GENOMICS

18,

797-802

(19%)

Mapping of the Motor Neuron Degeneration (Mnd) Gene, a Mouse Model of Amyotrophic Lateral Sclerosis (ALS) ANNE

MESSER,*‘t

JULIE PLUMMER,*‘t

PAUL

MASKIN,*

JOHN

M.

COFFIN,t

AND WAYNE

N.

FRANKELt,’

*Wadsworth Center for Laboratories and Research, New York State Department of Health, and tDepartment of Biomedical Sciences, State University of New York at Albany, P.O. Box 509, Albany, New York 12201-0509; and Department of Molecular Biology, Tufts University School of Medicine, Boston, Massachusetts 027 11 Received

January

13, 1992;

revised

March

3, 1992

malities in: (1) motor neurons in the spinal cord (earlier in lumbar than in cervical); (2) cranial motor nerves (especially 10 and 12); and (3) some upper motor neurons (Messer et al., 1987). The nature of the pathology is not spongiform, and there are no obvious signs of inflammatory response. The pathology shows many similarities to motor neuron changes reported for ALS (Tandan and Bradley, 1985). The degenerating motor neurons are characterized by numerous inclusion bodies in the affected soma (Messer et al., 1987), some of which contain immunoreactive ubiquitin (Mazurkiewicz, 1991) and/or lysosomal enzymes (Callahan et al., in preparation). There is also some increase in the incidence of somatic phosphorylated neurofilaments found in such cells, although the redistribution of all forms of neurofilament protein to the margins of the cell bodies is a much more striking finding (Callahan et al., 1991). Some neurofilament margination can be seen even in the most mildly affected cases.The idenINTRODUCTION tity of the mutant gene product that sets this pathogenic chain of events in motion should yield important information on Mnd, with implications for aspects of the deAmyotrophic lateral sclerosis (ALS) is a progressive human neurological disorder with an unknown, and generative process in motor neurons of both hereditary probably heterogeneous, etiology (Mitsumoto et at., and spontaneous ALS. 1988; Rowland, 1987). A fraction (&lo%) of the cases We have established a set of genetic and environmenare hereditary, with similar pathology to spontaneous tal conditions under which the homozygous Mnd/Mnd forms of the disease (Calne and Eisen, 1989; Vassilopoumice show a consistent age of onset and progression of los, 1989). Fully involved ALS affects both upper and symptoms clearly distinguished from +/+ or +/Mnd. To lower motor neurons. Given the difficulty of studying map Mnd, this subline was then used in a classical outthe early stages of the pathologic processesin ALS itself, cross/intercross breeding to generate F2 progeny that a number of induced and naturally occurring animal were segregating strain-specific markers along with the models have been investigated (Sillevis Smitt and De neurological phenotype. To avoid having to test a large Jong, 1989; Messer, 1992). Motor neuron degeneration number of individual chromosome-specific restriction (Mnd) in the mouse was originally identified as a spontafragment length polymorphisms (RFLPs), three sets of neous adult-onset neurological disease with symptoms mapped endogenous murine leukemia virus (MLV) fragbeginning at about 6 months of age, progressing to total ments were used initially as genetic markers (Frankel et spastic paralysis with premature death (Messer and Flaal., 1989a; Stoye and Coffin, 1988; Frankel et al., 1990, herty, 1986). Breeding studies show that the disease seg- 1989b). Once linkage to the Xmv-26 provirus on proxiregates as a single gene locus (Messer and Flaherty, mal Chr 8 was established, an Ank-1 RFLP (White et al., 1986). Basic histopathology reveals substantial abnor1990) and a polymerase chain reaction (PCR) assay for a simple sequence length polymorphism (SSLP) within 1 Present address: The Jackson Laboratory, Bar Harbor, ME 04609. the polymerase B (Polb) gene (Hearne et al., 1991) were The motor neuron degeneration mutation (Mnd) causes a late-onset, progressive degeneration of upper and lower motor neurons in mice. After establishing genetic and environmental conditions that distinguish the phenotypes of Mnd/Mnd from +/Mnd mice, Mnd was mapped to proximal Chr 8, using endogenous retroviruses as markers. The map location was confirmed with additional linked polymorphic markers. The outcross/intercross matings to the strain AKR/J, which were used to follow the segregation of the retroviral markers with respect to Mnd, also revealed the existence of a timing effect. Approximately one-fourth of the affected Mnd/Mnd F2 progeny showed accelerated disease. The Mnd mouse model should allow study of mechanisms affecting onset and progression of specific neuronal degeneration in both animal and human neu(c3 1992 Academic Press, Inc. rological disease.

