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[Cancer Research 60, 2492-2496, May 1, 2000]
© 2000 American Association for Cancer Research

Molecular Biology and Genetics

Genetic Instability and Hematologic Disease Risk in Werner Syndrome Patients and Heterozygotes1

Michael J. Moser, William L. Bigbee, Stephen G. Grant, Mary J. Emond, Richard G. Langlois, Ronald H. Jensen, Junko Oshima and Raymond J. Monnat, Jr.2

Departments of Pathology [M. J. M., J. O., R. J. M.] and Biostatistics [M. J. E.], University of Washington, Seattle, Washington, 98195; Center for Environmental and Occupational Health and Toxicology, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, Pennsylvania 15238 [W. L. B., S. G. G.]; Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, Livermore, California 94551 [R. G. L.]; and Department of Laboratory Medicine, Cancer Center, University of California, San Francisco, California 94143 [R. H. J.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Werner syndrome (WRN) is an uncommon autosomal recessive disease in which progeroid features are associated with genetic instability and an elevated risk of neoplasia. We have used the glycophorin A (GPA) somatic cell mutation assay to analyze genetic instability in vivo in WRN patients and heterozygotes. GPA variant frequencies were determined for 11 WRN patients and for 10 heterozygous family members who collectively carry 10 different WRN mutations. Genetic instability as measured by GPA Ø/N allele loss variant frequency was significantly increased, and this increase was strongly age-dependent in WRN patients. GPA Ø/N allele loss variants were also significantly elevated in heterozygous family members, thus providing the first evidence for in vivo genetic instability in heterozygous carriers in an autosomal recessive genetic instability syndrome. Our results and comparable data on other human genetic instability syndromes allow an estimate of the level of genetic instability that increases the risk of human bone marrow dysfunction or neoplasia.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
WRN3 (MIM no. 277700) is an uncommon autosomal recessive disease that results from mutational inactivation of a human RecQ helicase protein encoded by the chromosome 8p WRN locus (1) . Considerable interest has focused on the WRN phenotype, which resembles premature aging and includes genetic instability and an elevated risk of neoplasia (2 , 3) . To further examine the role of genetic instability in WRN pathogenesis (4 , 5) , we have used the GPA somatic cell mutation assay to quantify and characterize erythroid lineage genetic instability and to relate genetic instability to disease risk in WRN patients and heterozygotes.

The GPA assay quantifies somatic mutation by measuring the frequency of GPA variant peripheral blood red cells. GPA is a chromosome 4 locus that encodes the M/N blood group antigens. M and N allele GPA variant red cells that arise by mutation are detected and quantified by flow cytometric analysis of M/N-heterozygous peripheral blood erythrocytes that have been immunolabeled with M and N allele-specific monoclonal antibodies (6) . GPA Ø/N and M/Ø variant red cells can arise by GPA gene mutation, by chromosome 4 loss, or by the epigenetic silencing of GPA alleles. GPA N/N and M/M variant red cells that have lost expression of one allele and express the retained allele at a homozygous level may arise by mitotic recombination, by gene conversion, or by chromosome missegregation or loss and then reduplication (Fig. 1Citation and Ref. 7 ). GPA variant frequencies (Vf are reported as the number of variant red cells per 106 erythrocytes. N allele (Ø/N and N/N) Vf are most often reported because they can be reliably determined at frequencies as low as 1 in 106 (6) .



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  		Fig. 1. The GPA Vf assay. The GPA assay uses a combination of immunostaining and flow cytometric analysis to detect and quantify M/N variant erythrocytes that arise by somatic mutation in the erythroid lineage of M/N heterozygous donors. The M/N antigens are encoded by the autosomal GPA locus on chromosome 4. GPA gene mutations, chromosome loss, or the epigenetic silencing of one GPA allele can give rise to M/Ø and Ø/N allele loss variants (top, center and right panels). GPA M/M and N/N variants (bottom, center and right panels) can arise by mitotic recombination, gene conversion, or chromosome 4 missegregation or loss and then reduplication of the remaining chromosome 4. Ø/N and N/N Vf are most often reported because they can be determined at low background frequencies (>=1 in 106; Ref. 6 ).

