 1997 Oxford University Press

Human Molecular Genetics, 1997, Vol. 6, No. 4

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Mapping of the familial infantile myasthenia (congenital myasthenic syndrome type Ia) gene to chromosome 17p with evidence of genetic homogeneity Kyproula Christodoulou, Marios Tsingis, Feza Deymeer1, Piraye Serdaroglu1, Coskun Ozdemir1, Ahmad Al-Shehab2, Chrysostomos Bairactaris3, Ioannis Mavromatis3, Ioannis Mylonas3, Amelia Evoli4, Kyriacos Kyriallis and Lefkos T. Middleton* The Cyprus Institute of Neurology and Genetics, 1683 Nicosia, Cyprus, 1Department of Neurology, Istanbul University, Istanbul, Turkey, 2Faculty of Medicine, University of Jordan, Amman, Jordan, 3B’Neurology Clinic, Aristotelion University, Thessaloniki, Greece and 4Institute of Neurology, Universita Cattolica del Sarco Cuore, Rome, Italy Received January 2, 1997; Revised and Accepted January 31, 1997

Familial infantile myasthenia is an autosomal recessive disorder, recently classified as congenital myasthenic syndrome type Ia. Onset of symptoms is at birth to early childhood with significant myasthenic weakness and possible respiratory distress, followed later in life by symptoms of mild to moderate myasthenia. Thirty-six patients of 12 families, seven of them consanguineous, were used to map the familial infantile myasthenia gene. A combination of linkage search through the genome, DNA pooling and homozygosity mapping were employed resulting in the localisation of this disease locus to the telomeric region of chromosome 17p. A maximum lod score of 9.28 at θ = 0.034 was obtained between the disease locus and marker locus D17S1537. Haplotype analysis showed all families to be consistent with linkage to this region thus providing evidence for genetic homogeneity of familial infantile myasthenia. Multipoint linkage analysis mapped the disease gene in the ∼4.0 cM interval between marker loci D17S1537 and D17S1298 with a maximum multipoint lod score of 12.07. Haplotype analysis and homozygosity by descent in affected individuals of the consanguineous families revealed results in agreement with the confinement of the familial infantile myasthenia region within the interval between marker loci D17S1537 and D17S1298. INTRODUCTION Congenital myasthenia syndromes (CMS) form a group of inherited congenital disorders affecting the neuromuscular junction. CMS have recently been classified, based on their genetic *To whom correspondence should be addressed

and clinical features, into types Ia familial infantile myasthenia, Ib limb girdle myasthenia, Ic acetylcholine esterase deficiency, Id acetylcholine receptor deficiency; type II is the autosomal dominant ‘classic slow channel syndrome’; and type III groups sporadic cases with no family history, excluding myasthenia gravis (1). Familial infantile myasthenia (FIM) is an autosomal recessive disorder, initially identified by Greer and Schotland in 1960 (2). The phenotype was further delineated on clinical observations of additional patients (3–7). Diagnostic criteria (1) include an onset at birth to early childhood, with fluctuating ptosis, poor cry and suck, feeding difficulties and possible respiratory distress. In childhood, patients present with mild to moderate myasthenia, variable ptosis and ophthalmoparesis, with occasional episodic exacerbations which may result in respiratory distress and apnea. Later in life, symptoms are less pronounced, with mild to moderate fatiguable weakness of ocular, facial and less frequently bulbar and limb muscles. Patients are improved by anti-cholinesterase medication but not by thymectomy, plasma exchange or immuno-suppressive treatment. A decremental response at 2–3 Hz stimulation in repetitive studies of affected muscles is noted and anti-AChR antibodies are absent. A presynaptic defect of acetylcholine resynthesis or mobilisation was proposed by in vitro electrophysiological and morphological studies in patients with FIM (8,9) and an abnormality of synaptic vesicle metabolism was incriminated (9). We hereby present molecular genetic studies on the largest reported series of patients from the Mediterranean region, conforming to the diagnostic criteria of FIM (1). Using linkage search through the genome, DNA pooling and homozygosity mapping, we mapped the FIM gene to the telomeric region of chromosome 17p. All families originating from different countries and ethnic backgrounds linked to this region, suggesting genetic homogeneity of the disorder. Haplotype analysis and homozygosity by descent in affected individuals of consanguineous families enabled localisation of the FIM gene within an ∼4.0 cM interval. A candidate gene previously located at the