O&388-7543/92

$5.00

Copyright Q 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

MESSER

used to confirm more precisely.

linkage

MATERIALS

and to localize the Mnd gene

AND

METHODS

Animals. All mice came from the breeding colonies maintained at the Wadsworth Center Laboratories. For the Fl outcrosses, Mnd/ Mnd mice of the C57BL/G.KB2-Mnd/Msr subline were mat,ed to AKR/J or C3H/HeJ mice, using mutant mice of both sexes to generate different Fl stocks. The Fl mice were then brother-sister mated to generate the intercross F2 generation. Mice were tested behaviorally, using established measures of motor parameters (Callahan et a/., 1991), starting at 6 months in the first cross and at 4.5 months in later crosses. Animals were scored as affected if the disease progressed to “moderately severe” as previously described in detail (Messer and Flaherty, 1986; Callahan et al., 1991). Briefly, this consists of moderate hindlimb clutching when the animals are held up by the tail, plus a stiff gait characterizing the mild stage; progressing to strong hindlimb clutching with hindlimb weakness (for grabbing or locomotion) at the moderate stage; with further progression to loss of use of the hindlimbs plus moderate clutching of the forelimbs in the severe stage. At this point, the mice are sacrificed. Mice are checked daily for births and at least weekly for general health. Animal procedures have been approved by the Institutional Animal Care and Use Committee. DNA hybridization analysis of dried agarose gels. High-molecularweight genomic DNA was prepared from mouse liver as previously described (Messer, 1988). Genomic DNA was digested with the restriction enzyme P&I, precipitated in EtOH, resuspended in 10 mM Tris, pH 7.5, 1 mM EDTA, and electrophoresed in a 0.8% Tris-borate EDTA, SeaKem LE (FMC Corp.) agarose gel until a 2.0.kb molecular weight standard reached about 20 cm. Dried gels were then prepared according to the method of Wallace and Miyada (1987). The stained gel was denatured in 0.5 N NaOH, 1.5 M NaCl for 45 min and neutralized in 1.0 M Tris, pH 8.0, 1.5 M NaCl for 60 min, then dried under vacuum on a slab gel dryer at 25°C (Hoeffer Scientific) until flat, followed by an additional 30 min at 60°C. The dried gel was then hybridized for 4-16 h at 62°C in 5~ SSPE (Sambrook et al., 1989), 0.1% SDS, 10 pg/ml denatured salmon sperm DNA, plus 0.5 X lo6 CPM/ml oligonucleotide probe (JS-4, JS, 5, or JS-6+10;), which was 5’ labeled with 32P using T4 polynucleotide kinase. Dried gels were then washed in 2X SSC (Sambrook et al., 1989), 0.5% SDS twice at 25°C for 30 min, and twice at 62°C for 30 min each, then briefly airdried and exposed to X-ray film for 1-5 days using one intensifying screen. For dried gel reuse, probe was removed by denaturation in 0.5 N NaOH, 1.5 M NaCl for 15 min and neutralized in 1.0 M Tris. pH 8.0, 1.5 M NaCl for 30 min. Linkage analysis. Proviral loci were scored in the C57BL/GJ-Mnd X AKR/J F2 intercross in the following manner: All fragments hybridizing to JS-5, JS-4, or JS 6+10 in the two parental strains were readily identified as Pmv, Mpmv, and Xmv loci, respectively, with no apparent deviation from work previously published (Frankel et al., 1990). Proviruses present in C57BL/6J but absent from AKR/J and those present in AKR/J but absent from C57BL/6J were scored by gene dosage, using proviruses common to both strains as internal copy number standards. Genotype data were entered into a Microsoft Excel spreadsheet, sorted by chromosome, printed, and proofread for entry errors. Data were then uploaded into the computer program MAPMAKER (Lander et al., 1987). Data for some DNAs are partial, since not all loci are informative in all crosses. One provirus (Pmu-32), previously unmapped, was positioned on proximal Chr 16 (20.1% recombination with Mpmv-17). For Ank-1 allele analysis, standard Southern blots (Sambrook et al., 1989) of HindIII-digested genomic DNAs were probed with a ‘*P-labeled ANKl DNA probe described by Lambert et al. (1990). Fragments of 3.6 and 3.8 kb were recognized in AKR or C3H and B6, resnectivelv. Polymerase chain reaction assay for microsatellite assays were performed in lo-r1 reaction mixtures

loci. SSLP PCR in 0.5.ml microcen-

ET

AI,.