 
The GPA assay has been well-validated as a robust and quantitative assay for somatic mutation (6 , 7) . Background or spontaneous GPA Vf parallel mutation frequencies determined using the X-linked HPRT and autosomal TCR and HLA loci (reviewed in Ref. 8 ). GPA Vf increases with age, and an increased GPA Vf has been observed in cigarette smokers (8, 9, 10) . Patients exposed to ionizing radiation or receiving chemotherapy show GPA Vf elevations that parallel HPRT or HLA mutation frequency increases. These induced GPA Vf elevations are dose-dependent, and in many instances decay with kinetics that parallel erythrocyte turnover (for example, see Refs. 11, 12 ).

Patients with heritable genetic instability syndromes have persistently elevated GPA Vf. Syndromes that have been analyzed include ATM (MIM no. 20890; Refs. 13 and 14 ), BLM (MIM no. 210900; Refs. 9 and 15 ), and FAN (MIM nos. 227645, 227646, 227650, 227660, 600901, 603467, and 603468; Refs. 16 and 17 ). In each of these syndromes, the pattern of GPA Vf elevation is consistent with our understanding of the mechanistic basis for genetic instability. For example, the high level of N/N GPA variants in BLM patients is consistent with the idea that BLM modulates mitotic recombination (18) . The availability of GPA Vf data on large numbers of control donors facilitates these types of patient, disease-specific, and population-based analyses (10 , 19) .

Our results document genetic instability in vivo in WRN patients and, surprisingly, in WRN heterozygotes. These analyses used red cells from patients and heterozygotes carrying 10 different WRN mutations, including two mutations independently reported to lead to an elevated GPA Vf (20) . We have also been able to use our data on WRN and comparable results from the study of other genetic instability syndromes to estimate the level of genetic instability that confers a high risk of human bone marrow dysfunction or neoplasia.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Werner Pedigree and Control Blood Samples.
A total of 90 blood samples collected by the International Registry of Werner Syndrome at the University of Washington (21) were analyzed. Samples were serotyped for MN blood group antigens using commercial anti-M and anti-N typing sera (Ortho Diagnostics, Raritan, NJ) according to the manufacture’s protocol. Of the 90 samples, 28 (31%) were M/N heterozygous and informative. Genetically characterized blood samples from 22 M/N heterozygous donors who were WRN patients or family members were used for GPA Vf determinations. WRN mutation analyses were performed using nucleic acids isolated from lymphoblastoid cell lines established from each sample (1) . Control donors with the same age range as WRN patients (n = 283; ages 21–58) or heterozygotes (n = 362; ages 14–72) were chosen from the Lawrence Livermore National Laboratory (Livermore, CA) employee cohort (19) . There was no significant difference in the median or mean ages or in the age distribution of control donors and WRN patients or heterozygotes (Table 1Citation and Table 2Citation and additional results not shown).


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  		Table 1 GPA Vf and genotype of WRN patients and family members

 

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  		Table 2 GPA Vf in WRN patients and heterozygotes versus control donors

 
GPA Vf Determinations.
RBC immunolabeling and flow cytometic analysis to determine GPA Vf were performed as previously described (6) . In brief, erythrocytes were swollen in hypotonic buffer containing SDS to generate spheroplasts that were fixed with formaldehyde. Fixed cells were immunolabeled with a phycoerythrin-6A7 mouse monoclonal antibody to the GPAM epitope and a fluorescein-BRIC157 mouse monoclonal antibody to the GPAN epitope before high-speed (3000–4000 cells/s) flow cytometry on a FACScan flow cytometer (Becton Dickinson, San Jose, CA). A total of 5 x 106 cells were analyzed for each sample using a rectangular gate of forward scatter versus log side scatter to discriminate against antibody-induced cell aggregates. GPA Vf were determined from the number of red cells falling within defined variant regions of the histogram divided by the total number of single cells analyzed.

