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Lafora Progressive Myoclonus Epilepsy: Narrowing the Chromosome 6q24 Locus by Recombinations and Homozygosities

1997; Elsevier BV; Volume: 61; Issue: 5 Linguagem: Inglês

10.1086/301596

1537-6605

Jesús Sainz, Berge A. Minassian, José M. Serratosa, Manyee Gee, Lise M. Sakamoto, R. Iranmanesh, Saeed Bohlega, Robert J. Baumann, Steve Ryan, Robert S. Sparkes, Antonio V. Delgado‐Escueta,

Genetics and Neurodevelopmental Disorders

To the Editor: Lafora disease (LD) is an autosomal recessive and rare but fatal epilepsy syndrome characterized by stimuli-sensitive myoclonus, absence and grand mal seizures, progressive intellectual and neurological deterioration, and periodic acid Schiff (PAS) stain–positive intracellular inclusion bodies. Eighty-four years after Gonzalo Lafora (Lafora, 1911aLafora GR The presence of amyloid bodies in the protoplasm of the ganglion cells: a contribution to the study of the amyloid substance in the nervous system.Bull Gov Hosp Insane. 1911a; 3: 83-92Google Scholar, Lafora, 1911bLafora GR Über das Vorkommen amyloider Körperchen im Innern der Ganglienzellen: zugleich Ein zum Studium der amyloiden Substanz im Nervensystem.Virchows Arch A Pathol Anat Histopathol. 1911b; 205: 295-303Crossref Scopus (91) Google Scholar) first described such PAS-positive “intracellular amyloid bodies” in the CNS of a young adult who died from a progressive myoclonus epilepsy, we encountered extended areas of homozygosities in chromosome 6q23-25 in nine LD patients who were products of consanguineous marriages (families LD1, LD4, LD5, and LD9). We also detected significant linkage to chromosome 6q23-25 microsatellites in one large inbred family, LD9, and thus localized the LD gene to a 17-cM interval on chromosome 6q23-25, between D6S292 and D6S420 (Serratosa et al., 1995Serratosa JM Delgado-Escueta AV Posada I Shih S Drury I Berciano J Zabala JA et al.The gene for progressive myoclonus epilepsy of the Lafora type maps to chromosome 6q.Hum Mol Genet. 1995; 4: 1657-1663Crossref PubMed Scopus (100) Google Scholar). To reduce the size of the 17-cM candidate region, we have studied an expanded series of 39 biopsy-proved LD patients who belong to 26 unrelated families (12 inbred) from Spain, Canada, France, the United States, Palestine, Iran, Ecuador, and Saudi Arabia. We provide further proof for significant linkage of LD to chromosome 6q24 in a second and new large inbred family (LD33). Homozygosities and recombinations in six new informative families reduce the size of the previously reported 17-cM LD interval to 2.7 cM flanked centromerically by D6S1003 and telomerically by D6S311. The clinical diagnosis of LD was initially established by the referring physician and was corroborated by the senior epileptologist in this study. PAS-positive inclusion bodies were demonstrated in skin and/or muscle and in liver and/or brain biopsies of all affected family members, including affected individuals carrying recombinant chromosomes. High-molecular-weight DNA was extracted either from 10 ml of venous blood from living family members, by use of phenol/chloroform followed by isopropanol precipitation (Sambrook et al., 1989Sambrook J Fritsch EF Maniatis T Molecular cloning: a laboratory manual. 2d ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar), or from 200 μl of peripheral blood by use of the QUIAamp blood kit (Qiagen). DNA from deceased family members (LD9-10, LD9-12, LD9-16, LD18-3, and LD19-3) was extracted from paraffin-embedded archived autopsy specimens of liver, brain, and muscle (Jackson et al., 1990Jackson D Lewis F Taylor G Boylston A Quirke P Tissue extraction of DNA and RNA analyzed by the polymerase chain.J Clin Pathol. 