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Proteolipoprotein Gene Analysis in 82 Patients with Sporadic Pelizaeus

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Proteolipoprotein Gene Analysis in 82 Patients with Sporadic Pelizaeus Am. J. Hum. Genet. 65:360±369, 1999 Proteolipoprotein Gene Analysis in 82 Patients with Sporadic Pelizaeus-Merzbacher Disease: Duplications, the Major Cause of the Disease, Originate More Frequently in Male Germ Cells, but Point Mutations Do Not Corinne Mimault,1 GenevieÁve Giraud,1 Virginie Courtois,1 Fabrice Cailloux,1 Jean Yves Boire,2 Bernard Dastugue,1 Odile Boesp¯ug-Tanguy,1 and the Clinical European Network on Brain Dysmyelinating Disease* 1INSERM U.384, FaculteÂdeMeÂdecine and 2ERIM, FaculteÂdeMeÂdecine, Clermont-Ferrand, France Summary Introduction Pelizaeus-Merzbacher Disease (PMD) is an X-linked de- Pelizaeus-Merzbacher disease (PMD; MIM 312080), is velopmental defect of myelination affecting the central an X-linked defect of myelin formation affecting the nervous system and segregating with the proteolipopro- CNS. Originally described by Pelizaeus (1885), the clin- tein (PLP) locus. Investigating 82 strictly selected spo- ical syndrome was neuropathologically de®ned by Merz- radic cases of PMD, we found PLP mutations in 77%; bacher (1910) as a diffuse hypomyelination of the CNS, complete PLP-gene duplications were the most frequent associated with an abnormally low number of mature abnormality (62%), whereas point mutations in coding oligodendrocytes. The diagnosis is based on early/im- or splice-site regions of the gene were involved less fre- paired motor development (during the ®rst 3 mo of life) quently (38%). We analyzed the maternal status of 56 characterized by severe hypotonia associated with nys- cases to determine the origin of both types of PLP mu- tagmus and, later, the development of abnormal move- tation, since this is relevant to genetic counseling. In the ments and progressive spastic paraplegia. The severity 22 point mutations, 68% of mothers were heterozygous of the disease correlates with the level of motor per- for the mutation, a value identical to the two-thirds of formance acquired between 5 and 10 years of age; in- carrier mothers that would be expected if there were an tellectual development is generally less affected (Boesp- equal mutation rate in male and female germ cells. In ¯ug-Tanguy et al. 1996). Conduction velocity analysis sharp contrast, among the 34 duplicated cases, 91% of and magnetic resonance imaging (MRI) of the brain con- mothers were carriers, a value signi®cantly (x 2 5 9.20 , ®rm that the dysmyelinating process is limited to the P ! .01) in favor of a male bias, with an estimation of CNS (Boulloche et al. 1986). the male/female mutation frequency (k) of 9.3. More- Several lines of evidence led to the identi®cation of over, we observed the occurrence of de novo mutations the proteolipoprotein gene (PLP) as the causative locus between parental and grandparental generations in 17 in PMD. This gene maps to the long arm of the human three-generation families, which allowed a direct esti- X chromosome at Xq21-q22 (Mattei et al. 1986) and 5 mation of the k value (k 11 ). Again, a signi®cant male encodes the two major myelin proteins of the CNS, the mutation imbalance was observed only for the dupli- proteolipoprotein (PLP) and its isoform, DM20. Several cations. The mechanism responsible for this strong male point mutations in the coding and noncoding regions of bias in the duplications may involve an unequal sister the PLP gene (for references, see Nave et al. 1996) have chromatid exchange, since two deletion events, respon- been associated with the PMD phenotype but sible for mild clinical manifestations, have been reported account for only 10%±25% of the families analyzed. in PLP-related diseases. However, linkage analysis established apparent genetic homogeneity of families affected by PMD (Boesp¯ug- Received July 9, 1997; accepted for publication June 21, 1999; elec- tronically published July 15, 1999. Tanguy et al. 1994). In fact, in most families in which Address for correspondence and reprints: Dr. Odile Boesp¯ug- point mutations were not found, duplications of the en- Tanguy, INSERM U.384ÐFaculteÂdeMeÂdecine, 28 place Henri Du- tire PLP gene are the causative mutation (Ellis et al. nant, BP 38, 63001 Clermont-Ferrand Cedex, France. E-mail: Odile 1994; Inoue et al. 1996; Boesp¯ug-Tanguy et al. 1997; .boesp¯[email protected] ¤ Sistermans et al. 1998). Members' names are listed in the Acknowledgments. q 1999 by The American Society of Human Genetics. All rights reserved. Investigating 82 patients with sporadic PMD, we 0002-9297/99/6502-0012$02.00 found a PLP mutation in 63 (77%). Complete PLP-gene 360 Mimault et al.: Origin of PLP Mutations 361 duplication was the most frequent abnormality (62% of reaction and the DNA Dye Terminator Cycle Sequencing all mutations), and coding or splice-site point