Prenatal diagnosis of Fukuyama type congenital muscular dystrophy in eight Japanese families by haplotype analysis using new markers closest to the geneкод для вставкиСкачать
American Journal of Medical Genetics 77:310–316 (1998) Prenatal Diagnosis of Fukuyama Type Congenital Muscular Dystrophy in Eight Japanese Families By Haplotype Analysis Using New Markers Closest to the Gene Kayoko Saito,1* Eri Kondo-Iida,1 Yukiko Kawakita,1 Du Juan,1 Kiyoko Ikeya,1 Makiko Osawa,1 Yukio Fukuyama,1 Tatsushi Toda,2 Masao Nakabayashi,3 Tomoko Yamamoto,4 and Makio Kobayashi4 1 Department of Pediatrics, Tokyo Women’s Medical College, Tokyo, Japan Laboratory of Genome Medicine, Institute of Medical Science, University of Tokyo, Tokyo, Japan 3 Perinatal Division, Maternal and Perinatal Center, Tokyo Women’s Medical College, Tokyo, Japan 4 Department of Pathology, Tokyo Women’s Medical College, Tokyo, Japan 2 We conducted prenatal diagnosis by haplotype analysis, using newly developed microsatellite markers, in eight Fukuyama type congenital muscular dystrophy (FCMD) families. In addition to six new families, two previously reported families were reexamined by haplotype analysis including detection of an ancestral founder haplotype (138–183–301) for 3 microsatellite markers closest to the FCMD gene, designated D9S2105–D9S2107–D9S172, the distances of which from the FCMD gene are presumed to be ∼140, ∼20, and ∼280 kb, respectively. Five fetuses from five families were diagnosed as nonaffected, and were subsequently confirmed to be healthy. Three fetuses of the other three families were diagnosed as having a high probability of being affected by FCMD. In the prenatal diagnosis conducted for these eight families, the ancestral founder allele was observed in 13 of 16 (81%) FCMD-bearing chromosomes. Detection of the ancestral haplotype facilitated achieving accurate prenatal diagnosis of FCMD. The brains of all three fetuses prenatally diagnosed as FCMD-affected showed the initial stage of cortical dysplasia, strong evidence of FCMD. Am. J. Med. Genet. 77:310– 316, 1998. © 1998 Wiley-Liss, Inc. Contract grant sponsor: Ministry of Health and Welfare of Japan; Contract grant numbers: 8A-2, 8A-3; Contract grant sponsor: Ministry of Education, Science, and Culture, Japan; Contract grant number: 07670906. *Correspondence to: Dr. Kayoko Saito, Department of Pediatrics, Tokyo Women’s Medical College, 8-1 Kawadacho, Shinjuku, Tokyo 162, Japan. E-mail: email@example.com Received 12 September 1997; Accepted 10 February 1998 © 1998 Wiley-Liss, Inc. KEY WORDS: prenatal diagnosis; Fukuyama type congenital muscular dystrophy (FCMD); haplotype analysis; ancestral founder allele; cortical dysplasia INTRODUCTION Fukuyama type congenital muscular dystrophy (FCMD), first reported by Fukuyama et al.  and designated MIM 253800 in the McKusick catalog [McKusick et al., 1994], is the most common congenital muscular dystrophy in Japan [Fukuyama et al., 1981, 1996]. The incidence is 6.9–11.9/100,000 [Fukuyama and Ohsawa, 1984], and the incidence ratio of Duchenne muscular dystrophy to FCMD is 2.1:1, based on a multi-institutional study [Fukuyama et al., 1981]. The FCMD phenotype is characterized by dystrophic changes in the skeletal muscle and CNS migration disturbances, principally cerebral and cerebellar cortical dysplasia. The clinical manifestations are recognized in early infancy as psychomotor retardation with hypotonia and weakness. Peak motor function, in most patients, is no better than sitting without help or sliding along the floor on the buttocks. Brain malformations include agyria, pachygyria, and polymicrogyria. On the surface of the cerebral cortex, polymicrogyria and cloudy, thickened leptomeninges are apparent. There are three patterns of cellular migration disturbance in the cerebral and cerebellar cortex, as reported by Takada et al. , which are observable even in the 23-week-old FCMD fetal brain [Takada et al., 1987]. The cellular migration disturbance is the origin of the gyrus malformation characteristic of FCMD. FCMD is an autosomal recessive genetic defect, and the sib recurrence rate is 25% [Osawa, 1978; Fukuyama