trifuge tubes in a PTC-100 machine purchased from MJ Research (Cambridge, MA). Each reaction mixture contained 20 ng genomic DNA, approx 20 ng each oligonucleotide primer (5’-GCCTGGATTTCCTCATTGAA-3’; 5’.AGTTGGTTATCCCTGAAAATATACA-3’), 0.2 PM each dNTP, 0.1% gelatin (Sigma), 50 mM KCI, 10 mM Tris, pH 8.0.1.5 mM MgCl,, and 1 unit of Taq polymerase (Perkin-Elmer/ Cetus). A trace amount of one of the two primers was end-labeled with ‘{‘P using T4 polynucleotide kinase and added to the reaction mixture. The PCR conditions were 94”C, 5 min (1 cycle), 94”C, 1 min, 55”C, 2 min, 72°C. 3 min (25 cycles), under a drop of mineral oil. One-fifth of the reaction mixture was then analyzed by denaturing polyacrylamide gel electrophoresis (6% acrylamide). The gel was dried and exposed to X-ray film for 12 h. Figure presentations. The data shown in Figs. 1 and 2 are computer-scanned images of the original autoradiographs. Films were scanned to a Macintosh computer in a PICT file format using a Hewlett-Packard Scan Jet II. The images were then adjusted globally for brightness and contrast using the program Enhance (Microfrontier) and pasted into t,he drawing program Canvas 2.2 (Deneba Software) for labeling. Completed figures were then printed on a LaserWriter II NT printer (Apple Computer).

RESULTS Establishment

of an Mnd Subline

The Mnd disease, the result of a spontaneous mutation in a C57BL/6 stock, was initially characterized by substantial variability in age of onset and timecourse (Messer and Flaherty, 1986). While this variability is also characteristic of human ALS, it makes many kinds of experiments more difficult. We therefore have selected a subline of the original strain in which Mnd/Mnd homozygotes show the same neurological symptoms and pathology as the original line, but with a much more consistent and predictable timecourse. These mice always show the first symptoms just before 6 months of age, progress to forelimb symptoms by 7.5 months, and show severe symptoms by 9 months (Callahan et al., 1991). They seldom survive past 10 months of age. The subline has been designated C57BL/G.KB2-Mnd/Msr. In an effort to control skin lesions that were a severe husbandry problem (both in homozygotes and in obligate heterozygotes), the colony was also switched to sterilized cages, bedding, food, and water, with filter caps used on all cages. This regime has reduced the incidence of skin lesions to an occasional rare individual, which is promptly isolated and treated. Other secondary infections are reduced as well. With this strain in this environment, unambiguous symptoms are not observed in heterozygous (Mnd/+) mice less than a year old. Outcrosses

for Mapping

of Mnd

To map the Mnd gene, we outcrossed Mnd/Mnd to the AKR/J strain of mice and intercrossed (brother-sister mated) 20 of the hvbrid Fl’s. A total of 305 of these ” progeny were scored by testing for neurological symptoms starting at 6 months of age, with progression to forelimb involvement by 8 months; 69 were affected. A similar set of F2 intercross mice was generated from an outcross to C3H/HeJ. No neurological abnormalities

MAPPING (AK x BG-MndjF2

Progeny

I

I

OF

MURINE

MOTOR

AK BGmMnd I

Xmv-72

Xmv-26

MMMMM++M++

NEURON

799

DEGENERATION

(1) The gene Ank-I (restricted isoform of ankyrin) is polymorphic between C57BL/6J and AKR/J or C3H/ HeJ (White et al., 1990). Using a cDNA probe from the et al., closely related human ANKl gene (Lambert 1990), four obligate recombination events between Ank1 and Mnd were observed in a total of 76 F2 mice from the two crosses, giving a pairwise lod score of 17.0 (Fig. 2). (2) There is a PCR assay for strain-specific SSLP in the 5’ noncoding region of the Polb gene (Hearne et al., 1991). Both AKR/J and C3H/HeJ yield products of different sizes from C57BL/6. The data (Fig. 2) show five obligate recombination events between Mnd and Polb, for 76 F2 progeny (LOD = 15.8). The segregation data for all five loci were analyzed in a multipoint analysis, using the MAPMAKER computer program (Lander et al., 1987). With the double crossovers taken into account, the most likely gene order is Xmv-26-Mnd-Polb-Ank-1-Xmv-12 (Fig. 3a). The next best gene orders, shown in Fig. 3b, are 16 times and 363 times less likely, although still within 2.56 LOD units of each other. Recombination data have been submitted to