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telomeric region of chromosome 17p (10) is currently analysed for mutations in our series of patients. RESULTS We analysed 101 individuals from 12 FIM families, 36 of whom are affected, with microsatellite polymorphic marker loci spanning the genome at 30 cM distances (Research Genetics Inc., screening set 6a). Linkage search through the genome enabled the identification of a number of non-excluded regions with slightly positive lod scores. Further analysis of these regions with additional microsatellite polymorphic marker loci resulted in their subsequent exclusion. More than 70% of the genome was excluded but there was still a number of non-excluded gaps. We then employed homozygosity mapping using DNA pooled from all affected individuals from each family separately. We analysed these DNA pools with additional microsatellite poly-

morphic marker loci filling in the non-excluded gaps at 10 cM distances. We identified five regions for which most of the families had homozygous affected individuals. These regions were further investigated by linkage analysis in all families and resulted in the exclusion of the four and identification of linkage for the fifth region. A lod score of 6.48 at θ = 0.054 was obtained between the disease locus and marker locus D17S1298. Five additional microsatellite polymorphic marker loci from the region were then analysed (D17S578, D17S849, D17S926, D17S938 and D17S1537) and a maximum lod score of 9.28 was obtained at θ = 0.034 between the disease locus and marker locus D17S1537. Representative two-point linkage analysis results are shown in Tables 1 and 2. Analysis of the two-point data with the HOMOG program showed no significant evidence of heterogeneity (χ2 = 1.97, p = 0.16, likelihood ratio = 2.67 for marker locus D17S1298 and, χ2 = 0.00, likelihood ratio = 1 for the remaining marker loci).

Table 1. Two-point lod scores obtained between the disease and six microsatellite polymorphic marker loci from the telomeric region of chromosome 17p

Locus

θ values 0.00

0.01

0.05

0.1

0.2

0.3

0.4

D17S926 D17S849 D17S1298 D17S1537 D17S578 D17S938

–∞ –∞ –∞ –∞ –∞ –∞

1.78 -5.16 5.25 8.67 6.79 6.36

4.48 0.85 6.47 9.18 7.97 7.63

4.83 2.53 6.12 8.23 7.30 7.05

3.87 2.77 4.46 5.61 4.92 4.90

2.40 1.87 2.64 3.05 2.55 2.68

1.01 0.81 1.11 1.07 0.82 0.94

Zmax

θmax

4.84 2.91 6.84 9.28 7.98 7.63

0.091 0.156 0.054 0.034 0.046 0.048

The sum of lod scores of the twelve FIM families is presented. Table 2. Two-point lod scores between the disease and microsatellite polymorphic marker loci D17S1298 and D17S1357 θ values Locus

Fam #

0.00

0.01

0.05

0.1

0.2

0.3

0.4

D17S1298

3201 3202 3203 3204 3205 3206 3207 3208 3209 3210 3211 3213 3201 3202 3203 3204 3205 3206 3207 3208 3209 3210 3211 3213

0.89 –∞ 0.02 0.00 –∞ 0.30 2.18 0.60 0.03 –∞ 2.19 3.00 1.47 0.43 1.86 0.16 1.33 0.60 0.86 0.63 0.60 1.16 –∞ –0.10

0.86 –1.57 0.11 0.00 –1.21 0.29 2.13 0.59 0.03 –1.04 2.13 2.93 1.42 0.42 1.81 0.15 1.29 0.58 0.83 0.62 0.58 1.13 –0.57 0.41

0.73 –0.86 0.24 0.00 –0.57 0.26 1.96 0.52 0.02 –0.41 1.91 2.67 1.25 0.38 1.59 0.11 1.15 0.52 0.72 0.54 0.51 1.03 0.58 0.80

