An aggressive systemic mastocytosis preceded by ovarian dysgerminoma.

BACKGROUND: Aggressive systemic mastocytosis (ASM) is a rare malignant disease characterized by disordered mast cell accumulation in various organs. We here describe a female ASM patient with a previous history of ovarian dysgerminoma. METHODS: Molecular cytogenomic analyses were performed to elucidate an etiological link between the ASM and dysgerminoma of the patient. RESULTS: This patient was affected by ovarian dysgerminoma which was treated by chemotherapy and surgical resection. Having subsequently been in complete remission for 2 years, she developed symptoms of ASM. A somatic D816A mutation in the KIT gene was detected in her bone marrow, which facilitated the diagnosis of ASM. Unexpectedly, this KIT D816A variant was also detected in the prior ovarian dysgerminoma sample. Whole-exome sequencing allowed us to identify a somatic nonsense mutation of the TP53 gene in the bone marrow, but not in the dysgerminoma. Microarray analysis of the patient's bone marrow revealed a copy-number-neutral loss of heterozygosity at the TP53 locus, suggestive of the homozygous nonsense mutation in the TP53 gene. In addition, the loss of heterozygosity at the TP53 locus was also detected in the dysgerminoma. CONCLUSIONS: These results indicated that either the mast cells causing the ASM in this case had originated from the preceding ovarian dysgerminoma as a clonal evolution of a residual tumor cell, which acquired the TP53 mutation, or that both tumors developed from a common cancer stem cell carrying the KIT D816A variation.

Molecular analysis of low-level mosaicism of the IKBKG mutation using the X Chromosome Inactivation pattern in Incontinentia Pigmenti.

BACKGROUND: Incontinentia pigmenti (IP) is a rare X-linked disorder affecting the skin and other ectodermal tissues that is caused by mutation of the IKBKG/NEMO gene. Previous studies have reported that the overall mutation detection rate in IP is ~75%. We hypothesized that a low-level mosaicism existed in the remaining cases. METHODS: Genomic variations in the IKBKG gene were examined in 30 IP probands and their family members. Standard mutational analyses were performed to detect common deletions, nucleotide alterations, and copy number variations. To assess skewing of the X chromosome inactivation (XCI) pattern, a HUMARA assay was performed. We compared the results of this analysis with phenotype severity. RESULTS: Pathogenic variants were identified in 20 probands (66.7%), the rate of detection was suboptimal. The remaining 10 probands tended to manifest a mild phenotype with no skewed X chromosome inactivation that is generally observed in IP patients. Quantitative nested PCR and digital droplet PCR were performed for the 10 patients and mosaicism of the common IKBKG deletion were identified in five patients. CONCLUSION: Overall, we detected 25 IKBKG mutations (83.3%). Determination of the XCI value in advance of mutational analyses for IP could improve the mutation detection rate. Our improved detection rate for these mutations, particularly those with a low-level mosaicism, may present opportunities for appropriate genetic counseling.

Impact of DPYD, DPYS, and UPB1 gene variations on severe drug-related toxicity in patients with cancer.

Cancer treatment with a fluoropyrimidine (FP) is often accompanied by severe toxicity that may be dependent on the activity of catalytic enzymes encoded by the DPYD, DPYS, and UPB1 genes. Genotype-guided dose individualization of FP therapy has been proposed in western countries, but our knowledge of the relevant genetic variants in East Asian populations is presently limited. To investigate the association between these genetic variations and FP-related high toxicity in a Japanese population, we obtained blood samples from 301 patients who received this chemotherapy and sequenced the coding exons and flanking intron regions of their DPYD, DPYS, and UPB1 genes. In total, 24 single nucleotide variants (15 in DPYD, 7 in DPYS and 2 in UPB1) were identified including 3 novel variants in DPYD and 1 novel variant in DPYS. We did not find a significant association between FP-related high toxicity and each of these individual variants, although a certain trend toward significance was observed for p.Arg181Trp and p.Gln334Arg in DPYS (P = .0813 and .087). When we focused on 7 DPYD rare variants (p.Ser199Asn, p.IIe245Phe, p.Thr305Lys, p.Glu386Ter, p.Ser556Arg, p.Ala571Asp, p.Trp621Cys) which have an allele frequency of less than 0.01% in the Japanese population and are predicted to be loss-of-function mutations by in silico analysis, the group of patients who were heterozygous carriers of at least one these rare variants showed a strong association with FP-related high toxicity (P = .003). Although the availability of screening of these rare loss-of-function variants is still unknown, our data provide useful information that may help to alleviate FP-related toxicity in Japanese patients with cancer.

