A Patient with Kabuki Syndrome Mutation Presenting with Very Severe Aplastic Anemia.

Kabuki syndrome (KS) is a rare congenital disorder commonly complicated by humoral immunodeficiency. Patients with KS present with mutation in the histone-lysine N-methyltransferase 2D (KMT2D) gene. Although various KMT2D mutations are often identified in lymphoma and leukemia, those encountered in aplastic anemia (AA) are limited. Herein, we present the case of a 45-year-old Japanese man who developed severe pancytopenia and hypogammaglobulinemia. He did not present with any evident malformations, intellectual disability, or detectable levels of autoantibodies. However, B-cell development was impaired. Therefore, a diagnosis of very severe AA due to a hypoplastic marrow, which did not respond to granulocyte colony-stimulating factor, was made. The patient received umbilical cord blood transplantation but died from a Pseudomonas infection before neutrophil engraftment. Trio whole-exome sequencing revealed a novel missense heterozygous mutation c.15959G >A (p.R5320H) in exon 50 of the KMT2D gene. Moreover, Sanger sequencing of peripheral blood and bone marrow mononuclear cells and a skin biopsy specimen obtained from this patient identified this heterozygous mutation, suggesting that de novo mutation associated with KS occurred in the early embryonic development. Our case showed a novel association between KS mutation and adult-onset AA.

Comprehensive analysis for detecting radiation-specific molecules expressed during radiation-induced rat thyroid carcinogenesis.

Although the association between radiation exposure and thyroid carcinogenesis is epidemiologically evident, 'true' radiation-induced cancers cannot be identified from biological evidence of radiation-associated cases. To assess the individual risk for thyroid cancer due to radiation exposure, we aimed to identify biomarkers that are specifically altered during thyroid carcinogenesis after irradiation in a time-dependent manner in an animal model. Thyroid glands were obtained from rats (n = 175) at 6-16 months after local X-ray (0.1-4 Gy) irradiation of the neck at 7 weeks of age. The gene expression profile in thyroid glands was comprehensively analyzed using RNA microarray. Subsequently, the expression levels of the genes of interest were verified using droplet digital PCR (ddPCR). The expression level of candidate genes as biomarkers for irradiated thyroid was examined in a randomized, controlled, double-blind validation study (n = 19) using ddPCR. The incidence of thyroid cancer increased in a dose- and time-dependent manner and was 33% at 16 months after irradiation with 4 Gy. The Ki-67 labeling index in non-tumorous thyroid was significantly higher in the exposed group than in the control. Comprehensive analysis identified radiation-dependent alteration in 3329 genes. Among them, ddPCR revealed a stepwise increase in CDKN1A expression from early pre-cancerous phase in irradiated thyroid compared to that in the control. The irradiated thyroids were accurately distinguished (positive predictive value 100%, negative predictive value 69%) using 11.69 as the cut-off value for CDKN1A/ß-actin. Thus, CDKN1A expression can be used as a biomarker for irradiated thyroid glands at the pre-cancerous phase.

Progressive Massive Splenomegaly in an Adult Patient with Kabuki Syndrome Complicated with Immune Thrombocytopenic Purpura.

Kabuki syndrome is characterized by multiple systemic anomalies and intellectual disability. It is complicated with immunodeficiencies and autoimmune disorders. The syndrome is caused by a mutation in the KMT2D gene. We herein report a case of a Kabuki syndrome with developing immune thrombocytopenic purpura (ITP) and progressive splenomegaly. Laparoscopic splenectomy was performed and the patients' symptoms quickly disappeared with platelet recovery. After this operation, the patient had no severe complications. A sequence analysis of the KMT2D gene identified a pathogenic mutation frequently associated with ITP. Laparoscopic splenectomy is therefore considered to be a good therapeutic option for recurrent ITP and symptomatic splenomegaly with Kabuki syndrome.

TERT mRNA Expression as a Novel Prognostic Marker in Papillary Thyroid Carcinomas.

