A rapid diagnostic method for a retrotransposal insertional mutation into the FCMD gene in Japanese patients with Fukuyama congenital muscular dystrophy.

Fukuyama-type congenital muscular dystrophy (FCMD) is characterized by congenital muscular dystrophy in combination with central nervous system (CNS) abnormalities. Differential diagnosis of FCMD from Duchenne and Becker muscular dystrophies (DMD/BMD) or other types of congenital muscular dystrophy is occasionally difficult, because of their phenotypic similarity. The gene (FCMD) responsible for FCMD at 9q31 was isolated in 1998. In Japan, most FCMD-bearing chromosomes (87%) have a 3-kb retrotransposal insertion into the 3'-untranslated region (UTR) of the gene that could be derived from a single ancestral founder. Nine non-founder mutations have been identified in Japanese FCMD patients. Severe phenotype was significantly more frequent in patients who were compound heterozygotes for a point mutation and the founder mutation, than in homozygotes for the founder mutation. We developed a PCR-based diagnostic method for a rapid detection of the retrotransposal insertion mutation. Using this system, we screened 18 FCMD patients, and found 16 homozygotes and two heterozygotes for the insertion. We also evaluated the carrier frequency in the normal Japanese population. Six of 676 persons were recognized as a heterozygous carrier. Furthermore, we found three homozygotes for the FCMD founder mutation among 97 patients who had been said to have probable DMD/BMD without any DMD mutations. On the other hand, there were no FCMD homozygotes but four heterozygous carriers among 335 patients with DMD mutations. The diagnostic method we developed will provide a rapid and reliable diagnosis of FCMD, which can bring important information in genetic counseling, such as the accurate mode of inheritance, recurrence risk and a life expectancy.

Complex low-copy repeats associated with a common polymorphic inversion at human chromosome 8p23.

To characterize a submicroscopic, common 8p23 polymorphic inversion, we constructed a complete BAC/PAC-based physical map covering the entire 4.7-Mb inversion and its flanking regions. Two low-copy repeats (LCRs), REPD (approximately 1.3 Mb) and REPP (approximately 0.4 Mb), were identified at each of the inversion breakpoints. Comparison of the REPD and REPP sequences revealed that REPD showed high homology to REPP, with complex direct and inverted orientations. REPD and REPP contain six and five olfactory receptor gene-related sequences, respectively. LCRs at 8p23 showed multiple FISH signals from an Old World monkey to the human. Thus, multiplication of the LCR may have occurred at least 21-25 million years ago. We also investigated the frequency of the 4.7-Mb inversion in the general Japanese population and found that the allele frequency for the 8p23 inversion was estimated to be 27%.

Preferential paternal origin of microdeletions caused by prezygotic chromosome or chromatid rearrangements in Sotos syndrome.

Sotos syndrome (SoS) is characterized by pre- and postnatal overgrowth with advanced bone age; a dysmorphic face with macrocephaly and pointed chin; large hands and feet; mental retardation; and possible susceptibility to tumors. It has been shown that the major cause of SoS is haploinsufficiency of the NSD1 gene at 5q35, because the majority of patients had either a common microdeletion including NSD1 or a truncated type of point mutation in NSD1. In the present study, we traced the parental origin of the microdeletions in 26 patients with SoS by the use of 16 microsatellite markers at or flanking the commonly deleted region. Deletions in 18 of the 20 informative cases occurred in the paternally derived chromosome 5, whereas those in the maternally derived chromosome were found in only two cases. Haplotyping analysis of the marker loci revealed that the paternal deletion in five of seven informative cases and the maternal deletion in one case arose through an intrachromosomal rearrangement, and two other cases of the paternal deletion involved an interchromosomal event, suggesting that the common microdeletion observed in SoS did not occur through a uniform mechanism but preferentially arose prezygotically.

Duplication of 8p23.2: a benign cytogenetic variant?

We describe a duplication of the 8p23.2 band in seven individuals from four families. The duplication was recognizable as an enlarged 8p23.2 band on G-banded chromosomes at the 550 band level. It was transmitted from a parent to offspring in three of the four families in which both parents were karyotyped. Each proband in the four families had the enlarged band and showed various phenotypic abnormalities, but the abnormalities were inconsistent. Chromosomal and interphase fluorescence in situ hybridization (FISH) analysis of the enlarged band region defined a 2.5-Mb duplicated segment common to all seven individuals studied. Interphase FISH analysis of peripheral blood lymphocytes from 50 unrelated normal individuals showed the duplication in three individuals. In view of these findings, it is most likely that the 8p23.2 duplication we described is a normal variant.

A novel gene is disrupted at a 14q13 breakpoint of t(2;14) in a patient with mirror-image polydactyly of hands and feet.

Mirror-image polydactyly of hands and feet (MIP) is a very rare congenital anomaly characterized by mirror-image duplication of digits. To isolate the gene responsible for MIP, we performed translocation breakpoint cloning from an MIP patient with t(2;14)(p23.3;q13). We isolated a good candidate gene for MIP that was disrupted by the translocation of the patient. We had previously con structed a 1.2-megabase bacterial artificial chromosome (BAC)/P1-derived artificial chromosome (PAC) contig covering the 14q13 breakpoint of t(2;14)(p23.3;q13). From a 500-kb segment consisting of seven BAC/PAC clones in the contig, we isolated a novel gene (the mirror-image polydactyly 1 gene, designated as MIPOL1, GenBank Accession No. AY059470), in addition to the hepatocyte nuclear factor 3 alpha gene (HNF3A, GenBank Accession No. XM 007360). MIPOL1 spans about 350kb, comprises 15 exons, and encodes 442 amino acids. Northern blot analysis revealed that MIPOL1 expression is definite but very weak in adult heart, liver, skeletal muscle, kidney, and pancreas, and in fetal kidney. In view of the genome sequence and the contig map constructed, the 14q13 breakpoint of the patient was identified as located in intron 11 of MIPOL1, indicating that the gene was disrupted by the translocation, and that the breakage resulted in MIPOL1 protein truncation. Whole-mount in situ hybridization in mouse resulted in mouse Mipol1 signals all over E10.5-E13.5 mouse embryos. Two other unrelated patients with limb anomalies similar to MIP were subjected to mutation analysis of MIPOL1, but none had any mutations. We then isolated BAC clones from the other breakpoint, 2p23.3. A search for genes and expressed sequence tags in a more than 300-kb region around the 2p23.3 breakpoint found only the neuroblastoma-amplified protein gene (NAG, GenBank Accession No. NM 015909), which is located at least 50kb centromeric to the breakpoint and is not likely to be related to MIP. MIPOL1 is a good candidate gene for the MIP type of anomaly.