Unique PABP2 Mutations in “Cajuns” SuggestMultiple Founders of Oculopharyngeal MuscularDystrophy in Populations With French Ancestry

Peter C. Scacheri,1 Carlos Garcia,2 Richard Hebert,3 and Eric P. Hoffman1,4*1Department of Molecular Genetics and Biochemistry, University of Pittsburgh, Pittsburgh, Pennsylvania2Department of Psychiatry and Neurology, Tulane University Medical Center, New Orleans, Louisiana3Professional Genealogist, Montreal, Ontario, Canada4Research Center for Genetic Medicine, Children’s National Medical Center, Washington, District of Columbia

Oculopharyngeal muscular dystrophy(OPMD) is an adult-onset autosomal domi-nant myopathy found world-wide, but withthe highest incidence in French-Canadians.Short GCG expansions in the poly(A) bind-ing protein 2 (PABP2) gene were identifiedrecently as the molecular basis for OPMD inFrench-Canadians. All French-Canadiancases of OPMD have been traced to a singlefounder couple [Bouchard, 1997: Neuromus-cul Disord 7(Suppl):S5–S11]. Cultural linksbetween French-Canadians and Cajuns sug-gest that this same founder couple may havetransmitted the OPMD mutation to Cajunsas well. To determine if OPMD patients fromLouisiana share a founder effect withFrench-Canadian families, we collectedblood samples and muscle biopsies from sev-eral Cajuns with OPMD for mutation andlinkage studies. We found a unique ’GCAGCG GCG’ insertion mutation in Cajuns.Consistent with these sequence data, weidentified a disease haplotype in our Cajunfamilies that is different from the ancestralhaplotype defined in French-Canadians.These data prove that different founders in-troduced the PABP2 mutation to Cajuns andFrench-Canadians and lend support toemerging genealogical data suggesting thatFrench-Canadians and Cajuns representdistinct immigrant groups from France. Am.J. Med. Genet. 86:477–481, 1999.© 1999 Wiley-Liss, Inc.

KEY WORDS: oculopharyngeal musculardystrophy; linkage analysis;

linkage disequilibrium; tri-nucleotide insertion muta-tion

INTRODUCTIONOculopharyngeal muscular dystrophy is a late-onset

disease (>50 years) characterized by progressive dys-phagia and bilateral ptosis. Characteristic posturing ofthe head to compensate for ptosis (Hutchinson’s or as-trologist’s posture) may aggravate the dysphagia. Thedystrophic process later spreads to the pelvic girdleand distal limb muscles [Brais et al., 1995; Tome andFardeau, 1994; Schmitt and Krause, 1981].Until recentadvances in surgery aimed at treating pharyngeal dys-function [Fradet et al., 1988], patients often died fromstarvation or aspiration pneumonia.

Muscle biopsies from OPMD patients show abnor-malities common to many muscular dystrophies. Theseinclude loss of muscle fibers, abnormal variation in fi-ber size, increase in the number of central nuclei, in-creased interstitial fibrous and fatty connective tissue,small angulated fibers and rimmed vacuoles. Unique toOPMD are tubulofilamentous inclusions within musclefiber nuclei [Tome and Fardeau, 1980, 1994].

OPMD is an autosomal dominant trait. It is foundworld-wide, but with highest incidence in French Ca-nadians. Several authors [Barbeau 1966; Bouchard,1997; Tremblay-Tymczuk et al., 1992] reported that allFrench Canadian cases of OPMD could be traced backto a founder couple whose daughters migrated fromwestern France to Quebec province in 1634, suggestingthat a single OPMD mutation exists within the French-Canadian population.