Background: Telomerase reverse transcriptase (TERT) promoter mutations have been found in a subset of papillary thyroid carcinomas (PTCs) and are associated with tumor aggressiveness and worse prognosis. However, little is known about the status of TERT mRNA expression and its relationship between TERT promoter mutations and clinicopathological features. Methods: We analyzed 159 PTC samples for TERT promoter mutations using direct DNA sequencing. TERT expression was measured using quantitative reverse transcription polymerase chain reaction. To examine low allelic frequency of TERT promoter mutations with high sensitivity, we used droplet digital polymerase chain reaction (ddPCR). The relationship between the status of the TERT promoter mutation/expression and clinicopathological features including recurrence risk was statistically analyzed. Results:TERT promoter mutations were found in 20 cases (12.6%). However, TERT expression was observed not only in the mutation-positive tumors but also in 56 of 139 (40.3%) mutation-negative tumors. Among them, we detected low allelic frequency of TERT promoter mutations in three samples (5.4%) using ddPCR. We confirmed a significant association between TERT promoter mutations and aggressive clinicopathological features in this series. The risk of recurrence of TERT mutation-negative/expression-positive tumors was significantly higher than that of the mutation-negative/expression-negative tumors, suggesting that TERT expression even in absence of a mutation confers a negative influence on PTCs. Moreover, when we reclassified the mutation-negative cases into two groups based on the TERT expression levels: expression-negative/expression levels <80th percentile and expression levels >80th percentile because minimal expression may have a negligible clinical impact, a higher hazard ratio for recurrence was observed. Interestingly, TERT expression levels in the mutation-negative PTCs were inversely correlated with patient age and the presence of BRAF mutations. Conclusions: We confirm a strong correlation between the presence of TERT promoter mutations and aggressive clinicopathological features in this PTC series. In addition, there were PTCs showing high TERT mRNA expression even in the absence of TERT promoter mutations. These cases also showed a significantly higher recurrence rate. Since the TERT promoter mutations are observed only in elderly patients, TERT mRNA expression can be a useful prognostic marker especially in younger PTC patients.

Two consecutive successful live birth in woman with 17α hydroxylase deficiency by frozen-thaw embryo transfer under hormone replacement endometrium preparation.

17α-Hydroxylase deficiency is rare autosomal recessive disorder that manifested by hypertension, hypokalemia, delayed sexual development, primary amenorrhea and infertility. The information regarding infertility care and conception in women with this disorder are extremely limited. We report a 24-year-old Japanese woman with primary amenorrhea who was diagnosed as partial 17α-hydroxylase deficiency caused by homozygous 3 bp deletion in exon 1 of 17α-hydroxylase gene. In vitro fertilization with controlled ovarian stimulation was carried out and all viable embryo were frozen. During ovarian stimulation, serum progesterone levels were markedly elevated, and endometrial growth was impaired. Utilizing frozen-thaw embryo transfer under hormonal replacement (glucocorticoid, estradiol and progesterone), she had successfully given two consecutive live birth. Women with 17α-hydroxylase deficiency with residual ovarian reserve can afford reproductive success by appropriate diagnosis and treatment by assisted reproductive technology.

Ultra-sensitive droplet digital PCR for detecting a low-prevalence somatic GNAQ mutation in Sturge-Weber syndrome.

Droplet digital PCR (ddPCR), a method for measuring target nucleic acid sequence quantity, is useful for determining somatic mutation rates using TaqMan probes. In this study, the detection limit of copy numbers of test DNA by ddPCR was determined based on Poisson distribution. Peptide nucleic acid (PNA), which strongly hybridises to target lesions, can inhibit target amplification by PCR. Therefore, by combination of PCR with PNA and ddPCR (PNA-ddPCR), the detection limit could be lowered. We reanalysed a somatic GNAQ mutation (c.548G > A) in patients with Sturge-Weber syndrome (SWS) using ddPCR and PNA-ddPCR. Importantly, among three patients previously found to be mutation negative by next-generation sequencing, two patients had the GNAQ mutation with a mutant allele frequency of less than 1%. Furthermore, we were able to find the same mutation in blood leukocyte or saliva DNA derived from four out of 40 SWS patients. Vascular anomalies and blood leukocytes originate from endothelial cells and haemangioblasts, respectively, which are both of mesodermal origin. Therefore, blood leukocytes may harbour the GNAQ mutation, depending on the time when the somatic mutation is acquired. These data suggest the possibility of diagnosis using blood DNA in some patients with SWS.

Heterozygous mutations in cyclic AMP phosphodiesterase-4D (PDE4D) and protein kinase A (PKA) provide new insights into the molecular pathology of acrodysostosis.