and Ohsawa, 1984]. In an effort to elucidate the Prenatal Diagnosis of FCMD pathogenesis of FCMD, molecular genetic analysis has been undertaken. Toda et al.  located the FCMD gene at 9q31-q33 by a genetic linkage analysis. Furthermore, the FCMD gene was localized within a region of ∼5 cM between D9S127 and D9S2111 (CA246) [Toda et al., 1994], based on homozygous mapping and recombination mapping. Toda et al.  also found evidence for linkage disequilibrium between FCMD alleles and D9S306 (mfd220), the latter being located in the FCMD candidate gene region. We previously reported prenatal diagnosis of FCMD in two families using these microsatellite markers [Kondo et al., 1996]. Toda et al.  have since developed new microsatellite markers, D9S2105, D9S2107, and D9S172, the distances of which from the FCMD gene are presumed to be ∼140, ∼20, and ∼280 kb, respectively. These investigators indicated that most FCMD-bearing chromosomes were derived from a single ancestral founder in Japanese FCMD patients and emphasized the value of precisely diagnosing FCMD in order to ascertain the ancestral haplotype, because 75% of FCMD patients had the haplotype for D9S2105–D9S2107–D9S172, while control individuals did not. In this report we confirmed their data by detecting the ancestral haplotype in FCMD-bearing chromosomes compared with control chromosomes. Though the FCMD gene has not yet been isolated and the gene product remains unknown, prenatal diagnosis of the disease is now possible due to advances in the molecular genetic tools used to study FCMD. Herein, we describe prenatal diagnosis in eight FCMD families by haplotype analysis using the aforementioned newly developed microsatellite markers, D9S2105, D9S2107, and D9S172, closest to the FCMD gene. In addition to six new families, two previously reported families were re-examined by the haplotype analysis. MATERIALS AND METHODS Subjects Eight at-risk pregnancies from eight unrelated families (Families A to H) with at least one child afflicted with FCMD were studied. In Families A and B, examinations with the newly developed microsatellite markers were conducted to confirm our previous results [Kondo et al., 1996], the associated clinical findings of which were described previously. The diagnosis of FCMD in propositi was confirmed by us based on clinical findings, such as hypotonia, diffuse muscle weakness, and wasting since early infancy; facial muscle involvement; joint contractures; delay in psychomotor and speech development; high creatine kinase titers; gyrus abnormalities and demyelination on CT scan or MRI; and dystrophic change affecting muscle fibers with proliferation of perimysial connective tissue. For prenatal diagnosis of the eight pregnancies, fetal materials were obtained by mid-trimester amniotic fluid sampling in seven (Families A to F and H) and by chorionic villus sampling (CVS) in one (Family G). After the actual procedure, as well as the safety aspects and risks, had been explained and the confidentiality of all results guaranteed, these eight families expressed 311 their eagerness to utilize prenatal diagnosis. We obtained approval from the Intramural Ethical Committee, and this procedure was carried out according to the Prenatal Diagnosis Guidelines of the Japan Society of Human Genetics. Prenatal Diagnosis Procedures DNA was extracted from peripheral blood leukocytes of parents and siblings. In Family F, DNA was obtained from a frozen muscle specimen, because the propositus was deceased. Fetal materials were obtained by CVS and amniocentesis at 10 and 15–17 gestational weeks, respectively. DNA was extracted from the chorionic villi or amniocytes. Haplotypes of family members and fetuses were determined at D9S2105, D9S2107, and D9S172 loci [Toda et al., 1996]. The parents were informed of the results at 12–19 gestational weeks. Haplotype Analysis: Allele Typing We used the markers D9S2105 [Toda et al., 1996], D9S2107 [Toda et al., 1996], and D9S172 [Gyapay et al.,1994], as well as D9S127 [Lyall et al., 1992], D9S306 (mfd 220) [Weber, 1993], and D9S2111 (CA246) [Toda et al., 1995] which were mapped previously to