc types 1 and 2 in both groups, and (iii) lacks kuru plaques in the 1C-2K histotype.We normalized all samples to the same amount of PrP Sc (see methods).Kinetic profiles of 1C-2PL and 1C-2K are similar at 10 -4 and 10 -6 dilutions (Additional file 10: Fig. S9a and b).However, PrP Sc from 1C-2K seeds recHaPrP at a faster rate, and reaches the plateau earlier at 10 -8 dilution (Additional file 10: Fig. S9c).
Although 129MV heterozygosity and disease heterogeneity are coupled in sCJD, it should be emphasized that codon 129MV heterozygous alone does not always lead to phenotypical variability in other human prion diseases [20,44].With the exception of 1C, all other histotypes harbor both resPrP Sc types.1C is the only histotype of MV1, whereas the 1C-2PL, 1C-2K, and 1C-2C histotypes belong to a group of MV1-2 cases with long disease courses and under-representation of T2.The 1C-2C histotype has also been referred to as MV1 + 2C [7].In MV1-2, T2 is detected in subcortical structures (1C-2PL and 1C-2K), cerebellum (1C-2K), or cerebral cortex (1C-2C).The only case with the 1V histotype harbors a focal 2C component coupled with T2.We believe that in the few cases of 1C histotype with focal cPrP deposits (e.g., cases 7, 8, and 18, Additional file 1: Table S1), resPrP Sc T2 levels may be below the threshold of detectability.This discrepancy may be due to the molecular and histopathologic examination carried out in different cerebral hemispheres [39].Each histotype of sCJDMV shows a selective regional vulnerability to PrP Sc types resulting in different lesion profiles [18,26].In all histotypes, the cerebral cortex and subcortical structures (striatum and thalamus), are most severely affected; the substantia nigra is the least vulnerable of the subcortical structures, allowing replication of PrP Sc from patients with "PL" and "K" histotypic components.The least vulnerable region of the brain is the hippocampus, which is typically affected only in subjects with the 2K and 2C-K histotypes.
In a recent study, we demonstrated a novel mechanism of phenotypic determination [30] which had been previously explored by others [29].According to our study, in sCJDMV2 preferential conversion of PrP C -129 M to PrP Sc is associated with the 2C histotype, whereas a dominant conversion of PrP C -129V leads to the 2K histotype.Furthermore, T2 is associated with the 2C histotype in the cerebral cortex, whereas T1 20 -T2 is invariably detected in the 2K histotype (Additional file 1: Table S6) [1].In patients with the 2C-K histotype, PrP C -129M → PrP Sc occurs preferentially in the cerebral cortex, whereas PrP C -129V → PrP Sc preferential conversion takes place in the cerebellum [30].According to this proposition, we believe that the selective vulnerability across the broad spectrum of sCJDMV histotypes, including those reported in this study, is due to the relative ratios of PrP C -129M/-129V alleles converted into PrP Sc .For example, in cases with the 2C-PL histotype, a "pure T2" is predominantly found in the cerebral cortex, and is rare in subcortical regions.On the contrary, T1 20 -T2 is invariably detected in subcortical regions (Additional file 1: Table S6).Applying this logic to the 2C-PL histotype, we would expect a preferential conversion of (i) PrP C -129V into PrP Sc T1 20 -T2 in subcortical regions (harboring plaque-like PrP), and (ii) PrP C -129M → PrP Sc T2 in the cerebral cortex.A similar mechanism may be invoked in 1C-2PL and 1C-2K histotypes.In these groups, plaque-like/plaque PrP patterns and T2 co-distribute in subcortical regions and cerebellum, suggesting that PrP C -129V → PrP Sc preferential conversion occurs in these regions.We also hypothesize that misfolding and accumulation of a slower replicating PrP Sc T2 may start in subcortical structures, with the faster replicating PrP Sc T1 20 spreading throughout the brain later in the disease course.This hypothesis is supported by evidence that the disease epicenter is in the subcortical regions and cerebellum in patients with the 2K histotype [37].Furthermore, in bioassay with transgenic mice expressing the human PrP-129MV, incubation periods are significantly longer for sCJD MV2 than MV1 prions [6,15,28].Specifically, PrP Sc from MV2 of 2K and 2C histotypes show a 2-to 2.3-fold longer incubation period than MV1 (1C histotype) [15].These data suggest that prions propagate slowly in MV2.However, the preferential conversion of PrP C -129M or -129V into PrP Sc , known to be a major determinant of disease phenotype, does not necessarily translate into distinct incubation periods in bioassays experiments [43].Finally, the presence of medium-size vacuoles and ballooned neurons that typically feature in the VV1 subtype may be due to predominant expression of PrP C -129V in the 1V histotype [23].
We have determined histotype prevalences in MV2-1.It should not be surprising that 2C-K is the most common histotype, as the likelihood that both 129 alleles, rather than only one, are converted into PrP Sc is higher.Furthermore, the observation that 2C is the second most common histotype, and 2K the least common, suggests that PrP C -129M is preferentially converted into PrP Sc in agreement with the higher prevalence of 2C-K with high burdens of cPrP accumulating in the cerebral cortex.Indeed, the proportion of 2C-K cases with high burden of cPrP exceeds 60%, whereas the proportion of 2C-K cases with a low burden of cPrP is only 27%.Another intriguing observation is the co-occurrence of T1 20 and T1 21 in sCJD [4,11,14].T1 20 is by far the major T1 species in sCJDMV.We suggest that T1 20 detected in the cerebral cortex and subcortical region/cerebellum have divergent biochemical features in 1C-2K.At pH 6.9, T1 of 1C-2K appears as T1 21 in the cerebral cortex, and as T1 20 in the cerebellum.Under the same experimental conditions, only T1 21 is detected in 1C-2C and 1C histotypes.Furthermore, in patients with the 2K histotype (characterized by a marked and widespread PrP plaque pathology), a dominant T1 21 is never found.In summary, T1 of 1C and 1C-2C histotypes is sensitive to a change in pH, with a predominant T1 21 or T1 20 detected in all brain regions at pH 6.9 or pH 8.0; on the contrary, T1 20 is the only T1 variant in the cerebellum of 1C-2K regardless of the buffer pH.Another important piece of evidence on the effect of PrP C -129M/V to PrP Sc preferential conversion is highlighted by the T1 21 prevalence across the histotypes of MV2-1.A prominent T1 21 is never found in 2K histotype, but its prevalence increases progressively from 2C-K to 2C-PL/2C.Furthermore, T1 21 predominates in the cerebral cortex, the brain region where PrP plaque density is the lowest, and cPrP is the highest.It should also be mentioned that while the prevalence of T1 variants is likely to be driven by 129M/V preferential conversion in sCJDMV, a different mechanism must be involved in sCJDVV and sCJDMM cases with co-existing T1 variants [4,11,14].
We found that the burden of cortical cPrP is significantly higher in 2C-K patients with longer disease duration.We observed a similar phenomenon in sCJDMM1-2, and suggested that T2 accumulation would play a major role in determine disease duration [7].This hypothesis is corroborated by transmission studies in which incubation periods inversely correlate with prion titers [3], and levels of PrP Sc increase over time [42].
We believe that the 1C-2PL histotype does not represent an intermediate, immature form of the 1C-2K histotype.Two major observations support this hypothesis.The first is that 1C-2PL and 1C-2K have virtually identical disease durations.If 1C-2PL was the immature form of 1C-2K, 1C-2PL would be expected to have a significantly shorter disease duration than 1C-2K.The second is that PrP plaques are always found in the 2K histotype regardless of disease duration [1].The same principle could also apply to the 2C-PL and 2C-K histotypes, showing similar disease durations (14 ± 6 and 14 ± 8 months).
In conclusion, the highly heterogeneous histotypes and the number of co-existing PrP Sc types make sCJDMV the most heterogeneous human prion disease.This heterogeneity is likely to be driven by the tendency of both PrP C -129M and -129V polymorphic forms to convert to PrP Sc types 1 variants and type 2 in different proportions.Our results also suggest that T1 20 of the 2K histotype and T1 20 affecting the cerebellum in 1C-2K belong to the same prion strain.It is not surprising that 2 of the 3 novel histotypes were identified in MV1-2, as this case-cohort represents the least studied sCJDMV, accounting for ~ 22% of the 177 MV1 cases available at the NPDPSC.In addition to the identification of novel histotypes of sCJD [4], other approaches, such as multi-omics and digital pathology as well as cryo-EM and transmission studies are needed to further understand these complex disorders.

