Hidefumi Tonoki

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.

Spectrum of MLL2 (ALR) mutations in 110 cases of Kabuki syndrome.

Kabuki syndrome is a rare, multiple malformation disorder characterized by a distinctive facial appearance, cardiac anomalies, skeletal abnormalities, and mild to moderate intellectual disability. Simplex cases make up the vast majority of the reported cases with Kabuki syndrome, but parent‐to‐child transmission in more than a half‐dozen instances indicates that it is an autosomal dominant disorder. We recently reported that Kabuki syndrome is caused by mutations in MLL2, a gene that encodes a Trithorax‐group histone methyltransferase, a protein important in the epigenetic control of active chromatin states. Here, we report on the screening of 110 families with Kabuki syndrome. MLL2 mutations were found in 81/110 (74%) of families. In simplex cases for which DNA was available from both parents, 25 mutations were confirmed to be de novo, while a transmitted MLL2 mutation was found in two of three familial cases. The majority of variants found to cause Kabuki syndrome were novel nonsense or frameshift mutations that are predicted to result in haploinsufficiency. The clinical characteristics of MLL2 mutation‐positive cases did not differ significantly from MLL2 mutation‐negative cases with the exception that renal anomalies were more common in MLL2 mutation‐positive cases. These results are important for understanding the phenotypic consequences of MLL2 mutations for individuals and their families as well as for providing a basis for the identification of additional genes for Kabuki syndrome.

High frequency of p53 mutations in human oral epithelial dysplasia and primary squamous cell carcinoma detected by yeast functional assay

To determine the timing and actual incidence of p53 mutations in oral epithelial lesions, we examined 33 primary squamous cell carcinomas (SCCs), 14 dysplasias and six hyperplasias from Japanese patients by a combination of yeast functional assay and DNA sequencing. The assay detects mutations of p53 mRNA between codons 67 and 347 on the basis of the DNA-binding activity of the protein. Twenty-six SCCs (79%) and five dysplasias (36%) were positive for p53 mutation, while all six hyperplasias were negative for the mutation. Human papillomavirus type 16 E6 mRNA was detected in one of seven p53 mutation-negative SCCs by reverse transcription polymerase chain reaction (RT – PCR). We further examined p53 mutations in 17 Sri Lankan oral SCCs using the yeast functional assay and the single-strand conformation polymorphism analysis of PCR-amplified DNA fragments (PCR – SSCP) of exon 5 – 8. The mutations were confirmed by DNA sequencing and the detection sensitivity was compared between the two methods. Six samples (35%) were positive for p53 mutation in PCR – SSCP analysis, while nine samples (53%) were positive in yeast functional assay. This suggests that the incidence of p53 mutations has been considerably underestimated in the conventional SSCP analysis. The present data indicate that p53 mutations are extremely frequent in oral cancers in the Japanese, and suggest that the timing and significance of p53 mutation in oral tumor progression vary in different ethnic populations and areas.

New p57KIP2 mutations in Beckwith-Wiedemann syndrome.

Abstract Beckwith-Wiedemann syndrome (BWS) is characterized by numerous growth abnormalities and an increased risk of childhood tumors. The gene for BWS is localized in the 11p15.5 region, as determined by linkage analysis of autosomal dominant pedigrees. The increased maternal transmission pattern seen in the autosomal dominant-type pedigrees and the findings of paternal uniparental disomy reported for a subgroup of patients indicate that the gene for BWS is imprinted. Previously, we found p57KIP2, which is a Cdk-kinase inhibitor located at 11p15, is mutated in two BWS patients. Here, we screened for the mutation of the gene in 15 BWS patients.

Identification of an autoimmune enteropathy-related 75-kilodalton antigen.