1990; 43: 499-504Crossref PubMed Scopus (227) Google Scholar; Greer et al., 1991Greer C Petersen S Kiviat N Manos M PCR amplification from paraffin-embedded tissues.Anat Pathol. 1991; 95: 117-124Google Scholar). All primers for amplification were obtained from Research Genetics. The method of Weber and May, 1989Weber JL May PE Abundant class of human DNA polymorphisms which can be typed using the polymerase chain reaction.Am J Hum Genet. 1989; 44: 388-396PubMed Google Scholar was used to type highly polymorphic short tandem repeats or microsatellites (heterozygosity >.7) in 50 parents and in 39 affected and 56 unaffected individuals. Parametric or model-dependent two-point linkage analyses using LINKAGE 5.1 (Ott, 1974Ott J Estimation of the recombination fraction in human pedigrees: efficient computation of the likelihood for human linkage studies.Am J Hum Genet. 1974; 26: 588-597PubMed Google Scholar) were performed in eight multiplex families (LD3, LD4, LD6, LD9, LD12, LD27, LD28, and LD33) and in five simplex consanguineous families (LD1, LD5, LD7, LD22, and LD25). We estimated the frequency of the disease allele to be .001, and penetrance was set at 100%, assuming an autosomal recessive model. The gene mutation rate was set at 0. We calculated LOD scores at recombination fractions (θm=f). We performed multipoint linkage analyses in family LD33, using a new software package, GENEHUNTER (Kruglyak et al., 1996Kruglyak L Daly MJ Reeve-Daly MP Lander ES Parametric and nonparametric linkage analysis: a unified multipoint approach.Am J Hum Genet. 1996; 58: 1347-1363PubMed Google Scholar). We first looked for recombinations and homozygosities (Lander and Botstein, 1987Lander ES Botstein D Homozygosity mapping: a way to map human recessive traits with the DNA of inbred children.Science. 1987; 236: 1567-1570Crossref PubMed Scopus (660) Google Scholar) in families LD9 and LD33, because they provided independent proof for linkage to chromosome 6q24. We had previously published significant LOD scores obtained during two-point analyses in family LD9 (Serratosa et al., 1995Serratosa JM Delgado-Escueta AV Posada I Shih S Drury I Berciano J Zabala JA et al.The gene for progressive myoclonus epilepsy of the Lafora type maps to chromosome 6q.Hum Mol Genet. 1995; 4: 1657-1663Crossref PubMed Scopus (100) Google Scholar). We used the new generation of microsatellites in family LD9 but did not reduce the size of the 17-cM LD region, flanked centromerically by D6S292 and telomerically by D6S420, that we had reported in 1995. In family LD33, the LOD score for D6S1703 was 3.24 (θm=f = 0) during two-point analyses (Ott, 1974Ott J Estimation of the recombination fraction in human pedigrees: efficient computation of the likelihood for human linkage studies.Am J Hum Genet. 1974; 26: 588-597PubMed Google Scholar), exceeding the threshold for significance. We also computed 10-point LOD scores (Kruglyak et al., 1996Kruglyak L Daly MJ Reeve-Daly MP Lander ES Parametric and nonparametric linkage analysis: a unified multipoint approach.Am J Hum Genet. 1996; 58: 1347-1363PubMed Google Scholar) in family LD33, against a fixed genetic map with nine markers (D6S308, D6S409, D6S1003, D6S1010, D6S1703, D6S1042, D6S311, D6S978, and D6S420) in an 11-cM region surrounding the LD gene. During multipoint analysis, we obtained maximum location scores of 4.03 for markers D6S1010, D6S1703, and D6S1042, which are situated between D6S1003 and D6S311. Recombinations and homozygosities in LD33 were consistent with results of two-point and multipoint analyses and reduced the size of the LD-gene region to the interval flanked by D6S1003 and D6S1687 (fig. 1). Homozygosities in all three