0.57 –0.55 0.24 0.00 –0.33 0.21 1.73 0.43 0.02 –0.19 1.64 2.33 1.02 0.33 1.33 0.08 0.98 0.43 0.59 0.46 0.42 0.88 0.87 0.84

0.30 –0.25 0.12 0.00 –0.13 0.13 1.27 0.28 0.01 –0.04 1.13 1.64 0.61 0.22 0.84 0.04 0.64 0.27 0.35 0.30 0.26 0.59 0.85 0.64

0.11 –0.10 0.03 0.00 –0.05 0.06 0.80 0.15 0.00 –0.01 0.68 0.95 0.29 0.12 0.42 0.02 0.35 0.13 0.17 0.16 0.12 0.31 0.61 0.35

0.02 –0.02 0.00 0.00 –0.01 0.02 0.36 0.06 0.00 –0.00 0.31 0.38 0.08 0.03 0.12 0.01 0.13 0.03 0.06 0.06 0.03 0.09 0.32 0.10

D17S1537

Lod score values for each family are presented.

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Figure 1. Linkage map of the chromosome 17p region of interest showing the estimated distances between loci in cM.

Seven CEPH-type families from our DNA bank were analysed, in order to establish the linkage map of these marker loci. The resulting linkage map of the region is shown in Figure 1; the order of microsatellite polymorphic marker loci being: tel-D17S926D17S849-D17S1298-D17S1537-D17S578-D17S938-cen. We used this linkage map to perform multipoint linkage analysis. The disease locus was mapped against the fixed map of loci D17S1298-(4 cM)-D17S1537-(5 cM)-D17S578. A maximum multipoint lod score of 12.07 was obtained in the interval between marker loci D17S1298 and D17S1537. The multipoint lod score results are shown in Figure 2. We constructed the haplotypes of individuals in these families following the order in which the above marker loci were mapped on chromosome 17p. Haplotype analysis showed all families to be consistent with linkage to this region thus providing evidence for genetic homogeneity of FIM. Identification of recombination events and homozygosity by descent in two consanguineous families enabled restriction of the FIM region within a specific interval. In family 3213 (Fig. 3), affected individuals (V3, V4 and V6) share one common haplotype (6–5–2–3–4–4, for marker loci D17S926-D17S849-D17S1298-D17S1537-D17S578 - D17S93, respectively). Individuals V3 and V4 have inherited the above haplotype from their father (IV2). The haplotype they inherited from their mother (IV1) is the same from D17S926 to D17S1537 (6–5–2–3), but differs for D17S578, thus indicating a past generation recombination event between marker loci D17S1537 and D17S578. Individual V6 most probably inherited the 6–5–2–3–4–4 haplotype from his mother (IV3) and a 6–5–2–4–9–10 haplotype from his father (IV4). A past generation recombination event has probably occurred between marker loci D17S1298 and D17S1537. Haplotype analysis and homozygosity by descent results of family 3213 indicate that the FIM gene is most likely located distal to marker locus D17S1537. Similarly, in family 3203 (Fig. 3) the three affected individuals (IV3, IV4 and IV6) are homozygous for the D17S1537 to D17S938 haplotype, thus indicating that the FIM gene most likely lies

Figure 2. Multipoint linkage analysis results. The multipoint lod score of the disease locus is plotted against the fixed map of loci D17S1298, D17S1537 and D17S578.