++M

*

FIG. 1. Dried gel of PuuII-digested genomic DNA from AKR x BG-Mnd F2 intercross progeny and AKR/J and C57BL/6J parent strains, hybridized with oligonucleotides JS-6 + JS-IO, to identify Xmv proviruses. The Xmv-26 and Xmv-12 fragments are indicated at the right. Below the blot is the Mnd phenotype: M for affected; + for wildtype. The asterisk shows one obligate recombinant between Mnd and Xmv-26.

were observed in 8 to lo-month-old cross.

A B6AK

(AK x BG-MndjF2

Mnd

I

Progeny

I

- 23.5 kb

Fl mice from either

- 9.4 kb B6 allele AK allele

- 6.6.kb

’

Linkage of Mnd to the Endogenous Provirus Xmv-26 +MM+MMMMM++M+

Assays of 54 F2 DNAs from the AKR/J cross, digested with PvuII, gave the results shown in Fig. 1. Tight linkage to Xmv-26 on proximal Chr 8 was apparent from this study (lod score = 14.5), as well as weaker linkage to Xmv-12 (LOD = 6.9), which is more distal on the same chromosome. Xmv-26 is present in AKR/J and absent from C57BL/6; all but one affected F2 intercross mouse lacked the Xmv-26-specific fragment. No other chromosome showed significant linkage, using probes that identified the following loci: 15 Xmv, 18 Pmv, 10 Mpmv, and 20 additional endogenous retroviruses, covering 16 different chromosomes. There was no evidence that any unusual MLV fragments, insertions, or rearrangements are associated with the C57BL/6-Mnd genome. The C3H cross was not typed for endogenous provirus markers, since it is not informative at the Xmv-26 locus. Linkage Confirmation and Gene Ordering

To confirm the results and map the gene more precisely, two additional markers linked to Xmv-26 on Chr 8 were used:

l

B (AK x BG-Mnd)F2

AK I

I

86 allele AK allele

Progeny

310 bp 300 bp

’

+

M+MMMMM++M+ *

FIG. 2. (A) Ark-1 RFLP assay. Southern blots of HindIII-digested genomic DNA from AKR/J (AK) C57Bl/6J-Mnd (BG-Mnd) and 11 F2 progeny were hybridized with the human ANKl cDNA clone ANK25A. Positions of the AK and B6 alleles are shown on the left. The Mnd phenotype and an obligate recombinant (*) are shown below the blot, as in Fig. 1. (B) Polb SSLP assay. Genomic DNA from AK, BG-Mnd (not shown), and F2 progeny were PCR amplified using radiolaheled Polb-specific primers and were analyzed by polyacrylamide electrophoresis. Allele sizes were 310 bp for B6 and 300 bp for AK. The fuzziness reflects a slight stuttering which is characteristic of SSLP assays. Labeling is the same as that in Fig. 2A.

800

MESSER

A

Polb

(11.7)

An&-l

(14.9)

Xmv-72

(24.8)

1: 2: 3:

Xmv-26

Mnd Xmv-26 Xmv-26 Polb Mnd

PO/b Ank-1 PO/b Ank-7 Ank-1 Mnd

Xmv-12 Xmv-i2 Xmv-12

log-likelihood: log-likelihood: log-likelihood:

-166.71 -187.93 -169.27

FIG. 3. Localization of Mnd on proximal Chr 8. (A) Multipoint linkage map constructed using MAPMAKER 2.0 (maximum likelihood estimate), with the distance of each locus from the centromere shown at the right (in centimorgans). (B) Best three orders, as calculated using the MAPMAKER 2.0. Note that these are within 2.5 LOD units of each other.

A complete

haplotype

to C3H/HeJ early symptoms.

DISCUSSION

(3.9)

B

GBASE. quest.