The OPMD locus was mapped to chromosome region14q11.2-q13 in four large French-Canadian families(D14S283: max LOD 4 14.73 at (u 4 0.03)[Brais et al.,1995]. Linkage to this locus was also reported in fami-lies from six other ethnic backgrounds. Recently, shortGCG repeat mutations in the coding sequence of thepoly(A) binding protein 2 (PABP2) gene were found tocause OPMD [Brais et al., 1998]. A (GCG)6 in the cod-

Contract grant sponsor: American Heart Association.*Correspondence to: Eric P. Hoffman, Director, Research Cen-

ter for Genetic Medicine, Children’s National Medical Center,Washington, DC 20010. E-mail: ehoffman@cnmc.org

Received 2 March 1999; Accepted 16 June 1999

American Journal of Medical Genetics 86:477–481 (1999)

© 1999 Wiley-Liss, Inc.

ing sequence of PABP2 was found to be expanded to(GCG)8–13 in 144 OPMD families from 15 differentcountries (Armenia, Australia, Belgium, Canada,France, Germany, Isreal, Italy, Japan, Netherlands,Spain, Switzerland, United Kingdon, United States,and Uruguay). The vast majority of French CanadianOPMD families have a (GCG)9 mutation. These inser-tion mutations cause increased numbers of poly-alanines in the N-terminus of the PABP2 protein fromthe normal 10 alanines to 12–17 alanines. Thesestretches of hydrophobic alanines are much shorterand differ in charge from the polyglutamines seen inmost other dominantly inherited trinucleotide repeatdisorders. Thus, the pathogenesis of OPMD is clearlydifferent from glutamine toxicity seen in Huntingtondisease, and the spinocerebellar ataxias. Moreover, themolecular mechanisms that generate the larger DNAsequences may be different.

Historically, there have been several migrationsfrom France to North America. In 1604, French settlersmigrated to the territory now known as Nova Scotiaand founded “Acadia.” Several attempts at occupationwere made by the English (in 1654, 1690, 1710), and in1713 the colony was ceded to England. Despite oppres-sive English rule, the French population grew to 18,000by 1749. Between 1749 and 1752, nearly 6,000 Acadi-ans emigrated from Nova Scotia, and by 1755 all re-maining Acadians were forcibly ejected for refusing topledge allegiance to England (“Le Grand Derangementdes Acadiens”). Many Acadians were dispersed toFrance, Canada, and various Anglo American colonies:Two thousand went to Massachusetts, 700 to Connecti-cut, 300 to New York, 500 to Pennsylvania, 1,000 toVirginia, 500 to North Carolina, 500 to South Carolina,and 400 to Georgia. After the Treaty of Paris in 1763,many of the survivors from Pennsylvania, Maryland,Virginia, Georgia and the Carolinas migrated to Loui-siana, and rejoined over 3000 Acadian friends and rela-tives that were deported from France [Barbeau et al.,1984].

Despite this established ancestral link between Aca-dian French Canadians and Acadians in Louisiana(“Cajuns”) many, if not most Cajuns and French Cana-dians may not have originated from Acadia. Recent ge-nealogical data suggested, contrary to popular belief,that many Cajuns and French Canadians are descen-dants of Frenchmen who migrated directly to Louisi-ana and Canada [Barbeau et al., 1984]. In addition,Cajuns in Louisiana have lived among Spaniards,Blacks, Germans, and Americans, thereby further di-versifying their gene pool. Thus, although French-Canadians and Cajuns (Acadians) are geographicallydistinct populations with presumed common culturaland genetic heritage via Acadia, a significant portion ofthese populations has mixed ancestral roots [R. Hebert,personal communication].

To determine if OPMD patients from Louisianashare a founder effect with French-Canadian families,we studied muscle biopsies and DNA samples from sev-eral Cajuns with OPMD. We found that the insertion inthe Cajuns is not a “pure” sequence, and that the Cajunand French-Canadian patients do not share a commonancestor as the origin of their mutations.

PARTICIPANTS, MATERIALS, AND METHODSParticipants

Blood samples were obtained from 20 members ofthree Cajun OPMD families (9 individuals from FamilyA, 7 from Family B, and 4 from Family C) from south-west Louisiana. Muscle biopsies were obtained from 4individuals from southwest Louisiana. Three of thesepatients were Cajun whereas 1 of these patients wasItalian. The diagnosis of OPMD was based upon clini-cal symptoms of late-onset ptosis and dysphagia inher-ited in an autosomal dominant fashion.