Unexpected Mutations by CRISPR-Cas9 CTG Repeat Excision in Myotonic Dystrophy and Use of CRISPR Interference as an Alternative Approach.

Myotonic dystrophy type 1 is the most common type of adult-onset muscular dystrophy. This is an autosomal dominant disorder and caused by the expansion of the CTG repeat in the 3' untranslated region of the dystrophia myotonica protein kinase (DMPK) gene. Messenger RNAs containing these expanded repeats form aggregates as nuclear RNA foci. Then, RNA binding proteins, including muscleblind-like 1, are sequestered to the RNA foci, leading to systemic abnormal RNA splicing. In this study, we used CRISPR-Cas9 genome editing to excise this CTG repeat. Dual cleavage at the 5' and 3' regions of the repeat using a conventional Cas9 nuclease and a double nicking with Cas9 nickase successfully excised the CTG repeat. Subsequently, the formation of the RNA foci was markedly reduced in patient-derived fibroblasts. However, contrary to expectations, a considerable amount of off-target digestions and on-target genomic rearrangements were observed using high-throughput genome-wide translocation sequencing. Finally, the suppression of DMPK transcripts using CRISPR interference significantly decreased the intensity of RNA foci. Our results indicate that close attention should be paid to the unintended mutations when double-strand breaks are generated by CRISPR-Cas9 for therapeutic purposes. Alternative approaches independent of double-strand breaks, including CRISPR interference, may be considered.

The involvement of U-type dicentric chromosomes in the formation of terminal deletions with or without adjacent inverted duplications.

An inverted duplication with a terminal deletion (inv-dup-del) is one of the complex constitutional structural rearrangements that can occur in a chromosome. Although breakages of dicentric chromosome have been suggested, the precise mechanism of this is yet to be fully understood. In our present study, we investigated the genomic structure of 10 inv-dup-del cases to elucidate this mechanism. Two recurrent 8p inv-dup-del cases harbored a large copy-number-neutral region between the duplication and deletion in common. Although the other non-recurrent cases did not appear to have this copy-number-neutral region, refined sequencing analysis identified that they contained a small intervening region at the junction between the inverted and non-inverted segment. The size of this small intervening region ranged from 1741 to 3728 bp. Combined with a presence of microhomology at the junction, a resolution of the replication fork stalling through template switching within the same replication fork is suggested. We further observed two cases with mosaicism of the dicentric chromosome and various structural rearrangements related to the dicentric chromosome. Refined analysis allowed us to identify different breakpoints on the same chromosome in the same case, implicating multiple rounds of U-type formation and its breakage. From these results, we propose that a replication-based mechanism generates unstable dicentric chromosomes and that their breakage leads to the formation of inv-dup-dels and other related derivative chromosomes.

Novel mutation in the KITLG gene in familial progressive hyperpigmentation with or without hypopigmentation.

We herein report a novel mutation in familial progressive hyper- and hypopigmentation (FPHH). The KITLG gene encoding the KIT ligand protein is a disease-causing gene for FPHH. Various disease-causing gain-of-function mutations, which reside within or adjacent to the conserved VTNN motif of this gene, have been described to date. We have now identified a novel KITLG mutation, c.337G>A (p.Glu113Lys), in FPHH which is located within another ligand-receptor interaction site.