living affected members (see haplotypes of LD33-3, LD33-5, and LD33-6; fig. 1) involved 20–27 microsatellites, covering 13–17 cM. These homozygosities indicated that the three affected individuals inherited two copies of the same mutation from a common ancestor—in this case, a grandmother—six generations earlier. A recombination between D6S1553 and D6S1687 in LD33-6 determined that the telomeric border of the LD region is D6S1687. In addition, a recombination centromeric to the LD locus, between D6S1003 and D6S1010, in individual LD33-3 further identified the centromeric border of the LD region, as being D6S1003. These two recombinations (see fig. 1, arrows) effectively reduced the critical LD interval, to ∼7 cM flanked centromerically by D6S1003 and telomerically by D6S1687. Our second level of analyses looked at families whose extended regions of homozygosities strongly supported the presence of an LD locus in chromosome 6q24, even though the small sizes of their families precluded LOD scores from reaching significance. Homozygosities in families LD20 and LD22 show the centromeric flanking marker to be D6S308 and D6S403, respectively (see fig. 1). Data on LD22 are not shown. These observations verify the general vicinity of the centromeric border of the LD region, since D6S403 and D6S308 are <2 cM from D6S1003. They lend support to the observation, in family LD33, of D6S1003 as the centromeric flanking marker. Three proofs support D6S311 as the telomeric border of the LD gene. First, homozygosities in family LD15 identify the telomeric border as D6S1553, and results for family LD16 cut the LD region further and identify D6S311 as the telomeric flanking marker (see fig. 1). Second, another family, LD17, has loss of homozygosity at the telomeric end in D6S311, but we were unable to genotype for the new generation of markers in the interval spanned by D6S1003 and D6S311, because of the minute amounts of DNA obtained from archived paraffin-embedded tissues. Although the genotypes for these new microsatellites are missing, the existing data support D6S311 as the telomeric flanking marker in family LD17 (data not shown). Third, a recombination between D6S311 and D6S978 in family LD15 (see fig. 1, arrows) provides further proof that D6S311 is the telomeric border of the disease gene. In summary, we reduced the size of the LD interval to 2.7 cM flanked by D6S1003 and D6S311, by (a) correlations between recombinations and homozygosities in a new large family (LD33), which, by itself, independently proved linkage to chromosome 6q24 microsatellites, (b) extended area of homozygosities in affected members of smaller families (LD15, LD16, LD17, LD20, and LD22), and (c) a recombination in family LD15. What kind of gene might be responsible for Lafora progressive myoclonus epilepsy? If the gene responsible for LD is involved in the degradation pathways of glycoprotein metabolism (Lafora, 1955Lafora GR Myoclonus: physiological and pathological considerations.in: In: Proceedings of the 2d International Congress of Neuropathology. Part 1. Excerpta Medica, Amsterdam1955: 9-21Google Scholar; Schwarz and Yanoff, 1965aSchwarz GA Yanoff M Lafora bodies, corpora amilacea, and Lewy bodies: a morphological and histochemical study.Arch Neurobiol (Madrid). 1965a; 28: 800-818Google Scholar, Schwarz and Yanoff, 1965Schwarz GA Yanoff M Lafora's disease: distinct clinico-pathologic form of Unverricht's syndrome.Arch Neurol. 1965; 12: 172-188Crossref PubMed Scopus (83) Google Scholar; Yokoi et al., 1968Yokoi S Austin J Witmer F Sakai M Studies in myoclonus epilepsy (Lafora body form). I. Isolation and preliminary characterization of Lafora bodies in two cases.Arch Neurol. 