Fig. 1
Fig. 1 Schematic representation of the histotypes identified in this study.a The first raw identifies histotypes associated with MV1 and MV1-2; the second row refers to MV2-1.b Nomenclature used in this and other studies

Fig. 2
Fig. 2 Lesion profiles of 9 sCJDMV histotypes, and histopathology of 1C. a sCJD MV1/MV1-2 can be divided into two major groups.The first one includes 1C, 1V and 1C-2C histotypes, characterized by the sparing of the substantia nigra (SN) and less severe pathological changes in thalamic (TH) nuclei; the second group encompasses 1C-2PL & 1C-2K showing more severe lesions of SN and TH, and accumulation of plaque-like PrP deposits (1C-2PL) and PrP plaques (1C-2K).b Lesion profiles of MV2-1 show three major groups: 2C with sparing of SN, 2C-PL with mild lesions of SN, and 2C-K & 2K with overall different lesion profiles, and marked hippocampal pathology; the 2C-K histotype shows C and K histotypic features in the proportion of ~ 50% each.Dashed double arrows point to differences in severity in key brain regions.c-e, j Hematoxylin-eosin (H&E).f-i PrP immunostaining.c-e Spongiform degeneration affecting the neocortex (c) and subcortical regions (d) regions, but not the hippocampus (e).f-h Diffuse PrP; arrow, f a larger PrP granule.Mol.L.: Molecular layer; Grl.L.: granular layer.i A focus of coarse PrP.j H&E preparation stained for PrP in i; antibody: 3F4; dis.durat.: disease duration

Table 1
Clinical features of sCJDMV histotypes EEG Electroencephalogram; PSWC Periodic short-wave complexes; MRI Magnetic resonance imaging; CSF Cerebrospinal fluid MRI: 2C vs. 2C-K, P < 0.004 (Fisher's exact test) a Expressed as mean ± SD and b percentage.c Cases with the feature listed/total cases examined; d It compares MV1/MV1-2 and MV2-1 cases; e Student's t-test; f