Statistical Analysis of GPA Vf Data.
The age distributions of patient, heterozygote, and control donors were compared using the nonparametric Mann-Whitney test. GPA Vf data are not normally distributed in most study or control populations. Thus, we calculated exponentiated means and standard deviations of log-transformed data as descriptive statistics and for significance testing. A two-sided t test on log-transformed data and the Mann-Whitney test were used to determine the statistical significance of Vf differences. Regression analyses were used to study the relationship between GPA Vf data and donor age. Because of the nonnormality of the data, nonparametric tests were used for inference regarding the slopes of Vf measures versus donor age. Specifically, the Spearman rank correlation test was used to test the null hypothesis of a zero slope for Vf versus age within a group (the hypothesis of no correlation is equivalent to the hypothesis of zero slope), and a permutation test was used to test for equality of the Vf-versus-age slopes between groups (22) . Odds ratios and odds ratio confidence intervals and significance were calculated as previously described (Refs. 23 and 24 , respectively). Statistical analyses were performed using S-Plus (MathSoft, Seattle, WA), DataDesk 5.0 (Data Description, Ithaca, NY), and StatView 4.5 (Abacus Concepts, Berkeley, CA).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Somatic Mutation in WRN Pedigrees.
We analyzed GPA Vf in 11 WRN patients and 10 heterozygous family members who collectively carried 10 different WRN mutations (Table 1Citation and Fig. 2Citation ). The GPA Ø/N Vf was elevated in patients and in heterozygotes compared with matched control donors. In contrast, the N/N Vf of patients and heterozygotes did not differ from matched control donors (Fig. 3)Citation . The differences in mean GPA Ø/N Vf between WRN patients and controls, as well as between heterozygotes and controls, were highly significant (P = 10-9 and P <= 0.0001, respectively; Table 2Citation ). There was as much heterogeneity between patients with the same genotype as there was between patients with different WRN mutations (Table 1)Citation . WRN patients displayed higher median GPA Ø/N Vf than did heterozygotes, although this difference did not reach significance (Fig. 3Citation , left panel; P = 0.20). There was no significant difference in N/N Vf between patients and controls (P <= 0.50), between heterozygotes and controls (P <= 0.59), or between patients and heterozygotes (P = 0.50; Table 2Citation ).



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  		Fig. 2. Flow cytometric analysis of 106 erythrocytes from representative MN heterozygous Werner patient and control donor. Erythrocytes were immunolabeled for GPA M and N transmembrane proteins before flow cytometric analysis. The fluorescence intensity scales for GPA M (Y axis) and N (X axis) staining are given in log units. The major peak in each panel consists of M/N heterozygous cells. Rare Ø/N, N/N, and M/M-variant red cells are increased in the Werner patient (the 47-year-old female Werner patient listed in Table 1Citation ). Also noticeable in this patient is "tailing" between the M/N peak and the M/M and N allele variant boxes. This may arise due to somatic mutation during erythroid lineage expansion and differentiation.

 


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  		Fig. 3. GPA Vf as a function of WRN genotype. GPA N-allele Vf (x10-6) are shown for WRN patients (WRN-/-), heterozygous carriers (WRN+/-), and matched control donors (WRN+/+; see "Materials and Methods") as modified box plots. Boxes above each WRN genotype represent the central 50% of data points with the top and bottom of the box indicating 75th and 25th percentiles data, respectively. Median GPA N Vf for each WRN donor group are indicated by a horizontal line within the box, and the vertical lines extending from each box end indicate the 95th (top) and 5th (bottom) percentiles for Vf data for each donor group. Percentiles were determined directly for control donors and by interpolation for WRN patients and heterozygotes.

 
ORs calculated from comparing the frequency of individuals with high Vf in WRN patient and control populations were consistently significant at Vf >= 9 x 10-6 (OR = 38.8, i.e., the odds of being a WRN patient versus not being a patient are 38.8-fold higher at Vf >= 9 x 10-6 than at Vf < 9 x 10-6). GPA Vf >= 9 x 10-6 were observed in 10 of 11 (or 91%) of our WRN patients (Table 1)Citation . ORs calculated from N/N Vf data become significant at Vf >= 15 x 10-6, although at a much lower OR of 3.5. Among WRN heterozygotes, Ø/N Vf > 6 x 10-6 were significant with ORs >= 10.8. These values included 9 of 10 (90%) of our WRN heterozygotes.