1968; 19: 15-33Crossref PubMed Scopus (110) Google Scholar; Sakai et al., 1970Sakai M Austin J Witmer F Trueb L Studies in myoclonus epilepsy (Lafora body form). II. Polyglucosans in the systemic deposits of myoclonus epilepsy and in corpora amylacea.Neurology. 1970; 20: 160-176Crossref PubMed Google Scholar; Gambetti et al., 1971Gambetti P Di Mauro S Hirt L Blume RP Myoclonic epilepsy with Lafora bodies.Arch Neurol. 1971; 25: 483-493Crossref PubMed Scopus (63) Google Scholar; Schwarz, 1977Schwarz GA Lafora's disease: a disorder of carbohydrate metabolism.in: Goldensohn ES Appel SH Scientific approaches to clinical neurology. Lea & Febiger, Philadelphia1977: 148-159Google Scholar; Federico et al., 1980Federico A D'Amore I Palladini G Medolago-Albani L Guazzi GC Tomaccini D Lafora's disease: clinical, histological ultrastructural and biochemical study.Acta Neurol. 1980; 2: 466-475PubMed Google Scholar), the alpha fucosidase-2 gene (FUCA2), located on chromosome 6q24, would be a candidate gene. FUCA2 is tightly linked to the protein marker, plasminogen (Eiberg et al., 1984Eiberg H Mohr J Nielsen LS Linkage of plasma alpha-L-fucosidase (FUCA2) and the plasminogen (PLG) system.Clin Genet. 1984; 26: 23-29Crossref PubMed Scopus (31) Google Scholar), which, in turn, is genetically linked to chromosome 6q (Murray et al., 1987Murray JC Buetow KH Donovan M Hornung S Motulsky AG Disteche C Dyer K et al.Linkage disequilibrium of plasminogen polymorphisms and assignment of the gene to human chromosome 6q26-6q27.Am J Hum Genet. 1987; 40: 338-350PubMed Google Scholar). A second candidate gene that maps to chromosome 6q22.3-q24 is that for L-isoaspartyl/D-aspartyl protein methyltransferase, or protein carboxyl methyltransferase 1 (PCMT1) (MacLaren et al., 1992MacLaren DC O'Connor CM Xia YR Mehrabian M Klisak I Sparkes RS Clarke S et al.The L-isoaspartyl/D-aspartyl protein methyltransferase gene (PCMT1) maps to human chromosome 6q22.3-6q24 and the syntenic region of mouse chromosome 10.Genomics. 1992; 14: 852-856Crossref PubMed Scopus (18) Google Scholar), which is involved in repair of proteins (Ota et al., 1988Ota IM Gilbert JM Clarke S Two major isozymes of the protein D-aspartyl/L-isoaspartyl methyltransferase from human erythrocytes.Biochem Biophys Res Commun. 1988; 151: 1136-1143Crossref PubMed Scopus (21) Google Scholar). PCMT1 catalyzes the transfer of a methyl group from S-adenosyl-L-methionine to the free carboxyl groups of D-aspartyl and L-isoaspartyl residues, which represent sites of covalent damage to aging proteins. LD may represent a disorder of protein repair, and the “intracellular amyloid inclusion bodies” could be evidence of impaired protein repair (Tsai and Clarke, 1994Tsai W Clarke S Amino acid polymorphisms of the human l-isoaspartyl/d-aspartyl methyltransferase involved in protein repair.Biochem Biophys Res Commun. 1994; 203: 491-497Crossref PubMed Scopus (15) Google Scholar). We thank the families whose members have carried the burden of LD; without their cooperation this study would not have been possible. We also gratefully acknowledge the cooperation and assistance of Joan Spellman, Bernadette Sakamoto, and Susan G. Pietsch-Escueta, who helped recruit families and coordinate family studies. Our study was approved by the Human Subjects Protection Committee at the UCLA School of Medicine and the West Los Angeles DVA Medical Center. Each participating patient or, in the case of minors or deceased relatives, the responsible adult, signed an informed-consent form. Our project was supported by NIH-NINDS program project 5PO1-NS21908 (to A.V.D.-E.), by special contributions from Mrs. A. Malenfant and the Quebec Lafora's Disease Organization, and by Mrs. Vera Faludi of Sweden.