Acrodysostosis without hormone resistance is a rare skeletal disorder characterized by brachydactyly, nasal hypoplasia, mental retardation and occasionally developmental delay. Recently, loss-of-function mutations in the gene encoding cAMP-hydrolyzing phosphodiesterase-4D (PDE4D) have been reported to cause this rare condition but the pathomechanism has not been fully elucidated. To understand the pathogenetic mechanism of PDE4D mutations, we conducted 3D modeling studies to predict changes in the binding efficacy of cAMP to the catalytic pocket in PDE4D mutants. Our results indicated diminished enzyme activity in the two mutants we analyzed (Gly673Asp and Ile678Thr; based on PDE4D4 residue numbering). Ectopic expression of PDE4D mutants in HEK293 cells demonstrated this reduction in activity, which was identified by increased cAMP levels. However, the cells from an acrodysostosis patient showed low cAMP accumulation, which resulted in a decrease in the phosphorylated cAMP Response Element-Binding Protein (pCREB)/CREB ratio. The reason for this discrepancy was due to a compensatory increase in expression levels of PDE4A and PDE4B isoforms, which accounted for the paradoxical decrease in cAMP levels in the patient cells expressing mutant isoforms with a lowered PDE4D activity. Skeletal radiographs of 10-week-old knockout (KO) rats showed that the distal part of the forelimb was shorter than in wild-type (WT) rats and that all the metacarpals and phalanges were also shorter in KO, as the name acrodysostosis implies. Like the G-protein α-stimulatory subunit and PRKAR1A, PDE4D critically regulates the cAMP signal transduction pathway and influences bone formation in a way that activity-compromising PDE4D mutations can result in skeletal dysplasia. We propose that specific inhibitory PDE4D mutations can lead to the molecular pathology of acrodysostosis without hormone resistance but that the pathological phenotype may well be dependent on an over-compensatory induction of other PDE4 isoforms that can be expected to be targeted to different signaling complexes and exert distinct effects on compartmentalized cAMP signaling.

Transforming growth factor beta1 (TGFß1) polymorphisms and breast cancer risk.

Transforming growth factor ß1 (TGFß1) is suggested to be involved in the pathogenesis of and in complications with breast cancer (BC). Polymorphisms in TGFß1 gene (TGFß1) have been suggested by many investigators to have a role in susceptibility to BC; however, many discordant data have been reported. Considering the role of ethnic variations, we performed an association study between TGFß1 polymorphisms and BC among Iranian women. We sequenced DNA samples of 110 BC and 110 normal control women for the exons and their adjacent intronic regions of TGFß1 using PCR. The allele, genotype, and haplotype frequencies were calculated using PowerMarker V3.25 and R 3.0.2 softwares. Ten single nucleotide polymorphisms (SNPs) were detected. Statistical analysis on the frequency of seven most frequent SNPs, including the three coding SNPs (cSNPs) revealed no significant difference between BC and control women. Moreover, among 11 constructed haplotypes, "GTGCCGC" was significantly different between two study groups. In conclusion, we found no association between the studied SNPs of TGFß1 and BC among Iranian women, but a possible association between "GTGCCGC" haplotype and BC was seen. However, further studies are suggested to clarify this association.

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.

Coding region polymorphisms in the indoleamine 2,3-dioxygenase (INDO) gene and recurrent spontaneous abortion.

Indoleamine 2,3-dioxygenase (INDO) catalyzes degradation of the indole ring of indoleamines and locally depletes tryptophan. INDO expression suppresses T cell proliferation and activation. Genetic variation in the INDO gene may contribute to the variable INDO enzyme expression, activity and severity of some diseases. Recurrent spontaneous abortion (RSA) is a common pregnancy complication and the exact causes of RSA are not yet known. We performed an association study between INDO single nucleotide polymorphisms (SNPs) and RSA. To identify INDO SNPs we sequenced DNA samples for ten exons and adjacent intronic regions from 111 RSA patients. Consequently 10 SNPs were detected; four in exons (one in exon 4, two in exon 9 and one in exon 10) and six in intronic regions (one in intron 3, three in intron 6, one in intron 8 and one in intron 9). Three (IVS3+562 del C, IVS8+116 T→G and IVS9+2431 G→A) of these ten SNPs have been registered at the NCBI SNP database. Statistical analysis of allele, genotype and haplotype frequency distribution in the three most frequent SNPs (IVS3+562 del C, IVS6+61 G→A and IVS9+2431 G→A) showed no significant differences between the 111 RSA and 105 matched control women. CGA and CGG were the most frequent haplotypes in both the RSA and control groups. We conclude that there is no association between INDO polymorphisms and susceptibility of Iranian women to RSA.