Age Regression Analyses.
There was significant (nonzero) correlation between Ø/N Vf and age for both patient controls and heterozygote controls (P = 0.02 and P < 0.0001, respectively) as has been previously observed (for example, see Ref. 10 ). WRN patients demonstrated a steep increase in GPA Ø/N Vf with age compared with heterozygotes or controls (slope, 0.73 versus 0.25 and 0.09, respectively; Fig. 4Citation ), and this increase in patients differed significantly from controls (P = 0.02) as determined by permutation testing. Permutation testing was chosen as a relatively powerful nonparametric test because it requires no assumptions regarding the distribution of data in either the patient or control donor populations. Patients and heterozygotes did not have significantly different Ø/N Vf versus age slopes (P = 0.38), and neither did heterozygotes and controls (P = 0.82). N/N Vf was significantly correlated with age for patient controls (P < 0.0001) and for heterozygote controls (P < 0.0001). The N/N Vf age slopes for patients and heterozygotes did not differ from those for their respective control groups (P = 0.60 and P = 0.23, respectively), nor did they differ from each other (P = 0.62).



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  		Fig. 4. GPA Ø/N variant frequency as a function of age and WRN genotype. The age regression of GPA Ø/N Vf (x10-6) are shown for WRN patients (WRN-/-) and heterozygous mutation carriers (WRN+/-) together with matched control donors (WRN+/+; see "Materials and Methods"). Patient ({diamond}) and heterozygote (•) data points are shown together with corresponding regression line pairs for WRN patients and matched controls (dashed line) and for WRN heterozygotes and matched controls (solid lines). For clarity, data points for controls are not shown.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have used the GPA red cell Vf assay to quantify and characterize in vivo genetic instability in WRN patients and heterozygotes. WRN is an uncommon autosomal recessive disease in which progeroid features are associated with genetic instability and an elevated risk of neoplasia. Our study included 11 WRN patients and 10 within-pedigree heterozygotes who collectively carried 10 different WRN mutations. Three of these mutations, the 2-bp and 4-bp deletions identified in the CWW WRN pedigree and the CP6 splice-junction base subsitution, are reported for the first time (see Table 1Citation ). WRN patients and heterozygous family members had elevated GPA Ø/N Vf, indicating an elevated in vivo somatic mutation frequency.

The GPA Ø/N Vf elevations we observed in WRN patients are consistent with and corroborate our previous identification of an elevated mutant frequency in peripheral blood T lymphocytes from WRN patients (25) and a deletion mutator phenotype in WRN fibroblast cell lines (26) . These three findings in concert indicate that the loss of WRN function promotes genetic instability in multiple human somatic cell lineages. The steep increase in Ø/N Vf as a function of patient age is particularly intriguing because it parallels the rapid rise in clinical signs, symptoms, and disease risk that begin in WRN patients after puberty (2 , 27) . This suggests that an increased mutation rate and/or mutation accumulation could be important in the WRN pathogenesis. The high OR we observed for Ø/N Vf in patients, of 38.8 at Ø/N Vf >= 9 x 10-6 corresponding to a sensitivity of 91% and a specificity of 74%, indicates that the GPA assay may be useful in establishing a diagnosis of WRN in suspected patients. Although N/N Vf were not significantly elevated in WRN patients, Ø/N and N/N Vf covaried in patients at a level that approached significance (Pearson correlation = 0.6; P = 0.07). The only other patient population in which this type of Ø/N versus N/N Vf correlation has been observed is, intriguingly, BLM patients (9) ; BLM patients carry mutations in the BLM gene that encode a RecQ helicase related to WRN (18) .

The identification of in vivo genetic instability in WRN heterozygotes was surprising and unanticipated. These results are, to the best of our knowledge, the first demonstration of genetic instability in vivo in heterozygotes for a human autosomal recessive genetic instability syndrome. Clinically important heterozygote effects have long been postulated for other of the recessive genetic instability syndromes, e.g., ATM. ATM heterozygotes appear to be at increased risk for breast cancer and perhaps for other neoplasms. However, this increased risk is not clearly related to the modestly increased ionizing radiation sensitivity of ATM heterozygous cells, and there does not appear to be an excess of ATM heterozygotes among patients with a heightened or severe response to chemotherapy or ionizing radiation therapy (reviewed in Refs. 28, 29, 30 ). Moreover, no GPA heterozygote effect has been observed in ATM, BLM, or FAN pedigree analyses (9 , 13 , 15 , 16) .