proximal to marker locus D17S1298. The above results of families 3213 and 3203 enable restriction of the FIM gene region within the interval between marker loci D17S1298 and D17S1537 which has been estimated to be ∼4.0 cM. This result is in agreement with our multipoint linkage analysis results. DISCUSSION We mapped the locus for FIM at the telomeric region of chromosome 17p using a combination of linkage search through the genome, DNA pooling and homozygosity mapping. A maximum two-point lod score of 9.28 at θ = 0.034 was obtained between the disease and microsatellite polymorphic marker locus D17S1537. A maximum multipoint lod score of 12.07 within the 4.0 cM interval between loci D17S1298 and D17S1537 was obtained. Haplotype and homozygosity by descent analyses revealed recombination events that restrict the disease locus within the 4.0 cM interval between marker loci D17S1298 and D17S1537, in agreement with the multipoint linkage analysis results. The families included in this study represent the largest reported series of FIM. These were clinically evaluated and all conformed with the recently reported diagnostic criteria (1). Genetic heterogeneity within this group of FIM families could not be ruled out, thus, in our linkage analysis and homozygosity mapping on DNA pools, families were studied both as a group as well as individually. Despite their ethnic diversity all families linked to at least one of the marker loci in the region indicating genetic homogeneity within FIM, in contrast to the genetic heterogeneity observed in another congenital myasthenic syndrome, the slow-channel syndrome (CMS 2a) (11). In patients with this autosomal dominant type of CMS, pathogenic mutations have been identified in three (α, β, ε) of the four subunits of the

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Figure 3. Haplotype analysis in families 3213 and 3203. Affected individuals are designated by black symbols. The disease-bearing chromosomes are shown in boxes. The homozygous regions are shown as hatched areas.

acetylcholine receptor encoded by different genes on different chromosomes (11–14). Based on the genetic homogeneity demonstrated in our FIM families, we may suggest that mutations within a single gene are most likely involved in the pathogenesis of familial infantile myasthenia.

In vitro neurophysiological studies on external intercostal muscles of FIM patients showed findings similar to the effects of hemicholinium, an inhibitor of choline uptake by the nerve terminal (15). After prolonged stimulation at 10 Hz, the amplitude of the end plate potentials (EPP) was reduced due to an

639 Human Genetics, 1997, 6, No. NucleicMolecular Acids Research, 1994, Vol. Vol. 22, No. 1 4 abnormal decrease of amplitude of the miniature end plate potentials (MEPP), initially normal in rested muscle (8,9). Thus, a presynaptic defect of acetylcholine resynthesis, packaging or mobilisation was proposed (8,9,16). Morphological studies in three patients with FIM (9) showed no abnormalities of the neuromuscular junction; no histological or ultrastructural evidence for AChR deficiency was noted. In rested muscles of the patients, the synaptic vesicles were significantly smaller compared to rested control muscles. After 10 Hz stimulation, when the MEPP amplitude was significantly reduced in the patients, their synaptic vesicles increased or did not change in size, whereas in controls, the synaptic vesicles decreased or remained unchanged. A defect in the synaptic vesicle metabolism was incriminated (9). Synaptobrevin-2 (syb-2), a synaptic vesicle protein, is encoded by a gene that has been mapped to the telomeric region of chromosome 17p (10). We consider syb-2 a candidate gene to be involved in the pathogenesis of FIM. Synaptobrevin is a synaptic vesicle protein that probably participates in neurotransmitter release at a step between docking and fusion (17). Syb-2 forms a stable complex with syntaxin, synaptosomal-associated protein of 25 kDa (SNAP-25) and synaptotagmin that probably functions in synaptic vesicle exocytosis (18,19). Syb-2 also forms a distinct complex with synaptophysin (19,20). In addition, it has been reported that tetanus and botulinum neurotoxins types B, D, F and G block neurotransmitter release by cleaving synaptobrevin-2 at specific peptide bonds (20,21). The syb-2 gene is organised in five exons spanning a 3 kb region and encoding for 116 amino acids (10). We are in the process of screening our FIM patients for mutations in the syb-2 gene, a candidate gene that was previously mapped at the same chromosomal region that we mapped the FIM gene. Other genes that have been mapped at the telomeric region of chromosome 17p include pigment epithelium-derived factor, aspartoacylase, muscle specific beta-enolase, glycoprotein Ib, alpha-2-plasmin inhibitor, zinc finger protein-3, active BCRrelated gene, breast cancer-related regulator of TP53, avian sarcoma virus CT10 oncogene homolog, a cluster of 16 olfactory receptor genes, profilin-1, phosphatidylinositol transfer protein, retinitis pigmentosa-13 and replication protein A1. The position of the above genes relative to the ∼4 cM region of interest is unknown. MATERIALS AND METHODS Clinical data Twelve FIM pedigrees with two or more affected individuals conforming with the diagnostic criteria of FIM were included in the study. Seven families originate from Turkey, three from Greece, one from Jordan and one from Italy. The three Greek and one Turkish families are of Gypsy origin. Six of the seven Turkish and the Jordanian families are consanguineous. One hundred and one individuals from these families were included in the study and were clinically evaluated by at least two of us. Thirty-six individuals conformed with the diagnostic criteria of FIM (1), 21 male and 15 female, aged 6 to 46 years (mean 19.5). Linkage search One hundred and one individuals from the 12 FIM families were typed for microsatellite polymorphic marker loci spanning the human genome at on average 30 cM distances (Research Genetics

