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Determination of Carrier Status for the Wiskott-Aldrich Syndrome by Flow Cytometric Analysis of Wiskott-Aldrich Syndrome Protein Expression in Peripheral Blood Mononuclear Cells¹

Masafumi Yamada^*, Tadashi Ariga^2,, Nobuaki Kawamura^*, Koji Yamaguchi, Makoto Ohtsu^*, David L. Nelson, Tatsuro Kondoh^§, Ichiro Kobayashi^*, Motohiko Okano^*, Kunihiko Kobayashi^* and Yukio Sakiyama

^* Department of Pediatrics, Hokkaido University School of Medicine, Sapporo, Japan; Department of Human Gene Therapy, Hokkaido University School of Medicine, Sapporo, Japan; National Institutes of Health, National Cancer Institute, Metabolism Branch, Bethesda, MD 20892; ^§ Department of Pediatrics, Nagasaki University School of Medicine, Nagasaki, Japan

	Abstract

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The Wiskott-Aldrich syndrome (WAS) is caused by defects in the WAS protein (WASP) gene on the X chromosome. Previous study disclosed that flow cytometric analysis of intracellular WASP expression (FCM-WASP analysis) in lymphocytes was useful for the diagnosis of WAS patients. Lymphocytes from all WAS patients showed WASP^dim instead of WASP^bright. Here we report that FCM-WASP analysis in monocytes could be a useful tool for the WAS carrier diagnosis. Monocytes from all nine WAS carriers showed varied population of WASP^dim together with WASP^bright. None of control individuals possessed the WASP^dim population. In contrast, lymphocytes from all the carriers except two lacked the WASP^dim population. The difference of the WASP^dim population in monocytes and lymphocytes observed in WAS carriers suggests that WASP plays a more critical role in the development of lymphocytes than in that of monocytes. The present studies suggest that a skewed X-chromosomal inactivation pattern observed in WAS carrier peripheral blood cells is not fixed at the hemopoietic stem cell level but progresses after the lineage commitment.

	Introduction

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The Wiskott-Aldrich syndrome (WAS)³ is an X-linked recessive disorder characterized by severe recurrent infections due to immunodeficiency, tendency to bleeding due to thrombocytopenia with small platelets, and severe eczema (1, 2). The responsible WAS protein (WASP) gene was recently identified (3), and since then a lot of mutations in this gene have been reported with defective or reduced expression of WASP (4, 5). WASP is preferentially expressed in hemopoietic cells, such as lymphocytes, monocytes, and platelets (6). Although the definite WASP function remains to be determined, WASP seems to play roles in signal transduction pathways for cell growth (7) and in cytoskeletal organization in response to activation (8).

In a previous study, we have established the technique of flow cytometric analysis of intracellular WASP expression (FCM-WASP analysis) in lymphocytes (9) and demonstrated its clinical usefulness for the diagnosis of WAS patients. However, the results suggested that application of this technique for the carrier diagnosis is not possible because all the three carriers studied had only the WASP^bright population of lymphocytes.

In this study, we used monocytes for FCM-WASP analysis, and found that all the nine carriers studied had both the WASP^bright and WASP^dim population in varying degree. We conclude that this analysis could be a useful tool for the WAS carrier diagnosis. The mechanism of the skewed X-chromosomal inactivation pattern in WAS carriers was discussed, based on the difference of the WASP^dim population between monocytes and lymphocytes observed in WAS carriers.

	Materials and Methods

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Carriers studied

Nine WAS carrier mothers from different families and a WAS patient, who had received bone marrow transplantation (BMT) from his carrier mother, were included in this study. One noncarrier mother of a WAS patient was also studied. The results of mutations in the WASP gene belonging to each family, some of which were reported elsewhere (10, 11), are shown in Table I. Carrier diagnosis was performed by molecular methods as described previously (10). Carrier mothers in families 1 and 3 are the mothers of patients 1 and 2 in the previous study (9). Carrier mothers in families 3 and 4 are unrelated but had the same mutated allele. BMT was performed in the patient in family 5 from his mother 3 years previously. The severity of WAS-associated symptoms of patients in all the families was scored from 1 to 5, based on the criteria by Zhu et al. (12).

FCM-WASP analysis was performed as previously described (9) with minor modification. In brief, PBMC washed in PBS containing 1% FBS were treated with Cytofix/Cytoperm solution from CytoStain kit (PharMingen, San Diego, CA) at 4°C for 20 min. After washing twice with Perm/Wash solution, they were reacted with 1:200 diluted mouse anti-WASP mAb (3F3-A5) (6) or 1:5 diluted mouse IgG1 Ab (Becton Dickinson, San Jose, CA) at 4°C for 30 min. They were then reacted with FITC-labeled goat anti-mouse IgG1 Ab (Southern Biotechnology Associates, Birmingham, AL). Samples, thus processed, were analyzed on a FACSCalibur (Becton Dickinson, Mountain View, CA). Gating of lymphocytes or monocytes was made from a distribution pattern in forward and side scatter. A total of 20,000 events of each cell were studied.