Microsatellite Typing

Genomic DNA was extracted from leukocytes. Indi-viduals from all three OPMD families were genotypedfor the following STR markers linked to the OPMDlocus: D14S283, D14S990, MYH7.1, and D14S1041.The Genethon map reported the following sex-averaged distances between markers: D14S283-0.7cM-D14S990-8.8cM-D14S1041 [Dib et al., 1996]. The posi-tion of MYH7.1 was not reported by Genethon, butBrais et al. [1997] positioned MYH7.1 1cM telomeric toD14S990. STR marker primer sequences were identi-cal to those used by Brais et al. [1997]. Alleles sizeswere determined by genotyping CEPH individuals withknown allele sizes for each of these markers. PCR, elec-trophoresis, and detection of alleles were performed fol-lowing standard protocols. Genotypes were scored ac-cording to the size of the alleles.

Mutation Analysis

The following primers were designed and synthe-sized (GibcoBRL) to amplify the PABP2 mutated re-gion from genomic DNA: F-CATGGTGACGGGCAG-GCAGC, R-GCTCCTCAGGCTCCAGTTCC. PCR reac-tions were performed in a total volume of 12.5 mlcontaining 50 ng of genomic DNA, 150 mM of dATP,dCTP, dGTP, dTTP, 1 mCi [32P]dATP, 50 ng of eachprimer, 10% DMSO, 0.5 units of Taq Gold (Perkin-Elmer). 2.5 ml of a 5× concentrated mix containing 300mM Tris-HCl, 75 mM ammonium sulfate, 7.5 mMMgCl2, pH 8.5 was added as a buffer (Invitrogen). Theamplification procedure consisted of an initial denatur-ing/enzyme activation step at 94 °C for 10 min, followedby 40 cycles of 96 °C for 20 seconds, annealing at 60 °Cfor 20 sec, elongation at 75 °C for 40 sec and a finalelongation at 72 °C for 10 min. Reactions were per-formed on a MJ Research PTC-100 Thermal cycler.

RNA extraction, cDNA synthesis, and reverse tran-scription with oligo dT was performed on all fourmuscle biopsies. For RT-PCR on muscle cDNA, the fol-lowing reverse primer was substituted for the reverseprimer used to amplify genomic DNA: ACTCCAGGC-CGTTCCCGTA. The remainder of the PCR on musclecDNA was identical to that for genomic DNA.

Samples were loaded on 6% denaturing polyacryl-amide gels. Following electrophoresis, gels were driedand exposed to X-ray film. For DNA sequence analyses,allele-specific bands were directly excised from thedried gels, eluted in water, and reamplified for directautomated sequencing using cycle sequencing anddyedeoxyfluor kits (ABI, Foster City, CA). Expanded

478 Scacheri et al.

Fig. 1. OPMD families from Louisiana show a distinct haplotype from French Canadians. Linkage analysis on each family was conducted using themarkers listed in the right corner. Genotypes correspond to allele sizes. The disease haplotype is boxed. ( 4 mutation positive, but too young to showsymptoms.)

and normal alleles from all four muscle biopsies and atleast 1 member of each family were sequenced.

RESULTS

The results of genotyping all three Cajun familiesusing STR markers linked to 14q11.2-q13 were consis-tent with a localization of the disease gene to the 14q11.2-q13 OPMD locus (Fig. 1) (max LOD 4 2.41 @ theta 40.0). For markers D14S283, D14S990, MYH7.1, andD14S1041, a 143 bp, 149 bp, 102 bp, and 111 bp allelesegregated with the disease, respectively. The OPMDhaplotypes of the Cajun families were identical to eachother, but clearly different from those reported to seg-regate with the disease in French Canadian families[Brais et al., 1997] (Fig. 1).

The mutated region of PABP2 is highly GC rich andtherefore could not be readily amplified using standardPCR techniques. Specific conditions were developed toamplify a 269 bp fragment from genomic DNA and a245 bp fragment from muscle cDNA. PCR of the PABP2mutated region in all three Cajun families revealed alarger allele segregating with affected family members(Fig. 2). A larger allele was also detected in musclebiopsy cDNA from all four OPMD patients (Fig. 2). Di-rect sequencing of the alleles in Cajun patients re-

vealed a nine base pair insertion (Fig. 3). This muta-tion increases the number of alanine residues from 10to 13 in the N-terminus of the PABP2 protein. Sequenc-ing the expanded allele in the Italian patient revealeda 12 bp insertion of (GCG)4.