chromosome 9q31-q33 [Toda et al., 1994]. Primer sets for polymerase chain reaction (PCR) amplification of these markers were synthesized. Conditions for PCR and subsequent electrophoresis were as previously described [Toda et al., 1993, 1994, 1996]. PCR was performed in 25 ml reaction volumes containing 20 ng of genomic DNA, 20 pmol of one unlabeled primer and 20 pmol of one primer end-labeled with 1.0 mCi of [g-32P]ATP using T4 polynucleotide kinase, 1× PCR buffer (16.6 mM NH4SO4, 67 mM Tris-HCl, pH 8.8, 10 mM b-mercaptoethanol, 6.7 mM EDTA), 10% (v/v) dimethyl sulfoxide, 1.5 mM of each dNTP, 5 mM MgCl 2 , and 1.25 U of Taq DNA polymerase. For D9S127, D9S306, and D9S2111, samples were incubated in a DNA thermocycler (Perkin Elmer Cetus, Norwalk, CT) for 35 cycles under the following conditions: 94 °C for 1.5 min, 55 °C for 2 min, and 72 °C for 1.5 min. For D9S2105, D9S2107, and D9S172, samples were incubated for 38 cycles. The first denaturation and final elongation steps were extended to 3 and 10 min, respectively. The PCR products were analyzed on 6% polyacrylamide gel and visualized by autoradiography. Detection of Ancestral Haplotype in FCMD Patients and Control Individuals To confirm the existence of linkage disequilibrium and the ancestral founder haplotype (138–183–301 for D9S2105–D9S2107–D9S172), haplotype analyses were done in 39 FCMD patients (78 FCMD-bearing chromosomes), including the 8 probands, for prenatal diagnosis. The results were compared with those of 66 normal chromosomes from FCMD parents. Alleles from the 78 FCMD-bearing chromosomes and the 66 control chromosomes were classified according to the size of PCR products for the microsatellite DNA markers. 312 Saito et al. Neuropathological Analysis of the Fetuses Aborted fetuses of Families B, C, and E were autopsied at 20, 18, and 17 gestational weeks, respectively. The brains were fixed in 10% buffered formalin 40–60 min after termination of the pregnancy. Sections were stained with hematoxylin and eosin (HE) and periodic acid–methanamine–silver (PAM) stain. RESULTS Detection of Ancestral Haplotype in FCMD Patients and Normal Chromosomes From Their Parents First, we examined haplotypes of FCMD chromosomes from 39 FCMD patients at three of the closest loci, D9S2105, D9S2107, and D9S172. The results were compared with 66 normal chromosomes from FCMD parents in 33 families, i.e., one each normal chromosome of their homologs 9 (Table I). Sixty (77%) of 78 FCMD-bearing chromosomes carried the ancestral haplotype, while the same haplotype was seen in one of 66 normal chromosomes from FCMD parents. The remaining 18 chromosomes of the patients had haplotypes that may originate from previous recombinations within the ancestral haplotype, and 7 such chromosomes were found in the parents. Other haplotypes were observed in the remaining 58 normal chromosomes but were not seen in any of the FCMD-bearing chromosomes. Prenatal Diagnosis We conducted prenatal diagnosis of FCMD in eight families with at least one FCMD patient. In Families A and B, the examination with the three new markers was done to confirm the previous results [Kondo et al., 1996] (Fig. 1). In Family A, the fetus had a paternally derived haplotype which did not carry the FCMD gene mutation, while the other haplotype derived from the mother carrying the mutation, which did not show a crossover. The ancestral haplotype, 138–183–301, was observed on both chromosomes of the propositus (Fig. 1, A-II-2). The fetus was confirmed to be nonaffected by showing that the ancestral haplotype was derived only from the mother. The baby showed no signs of FCMD after birth. She is now 3 years old and healthy. TABLE I. Haplotypes Associated With FCMD and Normal Chromosomes Haplotype No. of chromosomes D9S2105–D9S2107–D9S172 FCMD Controla 138–183–301 138–183–297 130–183–295 138–183–291 134–183–301 138–183–295 139–183–297 130–183–297 138–193–301 140–183–301 Others Total 60 (77%) 6 4 2 1 1 1 1 1 1 0 78 1 0 1 0 0 0 0 1 1 4 58 (88%) 66 a Normal chromosomes from FCMD parents. In Family B, the fetus inherited a paternal haplotype carrying the mutation. On the maternal chromosome, a