	Results

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Significant difference of WASP expression in monocytes between normal individuals and WAS patients

As we have shown the significant difference of WASP expression in lymphocytes previously (9), the same difference of WASP expression in monocytes was observed between normal individuals and WAS patients. An example of a normal individual (A, WASP^bright) and a WAS patient from family 3 (B, WASP^dim) was shown (Fig. 1).

View larger version (31K): [in this window] [in a new window]   FIGURE 1. FCM-WASP analysis in monocytes from WAS patients. Results of a normal individual (A) and a WAS patient in family 3 (B) are shown. The WASP expression was significantly reduced (WASPdim) in the patient compared with that of the normal individual. The same results were obtained from patients in other families.

In all the carriers, the WASP^dim population was detected in monocytes in variable degree with the major population of WASP^bright. The pattern of the WASP^dim population in WAS carriers was largely divided into three groups according to the proportion of WASP^dim cells: skewed (A, <10%), moderately skewed (B, >10%, <30%), and random (C, >30%) groups (Fig. 2, A–C). Carriers in families 1 and 2 belong to group A, carriers in families 3, 4, 5, 6, and 10, and a patient in family 5 after BMT to group B, and carriers in families 7 and 8 to group C. The proportion of the WASP^dim population ranged from 3.5% to 50.7% (Table I). None of control individuals including a noncarrier mother in family 9 possessed the WASP^dim population (Fig. 2D).

View larger version (20K): [in this window] [in a new window]   FIGURE 2. FCM-WASP analysis in monocytes from three WAS carriers and one noncarrier. The pattern was largely divided into three groups according to the proportion of WASPdim cells: skewed (A, <10%), moderately skewed (B, >10%, <30%), and random (C, >30%) groups. A, B, and C are the results of carriers in families 1, 6, and 8, respectively. The proportion of cells in M1 area was shown in Table I as the percentage of WASPdim cells. The results of a noncarrier mother of family 9 were also shown (D).

In contrast to FCM-WASP analysis in monocytes, most of carriers (seven of nine) were shown to have only the WASP^bright population of lymphocytes (Fig. 3 and Table I). The WASP^dim population of lymphocytes was detected only in carriers in families 7 and 8 (the data of a carrier in family 8 is shown in Fig. 3C).

View larger version (21K): [in this window] [in a new window]   FIGURE 3. FCM-WASP analysis in lymphocytes. Results of a normal individual (A), a patient of family 8 (B), and a carrier in family 8 (C) are shown. Most of carriers (seven of nine) were shown to have only the WASPbright population of lymphocytes. The WASPdim population was demonstrated in lymphocytes from a carrier in family 8. The proportion of cells in M1 area was shown in Table I as the percentage of WASPdim cells.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In the previous study, we have established the technique of FCM-WASP analysis in lymphocytes (9) and demonstrated its clinical usefulness for the diagnosis of WAS patients. However, the application of this technique for the carrier diagnosis seemed not to be possible because all the three carriers studied had only the WASP^bright population of lymphocytes. We could not find any difference between WAS carriers and control females in these studies. This finding is not surprising, because a skewed X-chromosomal inactivation pattern is observed in female carriers of some X-linked genetic disorders including WAS in constitutional or in some specific lineage cells. The cause of this phenomenon is attributable to negative selection of cells, which have the growth disadvantage due to the mutant gene on the active X chromosome (13). Wengler et al. further reported that a skewed X-chromosomal inactivation pattern in WAS-obligate carriers occurs early during the hemopoietic differentiation, speculating that hemopoietic progenitors are affected by WASP mutations (14). In contrast, a random X-chromosomal inactivation pattern was reported in some carriers, although these reports are limited to carriers of X-linked thrombocytopenia or mild WAS with missense mutations (15).

In the present study, we performed FCM-WASP analysis in PBMC to study whether a skewed X-chromosomal inactivation pattern is universally observed in all hematological cells of WAS carriers. The results showed that all the WAS carriers have variable proportion of WASP^dim population of monocytes, together with the WASP^bright one. We then quantitatively evaluated the X-chromosomal inactivation pattern in monocytes from each carrier. The WASP^dim monocyte population detected in carriers in families 1 and 2, whose WASP gene mutation seemed to result in nearly null function of WASP, was 5.4% and 3.5%, respectively, whereas carriers in families 7 and 8 who have the missense mutations were revealed to have the WASP^dim population of 30.1% and 50.7%, respectively. These results may suggest the proportion of the WASP^dim monocyte population correlates with their genotypes. However, a carrier in family 5 who was supposed to have the most severe mutation, and a carrier in family 6 with missense mutation, both belong to the same B group of moderately skewed pattern (the WASP^dim population of 17.3% and 11.7%, respectively). Thus, genetic, stochastic, or other unknown factors might contribute to the skewed pattern in monocytes as well as types of WASP gene mutations.