Developmentally dynamic changes of DNA methylation in the mouse Snurf/Snrpn gene.

The mouse Snurf/Snrpn gene has two differentially methylated regions (DMRs), the maternally methylated region at the 5' end (DMR1) and the paternally methylated region at the 3' end (DMR2). DMR1, a region that includes the Snrpn promoter and the entire intron 1, has been thought to be a germline DMR, which inherits the parental-specific methylation profile from the gametes. DMR1 is not only associated with imprinted Snrpn expression, but implicated in imprinting control of other genes in the region. We have now characterized the highly conserved activator sequence (CAS) in the Snrpn intron 1 among human and rodents and demonstrate that the mouse CAS is not a germline DMR but shows developmentally dynamic changes of DNA methylation and has methylation-sensitive enhancer activity. The tissue-specific methylation of the mouse CAS and its methylation-sensitive enhancer activity may control tissue-specific expression of IC transcripts, resulting in the establishment and/or maintenance of imprinting in the Snrpn locus.

Mutations in CD96, a member of the immunoglobulin superfamily, cause a form of the C (Opitz trigonocephaly) syndrome.

The C syndrome is characterized by trigonocephaly and associated anomalies, such as unusual facies, psychomotor retardation, redundant skin, joint and limb abnormalities, and visceral anomalies. In an individual with the C syndrome who harbors a balanced chromosomal translocation, t(3;18)(q13.13;q12.1), we discovered that the TACTILE gene for CD96, a member of the immunoglobulin superfamily, was disrupted at the 3q13.3 breakpoint. In mutation analysis of nine karyotypically normal patients given diagnoses of the C or C-like syndrome, we identified a missense mutation (839C-->T, T280M) in exon 6 of the CD96 gene in one patient with the C-like syndrome. The missense mutation was not found among 420 unaffected Japanese individuals. Cells with mutated CD96 protein (T280M) lost adhesion and growth activities in vitro. These findings indicate that CD96 mutations may cause a form of the C syndrome by interfering with cell adhesion and growth.

Role of DNA methylation and histone H3 lysine 27 methylation in tissue-specific imprinting of mouse Grb10.

Mouse Grb10 is a tissue-specific imprinted gene with promoter-specific expression. In most tissues, Grb10 is expressed exclusively from the major-type promoter of the maternal allele, whereas in the brain, it is expressed predominantly from the brain type promoter of the paternal allele. Such reciprocally imprinted expression in the brain and other tissues is thought to be regulated by DNA methylation and the Polycomb group (PcG) protein Eed. To investigate how DNA methylation and chromatin remodeling by PcG proteins coordinate tissue-specific imprinting of Grb10, we analyzed epigenetic modifications associated with Grb10 expression in cultured brain cells. Reverse transcriptase PCR analysis revealed that the imprinted paternal expression of Grb10 in the brain implied neuron-specific and developmental stage-specific expression from the paternal brain type promoter, whereas in glial cells and fibroblasts, Grb10 was reciprocally expressed from the maternal major-type promoter. The cell-specific imprinted expression was not directly related to allele-specific DNA methylation in the promoters because the major-type promoter remained biallelically hypomethylated regardless of its activity, whereas gametic DNA methylation in the brain type promoter was maintained during differentiation. Histone modification analysis showed that allelic methylation of histone H3 lysine 4 and H3 lysine 9 were associated with gametic DNA methylation in the brain type promoter, whereas that of H3 lysine 27 regulated by the Eed PcG complex was detected in the paternal major-type promoter, corresponding to its allele-specific silencing. Here, we propose a molecular model that gametic DNA methylation and chromatin remodeling by PcG proteins during cell differentiation cause tissue-specific imprinting in embryonic tissues.

A -16C>T substitution in the 5' UTR of the puratrophin-1 gene is prevalent in autosomal dominant cerebellar ataxia in Nagano.