BACKGROUND & AIMS We have previously reported a 75-kilodalton autoantigen specific to X-linked autoimmune enteropathy (AIE) associated with tubulonephropathy. The aim of this study was to identify the autoantigen. METHODS Complementary DNA (cDNA) clones were isolated by immunoscreening a human duodenal cDNA-expression library with serum from a patient with AIE. RESULTS cDNA encoding the 75-kilodalton antigen (AIE-75) was identified. The composite nucleotide sequence of the cDNA for AIE-75 was 2214 base pairs long and encoded 552 amino acids. The genomic sequence of AIE-75 was found in Sequence DataBank, which consisted of 21 exons and was located on the chromosome 11p14.3. Recombinant AIE-75 specifically reacted with sera from 3 of 4 unrelated patients with AIE but not with 58 control sera. AIE-75 was predominantly distributed in the epithelial cells of the luminal surface and the upper half of the crypts of the intestine and in the proximal renal tubulus. Similarity searches revealed that the AIE-75 cDNA sequence was an authentic form of several colon cancer-related cDNAs of unknown function. The deduced amino acid sequence contained 3 conserved PSD-95/Dlg/ZO-1 (PDZ) domains. CONCLUSIONS AIE-75 is a PDZ domain-containing protein expressed in the differentiated epithelial cells of the intestine and kidney and may be involved in protein-protein interaction. The identification of the autoantigen may prove useful in the approach to the pathogenesis of this poorly understood disease.

MLL2 and KDM6A mutations in patients with Kabuki syndrome

Kabuki syndrome is a congenital anomaly syndrome characterized by developmental delay, intellectual disability, specific facial features including long palpebral fissures and ectropion of the lateral third of the lower eyelids, prominent digit pads, and skeletal and visceral abnormalities. Mutations in MLL2 and KDM6A cause Kabuki syndrome. We screened 81 individuals with Kabuki syndrome for mutations in these genes by conventional methods (n = 58) and/or targeted resequencing (n = 45) or whole exome sequencing (n = 5). We identified a mutation in MLL2 or KDM6A in 50 (61.7%) and 5 (6.2%) cases, respectively. Thirty‐five MLL2 mutations and two KDM6A mutations were novel. Non‐protein truncating‐type MLL2 mutations were mainly located around functional domains, while truncating‐type mutations were scattered through the entire coding region. The facial features of patients in the MLL2 truncating‐type mutation group were typical based on those of the 10 originally reported patients with Kabuki syndrome; those of the other groups were less typical. High arched eyebrows, short fifth finger, and hypotonia in infancy were more frequent in the MLL2 mutation group than in the KDM6A mutation group. Short stature and postnatal growth retardation were observed in all individuals with KDM6A mutations, but in only half of the group with MLL2 mutations.

Novel and recurrent TRPV4 mutations and their association with distinct phenotypes within the TRPV4 dysplasia family

Background Mutations in TRPV4, a gene that encodes a Ca2+ permeable non-selective cation channel, have recently been found in a spectrum of skeletal dysplasias that includes brachyolmia, spondylometaphyseal dysplasia, Kozlowski type (SMDK) and metatropic dysplasia (MD). Only a total of seven missense mutations were detected, however. The full spectrum of TRPV4 mutations and their phenotypes remained unclear. Objectives and methods To examine TRPV4 mutation spectrum and phenotype−genotype association, we searched for TRPV4 mutations by PCR-direct sequencing from genomic DNA in 22 MD and 20 SMDK probands. Results TRPV4 mutations were found in all but one MD subject. In total, 19 different heterozygous mutations were identified in 41 subjects; two were recurrent and 17 were novel. In MD, a recurrent P799L mutation was identified in nine subjects, as well as 10 novel mutations including F471del, the first deletion mutation of TRPV4. In SMDK, a recurrent R594H mutation was identified in 12 subjects and seven novel mutations. An association between the position of mutations and the disease phenotype was also observed. Thus, P799 in exon 15 is a hot codon for MD mutations, as four different amino acid substitutions have been observed at this codon; while R594 in exon 11 is a hotspot for SMDK mutations. Conclusion The TRPV4 mutation spectrum in MD and SMDK, which showed genotype−phenotype correlation and potential functional significance of mutations that are non-randomly distributed over the gene, was presented in this study. The results would help diagnostic laboratories establish efficient screening strategies for genetic diagnosis of the TRPV4 dysplasia family diseases.