In contrast to the observation in monocytes, we could not detect the WASP^dim population of lymphocytes in seven of nine carriers. Only two carriers with random X-inactivation pattern in monocytes were shown to have the WASP^dim population of lymphocytes. Even in the two carriers, the WASP^dim population of lymphocytes was smaller than those of monocytes were. Thus, WASP seemed to be more critical for the development of lymphocytes than that of monocytes. Alternatively, accelerated cell death in WASP^dim lymphocytes by spontaneous apoptosis (16) might be responsible for the lack of WASP^dim lymphocytes in most of WAS carriers. We speculate that the mutant WASP in these two families still remains to have a residual function that makes the carriers’ WASP^dim lymphocytes survive in their peripheral blood.

Although FCM-WASP analysis in monocytes is much simpler and faster than molecular methods for WAS carrier diagnosis, it is possible that some carriers will have undetectable percentages of WASP^dim monocytes. Therefore, we should carefully rule out the possibility of carriers when the WASP^dim population was not detected. And as was discussed for the diagnosis of WAS in the previous study (9), this method of carrier detection should be also limited to families where the affected boys have a WASP^dim population, although a normal amount of WASP expression has not been reported in WAS patients.

We had a chance to study the WAS patient (patient in family 5) who had received BMT from his carrier mother (carrier in family 5) 3 years previously. He was shown to have almost the same proportion of the WASP^dim population of monocytes as his mother (18.5% vs 17.3%). Also similar to his mother, he did not have WASP^dim lymphocytes, either. Because the karyotype of the patient had completely changed to 46, XX after BMT, both lymphocytes and monocytes analyzed were originated from the hematological progenitor cells of the carrier mother. The results indicate that transplantable hemopoietic progenitor cells in the carrier mother was not severely skewed, and the proportion of the reconstructed WASP^dim monocyte population was reproducible in vivo.

We previously demonstrated that most of the WAS carriers possessed lymphocytes and granulocytes expressing the mutant WASP message by allele-specific RT-PCR methods (17). These results were partly inconsistent with the present results, because seven of nine carriers did not have the WASP^dim lymphocyte population in this study. In the previous study, however, we did not separate monocytes from the lymphocyte fraction. Therefore, it was possibly monocytes that predominantly expressed the mutant WASP message because "lymphocytes" as described in the previous report included lymphocytes and monocytes. If it is the case, the previous results obtained by RT-PCR methods mostly agree with those by flow cytometric analysis in this study.

In conclusion, this study demonstrated the usefulness of FCM-WASP analysis in monocytes for the carrier diagnosis of WAS. The difference of the WASP^dim population between in monocytes and lymphocytes from WAS carriers suggests that a skewed X-chromosomal inactivation pattern observed in WAS carriers’ blood cells is not fixed at the hemopoietic stem cell level but progresses after the lineage commitment.

	Acknowledgments

 
We thank Drs. Mika Iwamura, Hirotaka Takahashi, Nobuyoshi Ishikawa, Tsuyosi Ito, and Michiya Anakura for allowing us to analyze their patients and their families.

	Footnotes

 
¹ This work was supported by a Health Science Research Grant from the Ministry of Health and Welfare of Japan.

² Address correspondence and reprint requests to Dr. Tadashi Ariga, Department of Human Gene Therapy, Hokkaido University School of Medicine, N-14 W-5, Kita-ku, Sapporo, 060-8638, Japan.

³ Abbreviations used in this manuscript: WAS, Wiskott-Aldrich syndrome; WASP, WAS protein; FCM-WASP, flow cytometric analysis of WASP expression; BMT, bone marrow transplantation.