crossover was identified between loci D9S2105 and D9S2111. The propositus (Fig. 1, B-II-3) and the fetus showed the ancestral haplotype on his both chromosomes. The fetus in Family B was demonstrated to have FCMD [Yamamoto et al., 1996]. In Family C, the fetus had both paternal and maternal chromosomes carrying the mutation and was thus homozygous, like the propositus (Fig. 2, C-II-1). The fetus was considered to have a high probability of having FCMD. The paternal chromosome showed the ancestral haplotype, while the maternal chromosome showed a haplotype of 138–183–297. The parents decided to terminate the pregnancy at 18 gestational weeks, and the fetus was neuropathologically demonstrated to have FCMD [Yamamoto et al., 1997]. In Family D, the fetus had a paternally derived ancestral haplotype that carried the FCMD gene mutation, while the other allele derived from the mother did not have the FCMD gene mutation. The fetus was diagnosed as nonaffected, and the pregnancy was continued. The baby girl was confirmed to be healthy at 2 months of age by one of the authors (K.S.). In Family E, both children, two boys, were afflicted with FCMD. The fetus was diagnosed as having a high probability of FCMD, because the haplotype was the same as those of the elder brothers, showing homozygosity for the ancestral pattern (138–183–301). The parents decided to terminate the pregnancy at 17 gestational weeks. In Family F, the fetus had a paternally derived haplotype which did not carry the FCMD gene mutation, while the other chromosome derived from the mother carried the mutation and also showed the ancestral haplotype. The fetus was diagnosed as nonaffected. The baby girl was confirmed to be healthy at 3 months of age by one of the authors (K.S.). In Family G, the paternal haplotype of the fetus carried the FCMD gene mutation, while the maternal haplotype did not. The paternal haplotype (134–183–301) was not the ancestral one. The fetus was diagnosed as nonaffected. The baby boy was confirmed to be healthy by a pediatrician at another hospital. In Family H, the propositus was homozygous for the ancestral haplotype. The fetus had neither the ancestral haplotype nor the affected alleles. We diagnosed the fetus as nonaffected, and a healthy girl was born. In these eight families seeking prenatal diagnosis, the results indicated that 13 (81%) of 16 FCMDbearing chromosomes shared an ancestral haplotype. Neuropathological Analysis of the Fetuses In Family B, the parents opted for an abortion at 20 gestational weeks. On macroscopic observation of the fetal brain, multiple small granular protrusions, up to 0.5 mm in diameter over the cerebral surface, were noted [Kondo et al., 1996]. Histopathologically, thick extracortical lesions, consisting of neuronal nodules, were detected. In Family C, on macroscopic observation of the brain from the 18-week-old fetus, the granular protrusions were smaller and much less numerous Prenatal Diagnosis of FCMD 313 Fig. 1. Upper: Eight-locus genotypes from members of the two pedigrees previously described [Kondo et al., 1996]. D9S306 (mfd220) was the closest marker available at that time. Crossover was observed in the fetal haplotype in each family. Lower: Six-locus genotypes from members of the two pedigrees. D9S2107 is the closest marker. In Family A, the fetus had a paternally derived wild-type allele which did not have the FCMD gene mutation, while the maternally derived chromosome carried the mutant allele, which did not show a crossover. The ancestral 138–183–301 haplotype for D9S2105– D9S2107–D9S172 was observed on both chromosomes of the propositus. In Family B, the fetus inherited a paternal haplotype carrying the mutation. On the maternal chromosome, a crossover was identified between loci D9S2105 and D9S2111. The propositus and the fetus showed the ancestral haplotype (138–183–301) on both their chromosomes. Each allele is shown according to PCR product sizes. Shaded and unshaded columns depict the regions carrying the mutant and wild-type FCMD alleles, respectively. The ancestral 138–183–301 haplotype is highlighted by the white square. than on the brain from