mutations Kit (Applied Biosystems). Sequence analysis was per- were found in 38% of all mutations. To determine the formed with an Applied Biosystems model 377 DNA origin of both types of PLP mutation, we analyzed the Sequencer. maternal status in 56 cases. In addition, in the 17 three- generation families in which the mother was a carrier but the grandmother was not, we used haplotype seg- regation analysis to determine the grandparental origin Detection of PLP Duplications in Patients with PMD of the mutations. We found that the de novo PLP du- plications are of male origin in the large majority of A multiplex PCR-based test was performed to cases, whereas point mutations occur with the same fre- coamplify exon IV from the PLP gene (Gencic et al. quency in male and female gametes. 1989) and exon IV from the CFTR gene (Dean et al. 1990). The use of one ¯uorescently labeled primer in Families, Material, and Methods each set allowed the quanti®cation of the multiplex PCR products on an automated sequencer: PLP SG50, Diagnosis and Selection of Families Affected by PMD 50-ACTCCAGGATCTCCCAGTTT-30 (labeled with 0 Patients with PMD were referred to us by neurope- 6FAM); PLP 4R, 5 -CGTACCCTAACTCACCATAC- 0 0 0 diatricians in several European countries because of our 3 ; CFTR 4A, 5 -AGTCACCAAAGCAGTACAGC-3 0 interest in this condition. PMD was diagnosed by the (labeled with 6FAM); and CFTR 4B, 5 -GCTATT- 0 combination of a clinical PMD syndrome (hypotonia CTCATCTGCATTCC-3 . and nystagmus of early onset, abnormal trunk and limb Preliminary experiments showed that, between 20 and movements, development of spastic paraplegia with py- 24 cycles, the amount of PCR product was directly pro- ramidal tract signs, and slow progression of the disorder) portional to the copy number of the target sequence. in a male patient, with neurophysiological and MRI ev- The reactions were performed in a PHC3 Techne ther- idence for a dysmyelinating process con®ned to the CNS malcycler in a ®nal volume of 50 ml containing 100 ng (Boesp¯ug-Tanguy et al. 1994). Appropriate informed of genomic DNA, 0.2 mM each dNTP, 1.5 mM mag- consent was obtained from all families. nesium chloride, 25 pmol of each primer, and 2 U of Two groups of subjects were used in this study: one Taq polymerase in the buffer provided by the manufac- group of individuals, used to determine conditions for turer (Gibco BRL). Cycling conditions for each of the the analysis of duplications, and one group of 82 families 21 cycles were 1 min at 917C, 45 s at 587C, and 1.30 affected by sporadic PMD, used to evaluate type and min at 707C, the ®rst cycle being preceded by one step origin of PLP abnormalities. The ®rst group of subjects of 5 min at 947C and the last cycle being followed by comprised 45 normal males, 34 noncarrier females, and an extension program at 707C for 10 min. One microliter 18 obligate carrier mothers. Because of the absence of of ampli®ed DNA, mixed with agonized formamide and any clinical signs in the great majority of females who loading buffer, was heat denatured and then loaded onto were heterozygous for PMD, the 34 noncarrier mothers a 12-cm 6.25% denaturing polyacrylamide gel. The in- were identi®ed by PLP haplotype segregation analysis tensities of the bands corresponding to exons IV of the in 24 large families with PLP-duplicated probands. In CFTR and PLP genes were analyzed by an Applied Bio- the same families, the 18 obligate carrier mothers were systems model 377 DNA Sequencer with the GENSCAN selected if they had both an affected son and an affected and GENOTYPER software (Applied Biosystems). The brother. PLP-gene copy number in each individual was estimated by comparison of the areas under the peaks for PLP DNA Preparation exon IV (PLP4) and CFTR exon IV (CFTR4). Genomic DNA was extracted by the guanidinium We evaluated the quality of each experiment and the chloride technique, as described elsewhere (Jeanpierre reproducibility of results between experiments by ana- 1987). lyzing three distinct control-DNA samples three times in each experiment: one control was a mixture of eight Detection of PLP Point Mutations DNA samples from normal males, one was a mixture DNA ampli®cation using intronic primers ¯anking of eight DNA samples from obligate carrier females, and each exon of the human PLP gene (Gencic et al. 1989) the third was a mixture of eight DNA samples from was performed under standard conditions adapted for noncarrier females. The ®nal result was the ratio of the each set of primers, as described elsewhere (Pham-Dinh PLP-gene dosage obtained for each PMD patient to the et al. 1991). Ampli®ed DNA was sequenced on both PLP-gene dosage of a mix of eight DNA samples from strands, with the same primers used in the ampli®cation normal males analyzed in the same experiment. 362 Am. J. Hum. Genet. 65:360±369, 1999 Figure 1 Detection of PLP duplication in 82 patients with sporadic PMD.
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