A female patient with retinoblastoma and severe intellectual disability carrying an X;13 balanced translocation without rearrangement in the RB1 gene: a case report.

BACKGROUND: Female carriers of a balanced X; autosome translocation generally undergo selective inactivation of the normal X chromosome. This is because inactivation of critical genes within the autosomal region of the derivative translocation chromosome would compromise cellular function. We here report a female patient with bilateral retinoblastoma and a severe intellectual disability who carries a reciprocal X-autosomal translocation. CASE PRESENTATION: Cytogenetic and molecular analyses, a HUMARA (Human androgen receptor) assay, and methylation specific PCR (MSP) and bisulfite sequencing were performed using peripheral blood samples from the patient. The patient's karyotype was 46,X,t(X;13)(q28;q14.1) by G-banding analysis. Further cytogenetic analysis located the entire RB1 gene and its regulatory region on der(X) with no translocation disruption. The X-inactivation pattern in the peripheral blood was highly skewed but not completely selected. MSP and deep sequencing of bisulfite-treated DNA revealed that an extensive 13q region, including the RB1 promoter, was unusually methylated in a subset of cells. CONCLUSIONS: The der(X) region harboring the RB1 gene was inactivated in a subset of somatic cells, including the retinal cells, in the patient subject which acted as the first hit in the development of her retinoblastoma. In addition, the patient's intellectual disability may be attributable to the inactivation of the der(X), leading to a 13q deletion syndrome-like phenotype, or to an active X-linked gene on der (13) leading to Xq28 functional disomy.

Clinical and genetic aspects of mild hypophosphatasia in Japanese patients.

BACKGROUND: Hypophosphatasia (HPP) is a rare inborn error of metabolism that results from a dysfunctional tissue non-specific alkaline phosphatase enzyme (TNSALP). Although genotype-phenotype correlations have been described in HPP patients, only sparse information is currently available on the genetics of mild type HPP. METHODS: We investigated 5 Japanese patients from 3 families with mild HPP (patients 1 and 2 are siblings; patient 4 is a daughter of patient 5) who were referred to Fujita Health University due to the premature loss of deciduous teeth. Physical and dental examinations, and blood, urine and bone density tests were conducted. Genetic analysis of the ALPL gene was performed in all patients with their informed consent. RESULTS: After a detailed interview and examination, we found characteristic symptoms of HPP in some of the study cases. Mobile teeth or the loss of permanent teeth were observed in 2 patients, and 3 out of 5 patients had a history of asthma. The serum ALP levels of all patients were 30% below the lower limit of the age equivalent normal range. ALPL gene analysis revealed compound heterozygous mutations, including Ile395Val and Leu520Argfs in family 1, Val95Met and Gly491Arg in family 2, and a dominant missense mutation (Gly456Arg) in family 3. The 3D-modeling of human TNSALP revealed three mutations (Val95Met, Ile395Val and Gly456Arg) at the homodimer interface. Severe collisions between the side chains were predicted for the Gly456Arg variant. DISCUSSION: One of the characteristic findings of this present study was a high prevalence of coexisting asthma and a high level serum IgE level. These characteristics may account for the fragility of tracheal tissues and a predisposition to asthma in patients with mild HPP. The genotypes of the five mild HPP patients in our present study series included 1) compound heterozygous for severe and hypomorphic mutations, and 2) dominant-negative mutations. All of these mutations were at the homodimer interface, but only the dominant-negative mutation was predicted to cause a severe collision effect between the side chains. This may account for varying mechanisms leading to different effects on TNSALP function.

Prostate Stem Cell Antigen Gene Polymorphism Is Associated with H. pylori-related Promoter DNA Methylation in Nonneoplastic Gastric Epithelium.