The genetic instability we observed in WRN heterozygotes may have parallels in intermediate sensitivity of WRN heterozygous B lymphoblastoid cell lines to killing by 4-nitroquinoline 1-oxide (31) and by the DNA topoisomerase I inhibitor camptothecin4 (32) . The most likely mechanistic basis for these effects and for the genetic instability we observed in vivo in WRN heterozygotes is haplo-insufficiency. WRN protein and immune-precipitable WRN helicase activity are both reduced in cell lines from WRN heterozygotes. Moreover, the truncated WRN proteins predicted by mutant alleles are present at very low levels or are undetectable in patient and heterozygote cells (33 , 34) .

WRN heterozygotes appear to be relatively common in both the United States and Japan, with estimated frequencies of 1/200–1/500 (1 , 35) . Thus, a WRN heterozygote effect, if expressed as genetic instability or an enhanced sensitivity to DNA damaging agents, could be a predisposition to neoplasia or to adverse therapeutic effects. One way to test this hypothesis would be to look for an excess of mutant WRN alleles in neoplasms, especially of the types observed in Werner patients or in patients with adverse responses to drugs such as camptothecin that are selectively toxic to WRN cells (32 , 36) . Although the WRN open reading frame is large, mutation screening may be readily tractable because all of the known, clinically important WRN mutations thus far identified should be readily detected by an in vitro protein truncation screening assay (1 , 5) .

Our results also provide additional insight into the relationship between in vivo genetic instability and disease risk. ATM, BLM, and FAN patients display GPA Vf that are 10- to 100-fold higher than those of healthy controls and are at high risk of developing immunodeficiency, leukemia, lymphoma, and marrow failure (37 , 38) . Werner patients, in contrast, display more modest GPA Vf elevations and have a correspondingly lower risk of developing bone marrow neoplasia or failure (2 , 3) . This comparison suggests that a 10-fold or greater increase in somatic mutation confers a high risk of developing bone marrow and/or lymphoid pathology. The rapid increase in GPA Ø/N Vf with age in WRN patients predicts that older patients should be at higher risk of developing marrow pathology. This appears to be the case: leukemia, myelodysplasia, and myelofibrosis collectively represent 20% of nonepithelial neoplasms and 11% of all neoplasms in WRN patients (3 , 39 , 40) . Moreover, patients who develop marrow dysfunction or neoplasia do so relatively late, with an average age of ~40 years at diagnosis (see Ref. 3 and additional references cited therein). Somatic mutation plays an important role in the genesis of at least one of the myelodysplastic syndromes, paroxysmal nocturnal hemoglobinuria (41, 42, 43) , and may play a role in the propensity of the myelodysplastic syndromes to evolve into acute leukemias (44) . Biochemical and functional analyses of WRN and the other human RecQ helicases should soon provide a better picture of in vivo RecQ helicase function and should indicate how the loss of function promotes genetic instability and the development of both neoplastic and nonneoplastic disease in specific human cell lineages (40 , 45) .


   ACKNOWLEDGMENTS
 
We thank George M. Martin and colleagues at the International Registry for Werner Syndrome (Seattle, WA) for their help in procuring WRN blood samples for this study.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by the National Cancer Institute (RO1 CA48022, R29 CA77607, and contract NO1-CP-50520), the National Institute on Aging (AG 14446 and T32 AG00057), and the United States Deparment of Energy (contract W-7405-ENG-48). Back

2 To whom requests for reprints should be addressed, at University of Washington, Box 357705, Seattle, WA 98195-7705. Phone: (206) 616-7392; Fax: (206) 543-3967; E-mail: monnat{at}u.washington.edu Back

3 The abbreviations used are: WRN, Werner syndrome; GPA, glycophorin A; MIM, Mendelian Inheritance in Man database; Vf, variant frequency; ATM, ataxia-telangiectasia; BLM, Bloom syndrome; FAN, Fanconi anemia; OR, odds ratio. Back

4 M. Poot, personal communication. Back

Received 11/ 4/99. Accepted 3/ 2/00.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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