Takeo Kubota

A new assay for the analysis of X-chromosome inactivation based on methylation-specific PCR

The pattern of X-chromosome inactivation in females is currently evaluated by assays of differential methylation in the genes between the active and the inactive X chromosomes, with methylation-sensitive enzymes. We report a new assay in the human androgen receptor (HUMARA) locus involving a methylation-specific polymerase chain reaction (M-PCR) technique, independent of the use of restriction enzymes. The assay involves the chemical modification of DNA with sodium bisulfite and subsequent PCR. By using the assay with specific primers for the methylated allele, we obtained an X-inactivation pattern based on the ratio of the maternal inactive X to the paternal inactive X. These patterns were consistent with those obtained by conventional PCR assay at the same locus in 48 female cases. We also obtained another X-inactivation pattern based on the ratio of the maternal active X to the paternal active X by using specific primers for the unmethylated allele. The latter pattern was complementary to the former pattern, and a combination of these patterns produced a reliable X-inactivation pattern. The assay revealed that 12 (11%) of the 105 normal females had non-random inactivation patterns (>80:20 or <20:80). Four patients with an X; autosome translocation showed extremely non-random patterns, and these results were consistent with those obtained by previous molecular/cytogenetic studies. We conclude that M-PCR provides an accurate assay for X-inactivation and that it can be performed on various DNA samples unsuitable for restriction digestion.

Validation studies of SNRPN methylation as a diagnostic test for Prader-Willi syndrome

Prader-Willi syndrome (PWS) is caused by absence of a paternal contribution of the chromosome region 15q11-q13, resulting from paternal deletions, maternal uniparental disomy, or rare imprinting mutations. Laboratory diagnosis is currently performed using fluorescence in situ hybridization (FISH), DNA polymorphism (microsatellite) analysis, or DNA methylation analysis at locus PW71 (D15S63). We examined another parent-of-origin-specific DNA methylation assay at exon alpha of the small nuclear ribonucleoprotein-associated polypeptide N gene (SNRPN) in patients referred with clinical suspicion of PWS or Angelman syndrome (AS). These included 30 PWS and 17 AS patients with known deletion or uniparental disomy status, and a larger cohort of patients (n = 512) suspected of PWS who had been analyzed previously for their methylation status at the PW71 locus. Results of SNRPN methylation were consistent with known deletion or uniparental disomy (UPD) status as determined by other molecular methods in all 47 cases of PWS and AS. In the larger cohort of possible PWS patients, SNRPN results were consistent with clinical diagnosis by examination and with PW71 methylation results in all cases. These data provide support for the use of SNRPN methylation as a diagnostic method. Because methylation analysis can detect all three major classes of genetic defects associated with PWS (deletion, UPD, or imprinting mutations), methylation analysis with either PW71 or SNRPN is an efficient primary screening test to rule out a diagnosis of PWS. Only patients with an abnormal methylation result require further diagnostic investigation by FISH or DNA polymorphism analysis to distinguish among the three classes for accurate genetic counseling and recurrence-risk assessment.

Mutation frequencies of EXT1 and EXT2 in 43 Japanese families with hereditary multiple exostoses

Hereditary multiple exostoses (EXT) is an autosomal dominant bone disease characterized by the formation of cartilage-capped prominences. EXT is genetically heterogeneous with at least four chromosomal loci. Among the four loci, the exostosis type 1 gene (EXT1) and type 2 gene (EXT2) have been cloned. Previous studies have shown that disease-type-specific frequency of mutations is different among various ethnic populations. To determine those frequencies in the Japanese, we conducted a large-scale mutation screening on both genes. In 23 of 43 Japanese families examined, we found 21 different mutations, of which 18 are novel. Seventeen (40%) of the 23 families had a mutation in EXT1 and six (14%) had a mutation in EXT2, suggesting that the former mutations are more frequent than the latter in Japanese EXT families. Of the 17 families with EXT1 mutations, 13 had those causing premature termination of the EXT1 protein and four showed missense mutations, whereas five of the six families with EXT2 mutations had those causing premature termination and one showed missense mutation. Interestingly, all four EXT1 missense mutations occurred in an arginine residue at codon 340 (R340) that is known as a critical site for expression of heparan sulfate glycosaminoglycans, suggesting that the region encompassing the arginine residue may play an important role in the function of the EXT1 protein. These results expand our knowledge of the ethnic difference of EXT and the structure-function relationship of the EXT genes.

Clinical applications of two-color telomeric fluorescence in situ hybridization for prenatal diagnosis: Identification of chromosomal translocation in five families with recurrent miscarriages or a child with multiple congenital anomalies

AbstractTwo-color fluorescence in situ hybridization (FISH) analysis using human chromosome arm-specific telomeric probes (telomeric probes) was used successfully to detect each derivative chromosome of a translocation carrier in five couples who requested a prenatal diagnosis in future pregnancies. Most of the human chromosome terminal bands are G-band-negative, and even FISH analysis using whole-chromosome painting (wcp) probes are often of insufficient complexity to detect subtle chromosomal changes. A complete set of human telomeric probes was developed to improve the sensitivity of diagnosis of microdeletions or other cryptic rearrangements in telomeric regions. Two-color telomeric FISH was the only possible method for precise prenatal diagnosis of one of the couples, because the carriers chromosomal aberration was too subtle to be detected by wcp FISH or conventional methods. We have demonstrated that two-color telomeric FISH has the potential to be a powerful new tool in the detection of cryptic chromosomal rearrangements involving telomeric regions in prenatal diagnosis precisely and in time.