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Inc., screening set 6a). Genotyping was performed as described by Christodoulou et al. (22). DNA pooling DNA samples from affected individuals of each family were mixed together at equal concentrations, resulting in 12 DNA pools. These DNA pools were analysed with microsatellite polymorphic marker loci at on average 10 cM distances at selected regions of the genome that were not excluded by our initial linkage search analysis. Typing was performed as described by Christodoulou et al. (22). The films were screened manually looking for those marker loci for which a single band was obtained for all or at least most of the pooled DNA samples. Haplotype analysis Haplotypes of individuals were constructed following the order in which marker loci were mapped at the chromosome 17p region of interest (tel-D17S926-D17S849-D17S1298-D17S1537-D17S578D17S938-cen). Homozygosity by descent enabled the identification of past generation recombination events. These and other recombination events that were also observed were used to flank the FIM region on both sides. Linkage analysis Two-point and multipoint linkage analyses were performed using the LINKAGE package of programs (23). Allele frequencies were estimated using the individuals married in the families. Equal male and female recombination fractions were assumed. A disease gene frequency of 10–3 was used. Families were tested for genetic heterogeneity with the HOMOG program (24), using the two-point lod scores for each family at marker loci D17S926, D17S849, D17S1298, D17S1537, D17S578 and D17S938. ACKNOWLEDGEMENTS This work was supported by a grant from the Muscular Dystrophy Association (USA) to L.T.M. We thank Mrs S. Demetriades, Mrs E. Polycarpou and Mrs E. Kyriacou for technical and secretarial assistance. REFERENCES 1. Middleton, L.T. (1996) Report of the 34th ENMC International Workshop Congenital Myasthenic Syndromes. Neuromusc. Disord., 6, 133–136. 2. Greer, M., Schotland, M. (1960) Myasthenia Gravis in the newborn. Paediatrics, 26, 101–108. 3. Conomy, J.P., Levisohn, M., Fanaroff, A. (1975) Familial infantile myasthenia gravis: A cause of sudden death in young children. J. Pediatr., 87, 428–429. 4. Fenichel, G.M. (1978) Clinical syndromes of myasthenia in infancy and childhood. Arch. Neurol., 35, 97–103. 5. Robertson, W.C., Chun, R.W.M., Kornguth, S.E. (1980) Familial infantile myasthenia. Arch. Neurol., 37, 117–119. 6. Seybold, M.E., Lindstrom, J.M. (1981) Myasthenia gravis in infancy. Neurology, 31, 476–480. 7. Gieron, M.A., Korthals, J.K. (1985) Familial infantile myasthenia gravis. Report of three cases with follow-up into adult life. Arch. Neurol., 42, 143–144. 8. Hart, Z.H., Sahashi, K., Lambert, E.H, Engel, A.G. (1979) A congenital familial myasthenic syndrome caused by a presynaptic defect of transmitter resynthesis or mobilisation. Neurology, 29, 556–557 (abstract). 9. Mora, M., Lambert, E.H., Engel, A.G. (1987) Synaptic vesicle abnormality in familial infantile myasthenia. Neurology, 37, 206–214.