AL.

from a total of 330 outcross/intercrosses and a BG/DBA hybrid stock developed Mnd

order order order

ET

table is available

on re-

Age of Onset Of the 69 mice from the AKR/J cross that developed clear neurological disease, 15 were already moderately affected by the time they were first tested at 6 months. There were 7 additional mice from the first cross that died between 6 and 6.5 months of age; death during this time period is quite unusual. Some or all of these probably died prior to detection as severe early onset, since we did not realize at first that there was an early-onset form of the disease that could kill the mice that young. We therefore scored 15-22 of the animals as showing early onset of symptoms. As noted above, the usual progression for Mnd/Mnd mice in the parent line proceeds from very mild symptoms at 6 months to moderate symptoms by 7-7.5 months, and death at 8.5-9.5 months (Callahan et al., 1991). Early-onset mice show mild symptoms by 4.5-5 months, moderate symptoms by 6 months, and death by 7 months. Comparisons of the clinical syndrome in the early-onset vs inbred C57BL/6 Mnd mice revealed no obvious differences in the types of symptoms seen or the order in which they appeared, however, the age of onset was earlier and the speed of progression increased. We have also seen rearrangements of neurofilament immunoreactivity in an examination of the spinal cord of an early-onset case (data not shown). No mice

Our current data on symptoms in Mnd mice imply that the mutation is semidominant with reduced penetrance. Since homozygous Mnd/Mnd mice show an even more distinct phenotype in this subline than in the original [in which heterozygotes and homozygotes differed sharply in progression of symptoms, but showed a variable although basically similar pattern for onset of mild symptoms (Messer and Flaherty, 1986)] we could use homozygosity mapping to map the Mnd gene. Most neurological mutants show some variation of symptoms with genetic background (Green, 1981). Mnd seems more extreme than most, with the heterozygote effect apparently disappearing as a result of a combination of selective breeding and stringent environmental controls. However, Mnd is a late-onset mutation that interacts with normal and strain-specific aging, an association that has not been well-investigated previously. Some combination of genetic and environmental background effects allowed clear expression of a slowly progressive motor neuron syndrome in 26 obligate heterozygotes (Mnd/Mnd X +/+ progeny), with motor neuron disease confirmed histologically in two of the cases (Messer and Flaherty, 1986). In a few cases, we did observe what appeared to be very mild, nonprogressive symptoms (mainly clutching when the animal is held by the tail) in F2 mice from the AKR cross. These may be examples of interaction of the early-onset gene(s) with +/Mnd. However, these examples are observed very rarely and are difficult to quantitate given the intrinsic variability and somewhat subjective nature of the mild symptoms. It was not possible to age all of the asymptomatic F2 mice beyond 8-9 months. When polymorphic markers make it feasible to type F2 mice for both Mnd and the putative timing gene(s) shortly after weaning, the effects of AKR on +/Mnd will be followed up further. Understanding how these background effects interact with the primary Mnd gene product may offer important clues as to the pathogenesis of Mnd. The use of polymorphic endogenous retroviruses (Frankel et al., 1990) and other multilocus DNA markers (Drivas et al., 1991; Siracusa et al., 1991) to screen for chromosomal location has been shown to have general application. Endogenous MLVs have been used recently to map an immunoglobulin transgene (Gerstein et al., 1990) and the genetic determinants of epilepsy in EL mice (Rise et al., 1991) Such multilocus markers also provide useful internal standards and genetic quality control. In this case the extensive data on nonecotropic retroviral fragments in the AKR X B6 cross confirmed that the Mnd background strain was a relatively “pure” C57BL/6J; it also effectively ruled out a hypothesis that the Mnd mutation was caused by an insertion of a provirus of this class into the Mnd gene (Contag and Plagemann, 1989; Frankel et al., 1989a).