DISCUSSION

Brais et al. [1998] reported insertion of GCG repeatsin 144 OPMD families from 15 different ethnic back-grounds. The insertions were generally detected byPCR, with two French-Canadian patients directly se-quenced. The PABP2 mutation reported in theseFrench-Canadian families is a (GCG)6 expanded to a(GCG)8–13; the most common being a (GCG)9. Devia-tions within the sequence of this mutation were notpreviously reported. Because all French-Canadiancases of OPMD can be traced to a single founder couple,deviations in the sequence of the PABP2 mutation arenot likely to exist in French Canadians. However, thepossibility remains that sequence variations exist innon-French-Canadian OPMD families.

Due to the historical links between French Canadi-ans and Cajuns, we hypothesized that the same found-er existed for both French Canadians and Cajun casesof OPMD. Unexpectedly, we found a different 9 bp in-

Fig. 2. Identification of an insertion in Cajun OPMD families. Shown is PCR of the PAPB2 mutated region on (A) genomic DNA from OPMD familiesand (B) cDNA from OPMD muscle biopsies. Arrows indicate expanded alleles in affected family members. C 4 Cajun, I 4 Italian, N 4 Normal.

Fig. 3. Cajun OPMD patients show a GCA(GCG)2 insertion. Chromatographs from sequencing both normal (top) and mutant (bottom) PABP2alleles. The ’GCA GCG GCG’ insertion identified in all Cajun OPMD patients is underlined.

480 Scacheri et al.

sertion consisting of a GCA codon in combination withtwo GCG codons in all Cajun patients tested (Fig. 3).This finding suggests that the slippage model of muta-tion generation in (CAG)n expansion disorders may notbe the same for OPMD, given the heterogeneous se-quence of the insertion we identified.

In an effort to fine map the OPMD gene, Brais et al.[1997] defined an ancestral haplotype segregating withthe disease in 11 French-Canadian families withOPMD. Consistent with our sequence data suggestinga distinct mutation in Cajuns, the disease haplotype inour Cajun families is discordant from the ancestralhaplotype defined in the French Canadians (Fig. 1).Furthermore, the disease haplotype is consistent be-tween all three Cajun OPMD families. These data sug-gest that different founders introduced the PABP2 mu-tation to Cajuns and French Canadians, and implicatea single founder of OPMD in the Cajuns.

The mutation studies on our Cajun families in com-bination with the polymorphism data show cases ofOPMD with French ancestry can inherit the diseasethrough distinct ancestors. Furthermore, these datasuggest that OPMD is caused by a recurrent mutationin the PABP2 gene. The repetitive GCG sequences inthe PABP2 gene may be susceptible to mutationscaused by polymerase “slippage” proposed for expan-sion mutations; however, the (GCAGCGGCG) inser-tions identified here argue against this mechanism. Re-cent studies have shown linkage to the French-Canadian locus at 14q11.2-q13 in OPMD families withHispanic [Grewal et al., 1998], Italian [Creel et al.,1998], English/Scottish [Stajich et al., 1997], and Ger-man [Porschke et al., 1997] origins. As with our Cajunfamilies, the German and English/Scottish families arereported to have different haplotypes from those de-fined in the French-Canadians, suggesting that mul-tiple founders have contributed to the world-wide fre-quency of OPMD. These families are likely to have in-sertion mutations in the PABP2 gene; however, theymay not be pure (GCG) insertions as originally de-scribed for French-Canadians. Future studies aimed atidentifying PABP2 mutations in these non-French-Canadian families may provide additional support forthis recurrent mutation hypothesis.

ACKNOWLEDGMENTS

This work was supported in part by the AmericanHeart Association. We also thank the family memberswho participated.

REFERENCES

Barbeau A. 1966. The syndrome of hereditary late-onset ptosis and dys-phagia in French Canada. In Kuhn E, editor. Progressive Muskeldys-trophie, Myotonie, Myasthenie. Berlin: Springer-Verlag. p 102–109.