the Family B fetus (Fig. 3a). Microscopically, there were small but numerous defects of the basal lamina observed by PAM staining. Neurites, glia, and granular cells were seen to erupt through defects into the leptomeninges (Fig. 3b). The 17 week abortus of Family E was also examined pathologically. On gross examination, small granular protrusions, a characteristic of the FCMD fetal brain, were seen (Fig. 4a). Microscopically, the linear structure stained with PAM showed irregular disruption. The pathological change was milder, presumably due to the earlier gestational age (Fig. 4b). Thus, the initial stage of cortical dysplasia was observed in these 17-, 18-, and 20-week-old fetuses, diagnosed as FCMD prenatally, and was milder at early gestational ages. DISCUSSION We conducted molecular genetic analyses, for the purpose of prenatal diagnosis, in eight FCMD families with at least one child afflicted with FCMD, carefully considering the ethical issues in each case. As a result, the fetuses in five families were diagnosed as being nonaffected, and all proved to be healthy after birth. Those in the other three families were revealed to be affected; the parents opted for abortion, and all three were confirmed to have FCMD by autopsy. In Families A and B, we used the microsatellite markers, including D9S306 [Kondo et al., 1996]. The analysis using this marker revealed a crossover in the fetal haplotype in each family. Thus, the possibilities of FCMD were 1% and 86% in Families A and B, respectively [Kondo et al., 1996]. The distance of D9S306 from the FCMD locus was presumed to be 1 Mb. We then conducted haplotype analysis using three new markers, D9S2105, D9S2107, and D9S172, the distances of which from the FCMD gene were estimated to be ∼140, ∼20, and ∼280 kb, respectively [Toda et al., 1996]. These markers revealed no crossover in Family A. In Family B, we diagnosed the fetus to be affected by demonstrating the fetus as having the same haplotypes as the affected sibling except for D9S2111. We identified a crossover between loci D9S2105 and D9S2111. As the D9S2107 and D9S172 alleles in the mother were homozygous, they were uninformative haplotypes and it was difficult to determine whether a crossover was 314 Saito et al. Fig. 2. Six-locus genotypes from members of the six most recent pedigrees. In Families C and E, the fetuses had both paternal and maternal chromosomes carrying the mutation and were thus homozygous, like the propositus. The paternal chromosome in Family C and both the paternal and maternal chromosomes in Family E showed the ancestral haplotype (138–183–301). The fetus was considered to have a high probability of having FCMD. In Families D, F, and G, the fetuses had a haplotype consistent with heterozygosity for the FCMD gene mutation, while the fetus of Family H was homozygous for the normal chromosome. All four fetuses were diagnosed as nonaffected. The ancestral haplotype was observed in one or both chromosomes of the propositus. present in these regions. However, in the maternal chromosome of the fetus, a crossover was assumed to have occurred between the D9S172 and D9S2111 loci, not between D9S2105 and D9S2107 or between D9S2107 and D9S172, because the distance between D9S172 and D9S2111 was much longer than that between D9S2105 and D9S172 [Toda et al., 1996; Miyake et al., 1997]. Thus, the fetal haplotype revealed homozygosity for the ancestral haplotype (138–183–301 for D9S2105–D9S2107–D9S172). This homozygosity also provided strong evidence that the fetus had FCMD. Whether FCMD occurs in populations of nonJapanese ethnicity, or is strictly limited to the Japanese, is an important issue which has yet to be resolved. FCMD is very rare outside Japan; only three patients from two Chinese (Taiwanese) families have been diagnosed as having FCMD [Jong et al., 1994]. The population of Japan has long been isolated genetically by geography, language, and culture. The isolation and population expansion have allowed Japan to develop a distinctive pattern of single-gene disorders, such as FCMD. The existence of the ancestral founder allele as the population-specific mutant