Molecular genetic study of Japanese patients with X-linked α-thalassemia/mental retardation syndrome (ATR-X)

X-linked alpha-thalassemia/mental retardation syndrome (ATR-X) is one of the many known X-linked mental retardation syndromes. Mutations in the ATR-X gene (ATRX) that encodes a putative global transcription factor have been identified in patients with ATR-X as well as those with other forms of X-linked mental retardation syndrome. To better understand the genetic basis of ATR-X, we investigated nine patients with the ATR-X phenotype from eight independent Japanese families for mutations in ATRX. We identified seven missense mutations, including six novel mutations, all of which were located either in the N-terminal region corresponding to the putative zinc finger domain (N179S, P190L, V194I, and R246C) or in the C-terminal region corresponding to the helicase domain (V1552F, L1645S, and Y1847C). R246C was found in two independent patients. Furthermore, we investigated the origin of the mutations in seven mothers. Five mothers were found to be carriers, and two were not, indicating de novo origin of the mutations. When we compared clinical manifestations with respective mutations, we could not find apparent phenotype-genotype correlation. Therefore, the putative zinc finger domain and the helicase domains may have similar functional significance for the function of ATRX.

A G to A transition at the last nucleotide of exon 6 of the γc gene (868G→A) may result in either a splice or missense mutation in patients with X-linked severe combined immunodeficiency

We report here that a defect of the interleukin common gamma subunit (γc) in X-linked severe combined immunodeficiency (XSCID) previously known as a missense mutation resulted instead in exon skipping in a Japanese XSCID patient. The phenotype of the patient was consistent with that of typical XSCID, and his Epstein-Barr virus-transformed B cells accordingly entirely lacked surface expression of γc. On analysis by the reverse transcription-polymerase chain reaction (RT-PCR), a single but small γc mRNA species was detected. Exon 6, which encodes the transmembrane domain of γc, was skipped in the mRNA. A G to A mutation was found at the last nucleotide of exon 6 of the γc gene (868G→A). The predicted consequence of the exon skipping is a frameshift resulting in a premature stop codon, and the mutated γc presumably loses association with the cell membrane. In XSCID, this mutation (868G→A) is known as a missense mutation that results in Q285A. Previously reported patients with the same mutation apparently had no aberrant or alternative splicing but did have the Q285A exchange. Similar mutations at the last nucleotide of an outskipped exon have been reported. However, such mutations do not always cause exon skipping. Analyses of RNA structural changes induced by the mutations supported the variability of consequences of the mutations. Taken together, our findings suggest that the 868G→A mutation of the γc gene may affect γc transcripts differently, i.e., generating missense or exon skipping, in XSCID patients with the same mutation. Patient-specific variation in splicing thus appears to occur.

Börjeson-Forssman-Lehmann syndrome in a woman with skewed X-chromosome inactivation.

Börjeson-Forssman-Lehmann (BFL) syndrome is an X-linked recessive disorder characterized by minor facial anomalies, obesity, epilepsy, and severe mental retardation. The phenotype of male patients is usually severe, whereas that of carriers is less severe, suggesting X-linked incompletely recessive inheritance. A recent linkage study mapped the BFL syndrome gene to Xq26-q27. The etiology of the condition in female patients with full manifestations is not known, although nonrandom X-chromosome inactivation has been considered. We recently developed an assay for X-inactivation studies based on the methylation-specific polymerase chain reaction (PCR) technique. Using the methylation-specific PCR assay, a woman with typical findings of this syndrome was shown to have an extremely skewed X-inactivation pattern. This finding suggests that the full manifestations of the BFL syndrome in carriers may be caused by skewed X inactivation with a high proportion of cells in which the X chromosome with a normal gene be inactivated, leaving the X chromosome with a mutant gene active.

Fetal polycystic kidney disease in oro-facio-digital syndrome type I

Abstract We report a girl with oro-facio-digital syndrome type I (OFD I) associated with polycystic kidney disease (PKD), which was identified on fetal US and fetal MRI. After birth, the diagnosis of this X-linked dominant disorder, which is lethal in males, was achieved by recognition of facial dysmorphism, lingual hamartomas, postaxial polydactyly, brain malformations, and the existence of her deceased male sibling with similar malformations. Adult PKD is a common feature in heterozygous females with OFD I. However, fetal PKD has been reported only in a lethal homozygous male. Our observation expands our knowledge about the phenotypic variations of PKD in OFD I.

Multiple Endocrine Neoplasia Type 1 Concomitant with Prader-Willi Syndrome: Case Report and Genetic Diagnosis

A case of multiple endocrine neoplasia type 1 (MEN 1) accompanied with Prader-Willi syndrome (PWS) was reported. Diagnosis of both diseases have been genetically confirmed. Delay in the diagnosis and management for PWS made surgery for endocrine tumors difficult. This is the first report on the concomitance of MEN 1 with PWS.