Four novel NIPBL mutations in Japanese patients with Cornelia de Lange syndrome

Cornelia de Lange syndrome (CdLS, OMIM #122470) is a multiple congenital anomaly syndrome characterized by dysmorphic facial features, hirsutism, severe growth and developmental delay, and malformed upper limbs [Ireland et al., 1993; Jackson et al., 1993]. The prevalence is estimated to be 1/10,000 [Opitz, 1985]. Recently, two independent groups proved that CdLS is caused by NIPBL mutations [Krantz et al., 2004; Tonkin et al., 2004]. NIPBL consists of 47 exons and encodes delangin, a 2,804 amino-acid protein, from exon 2 to 47. We analyzed 15 Japanese sporadic patients (CdL 1–15) with typical CdLS features (Table I) and their parents after obtaining written informed consent. All protocols in this study were approved by the Committee for the Ethical Issues on Human Genome and Gene analysis, Nagasaki University. Clinical geneticists diagnosed these patients based on mental and growth retardation, and characteristic facial features. Genomic DNA was extracted using a standard protocol. Fourty-six coding exons (from exon 2 to 47) of NIPBL were amplified by PCR as described previously [Krantz et al., 2004] except for exons 4, 33, 37, and 41, of which primers were originally designed (available on request). Sequence analysis was performed as described previously [Kurotaki et al., 2003]. We identified three novel nonsense mutations and one missense mutation in NIPBL among the 15 Japanese patients examined: 1885C>T (R629X) (CdL 4) and 1921G>T (E641X) (CdL 2) in exon 10, 3346G>T (E1116X) (CdL 15) in exon 12, and 5483G>A (R1828Q) (CdL 10) in exon 29. All the four mutations were not found in any of 97 normal Japanese controls or in the JSNP database (http://snp.ims.u-tokyo.ac.jp/). The altered amino acid (R1828Q) was de novo and located in the evolutionally conserved sequences at least in the human, rat, mouse, and fly homologs, thus the change is likely to be pathological. The C-terminal half 1500 amino acids of delangin is well conserved among homologs of flies, worms, plants, and fungi, and is expected to be biologically important [Tonkin et al., 2004], though it was not found to contain any obvious functional domains by analysis using PROSITE (http://kr. expasy.org/cgi-bin/prosite/PSScan.cgi). Three protein truncation mutations at amino acid positions 629, 641, and 1116 and a missense mutation at amino acid position 1828 could lose or impair the C-terminal half function. The Drosophila homolog of NIPBL, Nipped-B, is involved in activating the Ubx and Cut homeobox genes. Ubx suppresses the limb formation by repressing Dll that requires for the distal limb development, and Cut mutations cause leg and wing abnormalities [Tonkin et al., 2004]. Thus, it is plausible that reduced expression of human NIPBL may lead to limb anomalies in CdLS. Interestingly, limb abnormalities (oligodactyly and ulner deficiency) were observed in three of our four patients with a mutation, but only one of seven patients without any mutation whose clinical information was available did show some limb abnormality (oligodactyly), though Gillis et al. [2004] reported that severity of limb defects was not statistically different between mutation-positive and mutation-negative patients. Additionally, three single nucleotide polymorphisms (SNPs), 1151A>G (N384S) in exon 9, 2021A>G (N674S) in exon 10 and 5874T>C (S1958S) in exon 33, were identified, as they were found among normal controls and the second substitution (2021A>G) was previously reported as a SNP [Gillis et al., 2004]. Allele frequencies of the three SNPs in normal Japanese controls are 3.2% (6/186), 13.0% (25/192), and 64.5% (129/200), respectively. To exclude a submicroscopic deletion around NIPBL and its franking regions, fluorescence in situ hybridization (FISH) analysis was performed in 10 of 15 cases on their metaphase chromosomes using two BAC clones covering the NIPBL gene (Table I), RP11-14I21, and RP11-7M4, selected from the UCSC genome browser, 2003 July version (http://genome.ucsc.edu/ cgi-bin/hgGateway). FISH and subsequent photomicroscopy were performed as described previously [Miyake et al., 2004]. However, none of them showed any deletion. We also investigated core promoter regions in 11 affected individuals not having detectable point mutations in the coding regions. Two core promoter regions were identified, ranging 800 to 500 bp (CPR-A) and 400 to þ 200 bp (CPRB) from the beginning of NIPBL cDNA (NM_015384.3) using four different promoter prediction programs: neural network promoter prediction program (http://www.fruitfly.org/seq_ tools/promoter.html), human core-promoter finder (http:// rulai.cshl.org/tools/genefinder/CPROMOTER/human.htm), promoter 2.0 prediction server (http://www.cbs.dtu.dk/services/ promoter/), bioinformatics & molecular analysis section (http:// bimas.dcrt.nih.gov/molbio/proscan/). No nucleotide changes were detected among the 11 patients in the two core promoter regions except for a part of CPR-B sequence ( 60 þ 60), which was hardly determined due to high GC ratio (75.83%), suggesting that promoter mutations in NIBPL is less likely. In conclusion, we identified four novelNIPBLmutations and three SNPs. It is important to describe a full spectrum of phenotype in more patients with positive mutations and establish comprehensive diagnostic criteria. *Correspondence to: Dr. Naomichi Matsumoto, Department of Human Genetics, Yokohama City University Graduate School of Medicine, Fukuura 3-9, Kanazawa-ku, Yokohama 236-0004, Japan. E-mail: naomat@yokohama-cu.ac.jp