Barbeau A, Roy M, Sadibelouiz M, Wilensky MA. 1984. Recessive ataxia inAcadians and “Cajuns.” Can J Neurol Sci 11:526–533.

Bouchard JP. 1997. AndreBarbeau and the oculopharyngeal muscular dys-trophy in French Canada and North America. Neuromuscul Disord7(suppl):S5–S11.

Brais B, Bouchard JP, Gosselin F, Xie YG, Fardeau M, Tome FMS, RouleauG. 1997. Using the full power of linkage analysis in 11 French Cana-dian families to fine map the oculopharyngeal muscular dystrophygene. Neuromuscul Disord 7 Suppl 1:S70–S74.

Brais B, Bouchard JP, Xie YG, Rochefort DL, Chretien N, Tome FM, Lafre-niere RG, Rommens JM, Uyama E, Nohira O, Blumen S, Korczyn AD,Heutink P, Mathieu J, Duranceau A, Codere F, Fardeau M, RouleauGA. 1998. Short GCG expansions in the PABP2 gene cause oculopha-ryngeal muscular dystrophy. Nat Genet 18:164–167.

Brais B, Xie YG, Sanson M, Morgan K, Weissenbach J, Korczyn AD, Blu-men SC, Fardeau M, Tome FMS, Bouchard JP, Rouleau GA. 1995. Theoculopharyngeal muscular dystrophy locus maps to the region of thecardiac alpha and beta heavy chain genes on chromosome 14q11.2-q13.Hum Mol Gen 4:429–434.

Creel GB, Giuliani MJ, Lacomis D, Holbach SM. 1998. Oculopharyngealmuscular dystrophy: non-French-Canadian pedigrees. Muscle Nerve21:816–818.

Dib C, Faure S, Fizames C, Samson D, Drouot N, Vignal A, Millasseau P,Marc S, Hazan J, Seboun E, Lathrop M, Gyapay G, Morissette J, Weis-senbach J. 1996 A comprehensive genetic map of the human genomebased on 5,264 microsatellites. Nature 380:152–154.

Fradet G, Pouliot D, Lavoie S, FRCP, St-Pierre S. 1988. Inferior constrictormyotomy in oculopharyngeal muscular dystrophy: clinical and mano-metric evaluation. Jour Otolar 17:68–73.

Grewal RP, Cantor R, Turner G, Grewal RK, Detera-Wadleigh SD. 1998.Genetic mapping and haplotype analysis of oculopharyngeal musculardystrophy. Neuroreport 20:961–965.

Porschke H, Kress W, Reichmann H, Goebel HH, Grimm T. 1997. Oculo-pharyngeal muscular dystrophy in a northern German family linked tochromosome 14q, and presenting carnitine deficiency. NeuromusculDisord 7 Suppl 1:S57–S62.

Schmitt HP, Krause KH. 1981. An autopsy study of a familial oculopha-ryngeal muscular dystrophy (OPMD) with distal spread and neuro-genic involvement. Muscle Nerve 4:296–305.

Stajich LM, Gilchrist JM, Lennon F, Lee A, Yamaoka L, Rosi B, GaskellPC, Pritchard M, Donald L, Roses AD, Vance JM, Pericak-Vance MA.1997. Confirmation of linkage of oculopharyngeal muscular dystrophyto chromosome 14q11.2-q13 in American families suggests the exis-tence of a second causal mutation. Neurmuscul Disord 7 Suppl 1:S75–S81.

Tome FMS, Fardeau M. 1980. Nuclear incusions in oculopharyngeal dys-trophy. Acta Neuropathol 49:85–87.

Tome FMS, Fardeau M. 1994. Oculopharyngeal muscular dystrophy. InEngel AG, Franzini-Armstrong C, editors. Myology. New York: Mc-Graw-Hill. p 1233–1245.

Tremblay-Tymczuk S, Mathieu J, Morgan K, Bouchard JP, DeBraekeleerM. 1992. Genealogical study of oculopharyngeal dystrophy atSaguenay-Lac-St-Jean, Quebec, Canada. Rev Neurol 148:601–604.

Unique PABP2 Mutations in “Cajuns” 481