MAPPING

OF MURINE

MOTOR

The proximal region of mouse Chr 8 shows linkage conservation with proximal human Chr 8. No motor neuron diseases have been reported to map specifically to this region, although there are several reports of occasional human neurological disease, including motor neuron disease, in patients with hereditary spherocytosis (HS), which is now known to also map to Chr 8, with an ankyrin (ANKl) deficiency as its primary cause (Bennett and Lambert, 1991). However, the two most likely gene orders show significant genetic distance and one or two additional markers between Mnd and the mouse gene Ank-1. We have also excluded the gene encoding tissue plasminogen activator, Plat. It is closely linked to Ank-1 (White et al., 1990), and we have recently observed three obligate recombinants between Plat and Mnd in our F2 DNAs. Another attractive potential candidate gene on proximal human Chr 8, neurofilament light chain (NF-L), maps to Chr 14 in the mouse (Mattei et al., 1989). In outcrosses to mice carrying the early cerebellar mutation nervous (nr), which also maps to this region of Chr 8 in the mouse (Sidman and Green, 1970), Mndjnr heterozygotes showed neither cerebellar disease nor Mnd motor neuron symptoms at 7 months. Most human hereditary ALS is reported to be dominant, although there are no available cases of homozygotes in these families to establish whether a second copy of the mutant gene gives a much more severe phenotype. There is also a small number of families in which the inheritance is recessive. A recent report on mapping of the autosomal dominant forms of the human hereditary ALS gene in 23 human kindreds shows that some of the pedigrees show linkage to Chr 21. However, there is a very strong probability of heterogeneity (P < O.OOOl), with the best estimate of the proportion of families with Chr 21 linkage at 0.55 (95% confidence interval, 0.200.93) (Siddique et al., 1991). There are, therefore, still many families for which the chromosomal location of their hereditary ALS is undetermined. While there is more variability in the age of onset in the Mnd/AKR/J F2 mice than in the C57BL/6 Mnd subline, the data are consistent with a bimodal distribution of disease. Of a total of 305 intercross mice, 15 to 22 showed onset prior to 5 months and progression to very severe symptoms before 7 months, while the remaining 54 affected F2 casesfrom this cross clustered around the standard g-month age of onset, with a progression to severe after 9 months. The accelerated timing effect is strain specific, since it is seen in crosses with AKR/J, and not with crosses to either C3H/HeJ (108 F2s examined) or other strains used for other experiments (222 additional F2 mice, with no early-onset cases). The gene(s) for this timing effect must be segregating independently of Mnd itself, since only 15-22 of the 69 Mnd/ Mnd mice carry it. Additional genetic crosses are currently under way to establish the location(s) of the gene(s) responsible for this timing effect. There are reports of human kindreds in which the age of onset of ALS also seemsto follow a bimodal distribution (Mulder et al., 1986). Hereditary canine spinal muscular atrophy,

NEURON

DEGENERATION

801

an ALS model in dogs (Cork et al., 1979), also shows accelerated forms of disease, but the earlier onset form

seems to be due to a double dose of the original gene, rather than modifying genes (Sack et al., 1984). Possible mechanisms of the timing effect include: (1) Strain-specific differences in the wildtype number of motor neurons, with excess pressure on the lower number of remaining neurons accelerating the process as the disease progresses [see Goffinet (1990) for an analogous effect in the reeler mutant cerebellum]; (2) strain-specific differences in baseline levels of growth factor or growth factor receptors [preliminary studies suggest that treatment with ciliary neurotrophic factor (CNTF) can improve motor function in Mnd/Mnd mice (Lindsay et al., in preparation)]; and (3) some intrinsic difference in the properties of the motor neurons themselves, which alters interactions with the product of the Mnd gene [e.g., the Lurcher cerebellar mutant phenotype is not expressed within Purkinje cells altered by a staggerer mutant background (Messer et al., 1991)]. There is no evidence that the timing effect is influenced by either the sex of the affected mice or parental imprinting from the original mating (Moore and Haig, 1991). Identification of the chromosomal location of the Mnd gene is the first step toward the identification of its product. Examination of Chr 8 in human kindreds with heterogeneous forms of hereditary ALS, as well as related hereditary neurological diseases, may be fruitful. The fact that a combination of genetic and environmental modifiers can alter the time course of the phenotypic expression suggeststhat this mouse model can be used to examine both the process leading from the primary defect to the final cell dysfunction and the endogenous modifiers of the rate and severity of this process. ACKNOWLEDGMENTS We are grateful to John Todd for informing us of the Polb polymorphism and to William Dietrich and Eric Lander for providing Polb oligonucleotides and their PCR conditions, prior to publication. We also thank Steve Lambert for ANKl; Jane Barker and Connie Birkenmeyer for Ank-I probes; and Ben Taylor for helpful discussions. This work was supported by a research grant from the ALS Association (A.M.) and NIH Grant R35CA44385 (J.M.C.). Wayne N. Frankel is a Special Fellow of the Leukemia Society of America, Inc.

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