Distinct prognostic values of p53 mutations and loss of estrogen receptor and their cumulative effect in primary breast cancers.

A total of 76 primary breast cancers were screened for p53 mutations using the yeast p53 functional assay, and the mutations were determined by DNA sequencing. Clonal mutations of p53 were detected in 30 tumors (39%). Immunohistochemical staining for nuclear p53 accumulation performed on the yeast assay‐positive cases clearly differentiated missense mutations in the DNA binding domain (contact mutant; 17 cases) as positive stain and nonsense‐type mutations or missense mutations that may affect 3D‐structure of p53 protein (structural mutant; 13 cases) as negative stain. Enzyme immunoassay revealed loss of estrogen receptor in 36 tumors (50%). Prognostic values of p53 mutation and loss of estrogen receptor were evaluated after a median follow‐up period of 44 months. p53 mutations were associated with a short overall survival (log rank test, p = 0.0319), whereas it was not related to disease‐free (recurrence‐free) survival. Contact mutants were associated with slightly shorter survival compared with structural mutants. Inversely, loss of estrogen receptor was associated with early recurrence (p = 0.0461) but not with short overall survival. The patients with tumors harboring both p53 mutation and loss of estrogen receptor had the poorest outcome (p = 0.0019 and 0.0075 for overall and disease‐free survivals, respectively), suggesting independent and additive effects of the 2 factors. The independent role of the 2 factors was confirmed by a multivariate analysis using the Cox proportional hazard model stratified according to clinical tumor stages. Although preliminary, due to the small number of patients studied and the relatively short follow‐up time, our results suggest that p53 mutations and loss of estrogen receptor cooperatively affect the prognosis of primary breast cancer patients. Int. J. Cancer (Pred. Oncol.) 89:92–99, 2000.

Standard growth curves for Japanese patients with Prader‐Willi syndrome

We constructed the standard growth (length/height and weight) curves for Japanese individuals with Prader-Willi syndrome (PWS). Crude height and weight data were collected from 153 males and 99 females with the syndrome, and the collected data were arranged by a mathematical method to construct the curves. Height growth patterns were quite different between PWS and normal children. Mean height of individuals with the syndrome by puberty is -2 SD for normal children, and it drops off far below -2 SD value after puberty. Final mean height is 141.2 +/- 4.8 cm for females (n = 13) and 147.7 +/- 7.7 cm for males (n = 17), showing 15.8 and 21.9 cm below the average height for normal Japanese girls and boys, respectively. Thus, the degree of shortness is more pronounced in male than in female patients. There was no difference in height between those with chromosome 15q deletion and those without. Mean weight at birth of girls (n = 88) and boys (n = 131) were 2.70 +/- 0.45 Kg and 2.62 +/- 0.47 Kg, respectively. These values were smaller than those for normal neonates (P < 0.05, t-test). The weight of PWS children was under the mean value for normal infants by age 2 years, but gradually increase above the mean values for normal children around ages 2-4 years. Overweight in both males and females becomes obvious during prepuberty. Growth patterns are not different between Japanese and Caucasian children with the syndrome. Short stature is more prominent in boys of both ethnic groups, whereas the degree of overweight appears much more severe in Caucasians.