The molecular bases of autosomal dominant cerebellar ataxia (ADCA) have been increasingly elucidated, but 17-50% of ADCA families still remain genetically undefined in Japan. In this study we investigated 67 genetically undefined ADCA families from the Nagano prefecture, and found that 63 patients from 51 families possessed the -16C>T change in the puratrophin-1 gene, which was recently found to be pathogenic for 16q22-linked ADCA. Most patients shared a common haplotype around the puratrophin-1 gene. All patients with the -16C>T change had pure cerebellar ataxia with middle-aged or later onset. Only one patient in a large, -16C>T positive family did not have this change, but still shared a narrowed haplotype with, and was clinically indistinguishable from, the other affected family members. In Nagano, 16q22-linked ADCA appears to be much more prevalent than either SCA6 or dentatorubral-pallidoluysian atrophy (DRPLA), and may explain the high frequency of spinocerebellar ataxia.

BAC array CGH reveals genomic aberrations in idiopathic mental retardation.

Array using 2,173 BAC clones covering the whole human genome has been constructed. All clones spotted were confirmed to show a unique signal at the predicted chromosomal location by FISH analysis in our laboratory. A total of 30 individuals with idiopathic mental retardation (MR) were analyzed by comparative genomic hybridization using this array. Three deletions, one duplication, and one unbalanced translocation could be detected in five patients, which are likely to contribute to MR. The constructed array was shown to be an efficient tool for the detection of pathogenic genomic rearrangements in MR patients as well as copy number polymorphisms (CPNs).

Lack of association between the TGF-beta1 gene polymorphisms and recurrent spontaneous abortion.

Transforming growth factor-beta1 (TGF-beta1) is produced by T regulatory lymphocytes (Treg), which play an important role in the physiology of pregnancy. Several polymorphisms of the TGF-beta1 gene (TGFB1) have been reported, some with an important correlation with TGF-beta1 production and disease severity. We performed an association study between TGFB1 polymorphisms and recurrent spontaneous abortion (RSA). We first used a PCR-RFLP method to detect three known TGFB1 cSNPs (coding single nucleotide polymorphisms) among 111 RSA and 110 normal control women from Southern Iran, such as 29T-->C (Leu 10 Pro), 74G-->C (Arg 25 Pro) and 788C-->T (Thr 263Ile), and compared their frequencies between the two groups of subjects. To confirm results of the RFLP study and to identify new SNPs in the RSA women, we then sequenced their DNA samples for seven exons and adjacent intronic regions of TGFB1. Consequently, 10 SNPs were detected; one (-14G-->A) was located in the upstream region of exon 1, three in exons (two in exon 1 and one in exon 5) and six in intronic regions. Two (IVS5+18G-->C and IVS6+910G-->A) of the 10 SNPs were novel. Statistical analysis on the frequency of six most frequent SNPs, including the three cSNPs, as well as on the frequencies of genotypes and 13 haplotypes regarding the 6 SNPs, revealed no significant difference between RSA and control women. Therefore, this study concludes that there is no association between exonic and adjacent intronic polymorphisms of TGFB1 and RSA.

Neuron-specific relaxation of Igf2r imprinting is associated with neuron-specific histone modifications and lack of its antisense transcript Air.

The mouse insulin-like growth factor II receptor (Igf2r) gene and its antisense transcript Air are reciprocally imprinted in most tissues, but in the brain, Igf2r is biallelically expressed despite the imprinted Air expression. To investigate the molecular mechanisms of such brain-specific relaxation of Igf2r imprinting, we analyzed its expression and epigenetic modifications in neurons, glial cells and fibroblasts by the use of primary cortical cell cultures. In glial cells and fibroblasts, Igf2r was maternally expressed and Air was paternally expressed, whereas in the primary cultured neurons, Igf2r was biallelically expressed and Air was not expressed. In the differentially methylated region 2 (DMR2), which includes the Air promoter, allele-specific DNA methylation, differential H3 and H4 acetylation and H3K4 and K9 di-methylation were maintained in each cultured cell type. In DMR1, which includes the Igf2r promoter, maternal-allele-specific DNA hypomethylation, histones H3 and H4 acetylation and H3K4 di-methylation were apparent in glial cells and fibroblasts. However, in neurons, biallelic DNA hypomethylation and biallelic histones H3 and H4 acetylation and H3K4 di-methylation were detected. These data indicate that lack of reciprocal imprinting of Igf2r and Air in the brain results from neuron-specific relaxation of Igf2r imprinting associated with neuron-specific histone modifications in DMR1 and lack of Air expression. Our observation of biallelic Igf2r expression with no Air expression in neurons sheds light on the function of Air as a critical effector in Igf2r silencing and suggests that neuron-specific epigenetic modifications related to the lineage determination of neural stem cells play a critical role in controlling imprinting by antisense transcripts.