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10. Archer, B.T., Ozcelik, T., Jahn, R., Franke, U., Sudhof, T.C. (1990) Structures and chromosomal localizations of two human genes encoding synaptobrevins 1 and 2. J. Biol. Chem. 265, 17267–17273. 11. Engel, A.G., Ohno, K., Milone, M., Wang, H., Nakano, S., Bouzat, C., Ned Pruitt II, J., Hutchinson, D.O., Brengman, J.M., Bren, N., Sieb, J.P., Sine, S.M. (1996) New mutations in acetylcholine receptor subunit genes reveal heterogeneity in the slow-channel congenital myasthenic syndrome. Hum. Mol. Genet., 5, 1217–1227. 12. Ohno, K., Hutchinson, D.O., Milone, M., Brengman, J.M., Bouzat, C., Sine, S.M., Engel, A.G. (1995) Congenital myasthenic syndrome caused by prolonged acetylcholine receptor channel openings due to a mutation in the M2 domain of the ε-subunit. Proc. Natl. Acad. Sci. USA, 92, 758–762. 13. Gomez, C.M., Gammack J.T. (1995) A leucine-to-phenylalanine substitution in the acetylcholine receptor ion channel in a family with the slow-channel syndrome. Neurology, 45, 982–985. 14. Gomez, C.M., Maselli, R., Gammack, J., Lasalde, J., Tamamizu, S., Cornblath, D.R., Lehar, M., McNamee, M., Kuncl, R.W. (1996) A β-subunit mutation in the acetylcholine receptor channel gate causes severe slow-channel syndrome. Ann. Neurol., 39, 712–723. 15. Elmqvist, D., Quastel, D.M.J. (1965) Presynaptic action of hemicholinium at the neuromuscular junction. J. Physiol. (Lond.), 177, 463–482. 16. Engel, A.G. (1994) Congenital myasthenic syndromes. In Engel, A.G., Erduzini-Armstrong C. (ed.), Myology. McGraw-Hill, New York, Vol 1, pp. 1806–1835.

17. Hunt, J.M., Bommert, K., Charlton, M.P., Kistner, A., Habermann, E., Augustine, E., Betz, H. (1994) A post-docking role for synaptobrevin in synaptic vesicle fusion. Neuron, 12, 1269–1279. 18. Hayashi, T., McMahon, H., Yamasaki, S., Binz, T., Hata, Y., Sudhof, T.C., Niemann, H. (1994) Synaptic vesicle membrane fusion complex: action of clostridial neurotoxins on assembly. EMBO J., 13, 5051–5061. 19. El Far, O., Charvin, N., Leveque, C., Martin-Moutot, N., Takahashi, M., Seagar, M.J. (1995) Interaction of a synaptobrevin (VAMP)- syntaxin complex with presynaptic calcium channels. FEBS Lett., 361, 101–105. 20. Washbourne, P., Schiavo, G., Montecucco, C. (1995) Vesicle-associated membrane protein-2 (synaptobrevin-2) forms a complex with synaptophysin. Biochem. J., 305, 721–724. 21. Schiavo, G., Benfenati, F., Poulain, B., Rossetto, O., Polverino de Laureto, P., DasGupta, B.R., Montecucco, C. (1992) Tetanus and botulinum-B neurotoxins block neurotransmitter release by proteolytic cleavage of synaptobrevin. Nature, 359, 832–835. 22. Christodoulou, K., Kyriakides, T., Hristova, A.H., Georgiou, D.M., Kalaydjieva, L., Yshpekova, B., Ivanova, T., Weber, J.L., Middleton, L.T. (1995) Mapping of a distal form of spinal muscular atrophy with upper limb predominance to chromosome 7p. Hum. Mol. Genet., 4, 1629–1632. 23. Lathrop, G.M., Lalouel, J.M., Julier, C., Ott, J. (1985) Multilocus linkage analysis in humans: detection of linkage and estimation of recombination. Am. J. Hum. Genet., 37, 482–498. 24. Terwilliger, J.D., Ott, J. (1994) Handbook of human genetic linkage. Johns Hopkins University Press, Baltimore, pp 235–242.