allele has significance for understanding the origin of genetic disease and also provides opportunities for the detection of FCMD-specific alleles in an at-risk population. Toda et al.  examined the rate of the ancestral haplotype, and we added the FCMD cases and control subjects described herein. We found the rate of FCMDbearing chromosomes carrying the ancestral haplotype to be 60 (77%) of 78 chromosomes, while Toda et al.  obtained a rate of 84 (75%) of 112 chromosomes. The rates in control chromosomes in our study and theirs were one (1.5%) of 66 and 0 (0%) of 87, respectively. The frequency of one control chromosome having the 138–183–301 haplotype is equal to a value calculated from each allele frequency (0.1765 × 0.5185 × 0.2571 4 0.0235) [Toda et al.,1996; unpublished data: allele frequency of allele 138 for D9S2105 was 18/102 4 0.1765; allele 183 for D9S2107 was 56/108 4 0.5185; allele 301 for D9S172 was 27/105 4 0.2571]. Thus, 2% of normal chromosomes may show the same haplotype as the ancestral founder haplotype. In these eight families seeking prenatal diagnosis, the founder haplotype was observed in 13 (81%) of the 16 FCMD-bearing chromosomes from the propositi. In other words, confirming the diagnosis in each propositus and determining whether the fetuses had FCMD were facilitated, because there was at least one founder haplotype in each of these eight FCMD families. Prenatal Diagnosis of FCMD 315 Fig. 3. a: Macroscopic appearance of the Family C fetal brain at 18 gestational weeks. The granular protrusions were seen on the brain from the Family B fetus. b: Microscopically, there were small but numerous defects of the basal lamina. Neurites, glia, and granular cells were seen to erupt through defects into the leptomeninges (PAM ×250). In addition, crossover was seen in Families B and G between D9S172 and D9S2111, but was not problematic, because there was no crossover between the three microsatellite markers, D9S2105, D9S2107, and D9S172, and D9S2111 is approximately 3 cM apart from D9S172. The 17-, 18-, and 20-week-old fetuses, diagnosed as FCMD prenatally, were examined neuropathologically. Multiple small granular protrusions over the cerebral surface were composed of aberrant neuroglial clusters. As judged by microscopic examination, the neurites, granular cells, and glia migrated out through the discontinuous pial–glial barrier into the extracortical glial layer. These findings represent the initial stage of cortical dysplasia [Nakano et al., 1996; Yamamoto et al., 1996, 1997]. The pathological change was milder at earlier gestational ages. The eventual outcomes confirmed the prenatal predictions of FCMD to be correct in the three families who elected to terminate the pregnancies. We antici- Fig. 4. a: Macroscopic appearance of the Family E fetal brain at 17 gestational weeks. Arrowheads indicate small granular protrusions which were a characteristic feature of the FCMD fetal brain. These were smaller and much less numerous than on the brains from the Family B and Family C fetuses. b: Microscopically, the linear structure showed irregular disruption. The pathological change was milder, presumably due to the earlier gestational age (PAM ×400). 316 Saito et al. pate a growing demand for this service. We are moving ahead slowly, considering the many ethical issues inherent to prenatal diagnosis. We would like to emphasize that Families A, D, F, G, and H would most likely have opted for abortion without the assurance, provided by prenatal diagnosis, that their babies would be healthy. Family E also represents a special case in that having three afflicted children would be an enormous burden on one family. Ultimately, the decision as to whether to utilize prenatal diagnosis, and if it is utilized, what to do with the information obtained, must be made by the parents. Our job, as physicians, is to support these families both medically and educationally. Kondo E, Saito K, Toda T, Osawa M, Yamamoto T, Kobayashi M, Fukuyama Y (1996): Prenatal diagnosis of Fukuyama type congenital muscular dystrophy by polymorphism analysis. 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