Genome-wide association study identified two functional SNPs associated with gastric cancer especially the diffuse type. The first was a polymorphism (rs2294008) in prostate stem cell antigen (PSCA), and the other was a polymorphism (rs4072037) in mucin 1 (MUC1). DNA methylation is associated with gastric cancer and Helicobacter pylori (H. pylori)-induced gastritis, while hypermethylation of promoter CpG island (CGI) is a common characteristic of enlarged-fold gastritis induced by H. pylori, a risk factor of diffuse-type gastric cancer. We evaluated the association between PSCA and MUC1 polymorphisms with H. pylori--related promoter CGI methylation in the nonneoplastic gastric mucosa. PSCA rs2294008 C/T and MUC1 rs4072037 A/G polymorphisms were genotyped in 410 cancer-free subjects in relation to promoter CGI methylation status of three candidate genes, of which the methylation status is associated with H. pylori infection (IGF2, MYOD1, and SLC16A12). Methylation levels of all three genes were significantly higher in subjects with PSCA rs2294008 T/T compared with the PSCA rs2294008 C/C (all P < 0.05). Such associations were more enhanced in H. pylori-positive subjects (all P < 0.01). The multivariate analysis demonstrated that PSCA C/T [OR, 2.37; 95% CI (confidence interval), 1.06-5.29; P = 0.035] and T/T genotypes (OR, 3.2; 95% CI, 1.41-7.25; P = 0.005) were significantly associated with methylation-high gastric mucosa as independent factors. MUC1 rs4072037 A/G polymorphism was not associated with methylation status of all three genes. PSCA C/T and T/T genotypes are associated with H. pylori-related promoter DNA methylation in the gastric mucosa.Impact: Our observations provided the evidence that PSCA polymorphism influence the susceptibility to gastric cancer through DNA methylation induction.

DNA methylation accumulation in gastric mucosa adjacent to cancer after Helicobacter pylori eradication.

Molecular irreversibleness with Helicobacter pylori (H. pylori) infection might have a role in gastric tumorigenesis after H. pylori eradication. We performed comprehensive DNA methylation profiling of gastric mucosa after H. pylori eradication with or without gastric cancer. Using four different groups of biopsies obtained from gastric body without history of H. pylori infection (Hp-), gastric body without cancer after H. pylori eradication (cancer-free body), gastric body with early gastric cancer diagnosed after H. pylori eradication (EGC body) and their paired samples from adjacent mucosa of cancer (EGC ADJ), methylation status of five candidate genes (MYOD1, SLC16A12, IGF2, RORA and PRDM5) was examined by the bisulfite pyrosequencing. An Infinium Methylation EPIC BeadChip array was also used to characterize the methylation status of greater than 850,000 CpG sites. The EGC ADJ group showed highest methylation levels of five candidate genes among the four groups of biopsies. In the gastric body (cancer-free body + EGC body), methylation levels were significantly decreased in patients with longer period after eradication, while such association was not observed in EGC ADJ group. Hyper methylated samples were associated with shorter telomere, an indicator for rapid cell turnover, and higher DNMT1 protein expression, an enzyme related to methyl transfer reaction. The genome-wide methylation analysis demonstrated strikingly higher methylation levels especially at CpG islands in the EGC ADJ group. Exclusively hypermethylated promoter CpG islands in the same group frequently coded zinc finger proteins. Our data show that DNA methylation accumulation is associated with molecular irreversibleness and gastric carcinogenesis after H. pylori eradication.

FOXA2 gene mutation in a patient with congenital complex pituitary hormone deficiency.

We report a patient with congenital complex pituitary hormone deficiency (CPHD) with intestinal malrotation and anal atresia. We identified a de novo heterozygous mutation, c.664T > G (p.Cys222Gly), in the FOXA2 gene in this individual. This missense mutation had the potential to affect the DNA binding properties of the FOXA2 protein based on a protein structure prediction. Since a CPHD patient with another missense mutation and one other case with an entire gene deletion have also been reported, we speculated that a haploinsufficiency of the FOXA2 gene might be a genetic etiology for this disorder. Phenotypic similarities and differences among these three cases are also discussed.