Received for publication February 22, 2000. Accepted for publication April 26, 2000.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Wiskott, A.. 1937. Familiarer, angeborener Moubus Werlhofii?. Mschr. Kinderheilk 68:212.
2. Aldrich, R. A., A. G. Steinberg, D. C. Campbell. 1954. Pedigree demonstrating a sex-linked recessive condition characterized by draining ears, eczematoid dermatitis and bloody diarrhea. Pediatrics 13:133.[Abstract/Free Full Text]
3. Derry, J. M. J., H. D. Ochs, U. Francke. 1994. Isolation of a novel gene mutated in Wiskott-Aldrich syndrome. [Published erratum appears in 1994 Cell 79:922.]. Cell 78:635.[Medline]
4. Schwarz, K., S. Nonoyama, M. C. Peitsch, G. de-Saint Basile, T. Espanol, A. Fasth, A. Fisher, K. Freitag, W. Freidrich, S. Fugmann, et al 1996. WASPbase: a database of WAS- and XLA-causing mutations. Immunol. Today 17:496.[Medline]
5. Zhu, Q., C. Watanabe, T. Liu, D. Hollenbaugh, R. M. Blaese, S. B. Kanner, A. Aruffo, H. D. Ochs. 1977. Wiskott-Aldrich syndrome/X-linked thrombocytopenia: WASP gene mutations, protein expression, and phenotype. Blood 90:2680.[Abstract/Free Full Text]
6. Stewart, D. M., S. Treiber-Held, C. C. Kurman, F. Facchetti, L. D. Notarangelo, D. L. Nelson. 1966. Studies of the expression of the Wiskott-Aldrich syndrome protein. J. Clin. Invest. 97:2627.[Medline]
7. Cory, G. O. C., L. MacCarthy-Morrogh, S. Banin, I. Gout, P. M. Brickell, R. J. Levinsky, C. Kinnon, R. C. Lovering. 1966. Evidence that the Wiskott-Aldrich syndrome protein may be involved in lymphoid cell signaling pathways. J. Immunol. 157:3791.[Abstract]
8. Symons, M., J. M. J. Derry, B. Karlak, S. Jiang, V. Lemahieu, F. McCormick, U. Francke, A. Abo. 1996. Wiskott-Aldrich syndrome protein, a novel effector for the GTPase CDC42Hs, is implicated in actin polymerization. Cell 84:723.[Medline]
9. Yamada, M., M. Ohtsu, I. Kobayashi, N. Kawamura, K. Kobayashi, T. Ariga, Y. Sakiyama, D. L. Nelson, S. Tsuruta, M. Anakura, N. Ishikawa. 1999. Flow cytometric analysis of Wiskott-Aldrich syndrome (WAS) protein in lymphocytes from WAS patients and their familial carriers. Blood 93:756.[Free Full Text]
10. Ariga, T., M. Yamada, Y. Sakiyama. 1997. Mutation analysis of five Japanese families with Wiskott-Aldrich syndrome and determination of the family members’ carrier status using three different methods. Pediatr. Res. 41:535.[Medline]
11. Ariga, T., M. Yamada, S. Ito, M. Iwamura, M. Iseki, Y. Sakiyama. 1997. Characterization of deletion mutation involving exons 3–7 of the WASP gene detected in a patient with Wiskott-Aldrich syndrome. Hum. Mutat. 10:310.[Medline]
12. Zhu, Q., M. Zhang, R. M. Blaese, J. M. J. Derry, A. Junker, U. Francke, S. H. Chen, H. D. Ochs. 1995. The Wiskott-Aldrich syndrome and X-linked congenital thrombocytopenia are caused by mutations of the same gene. Blood 86:3797.[Abstract/Free Full Text]
13. Belmont, J. W.. 1996. Genetic control of X inactivation and processes leading to X-inactivation skewing. Am. J. Hum. Genet. 58:1101.[Medline]
14. Wengler, G., J. B. Gorlin, J. M. Williamson, F. S. Rosen, D. H. Bing. 1995. Nonrandom inactivation of the X chromosome in early lineage hematopoietic cells in carriers of Wiskott-Aldrich syndrome. Blood 85:2471.[Abstract/Free Full Text]
15. de Saint-Basile, G., R. D. Lagelouse, N. Lambert, K. Schwarz, B. Le Mareck, S. Odent, N. Schlegel, A. Fisher. 1996. Isolated X-linked thrombocytopenia in two unrelated families is associated with point mutations in the Wiskott-Aldrich syndrome protein gene. J. Pediatr. 129:56.[Medline]
16. Rawlings, S. L., G. M. Crooks, D. Bockstoce, L. W. Barsky, R. Parkman, K. I. Weinberg. 1999. Spontaneous apoptosis in lymphocytes from patients with Wiskott-Aldrich syndrome: correlation of accelerated cell death and attenuated Bcl-2 expression. Blood 94:3872.[Abstract/Free Full Text]
17. Ariga, T., M. Yamada, T. Wada, S. Saito, Y. Sakiyama. 1999. Detection of lymphocytes and granulocytes expressing the mutant WASP message in carriers of Wiskott-Aldrich syndrome. Br. J. Haematol. 104:893.[Medline]

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