Multicolour and lineage-specific interphase chromosome Flow-FISH: method development and clinical validation.

Flow cytometry can be applied in the detection of fluorescence in situ hybridisation (FISH) signals to efficiently analyse chromosomal aberrations. However, such interphase chromosome (IC) Flow-FISH protocols are currently limited to detecting a single colour. Furthermore, combining IC Flow-FISH with conventional multicolour flow cytometry is difficult because the DNA-denaturation step in FISH assay also disrupts cellular integrity and protein structures, precluding subsequent antigen-antibody binding and hindering concurrent labeling of surface antigens and FISH signals. We developed a working protocol for concurrent multicolour flow cytometry detection of nuclear IC FISH signals and cell surface markers. The protocol was validated by assaying sex chromosome content of blood cells, which was indicative of chimerism status in patients who had received sex-mismatched allogeneic haematopoietic stem cell transplants (allo-HSCT). The method was also adapted to detect trisomy 12 in chronic lymphocytic leukaemia (CLL) subjects. We first demonstrated the feasibility of this protocol in detecting multiple colours and concurrent nuclear and surface signals with high agreement. In clinical validation experiments, chimerism status was identified in clinical samples (n=56) using the optimised IC Flow-FISH method; the results tightly corresponded to those of conventional slide-based FISH (R2=0.9649 for XX cells and 0.9786 for XY cells). In samples from patients who received sex-mismatched allo-HSCT, individual chimeric statuses in different lineages could be clearly distinguished with high flexibility in gating strategies. Furthermore, in CLL samples with trisomy 12, this method could demonstrate that enriched trisomy 12 FISH signal was present in B cells rather than in T cells. Finally, by performing combined labelling of chromosome 12, X chromosome, and surface markers, we could detect rare residual recipient CLL cells with trisomy 12 after allo-HSCT. This adaptable protocol for multicolour and lineage-specific IC Flow-FISH advances the technique to allow for its potential application in various clinical contexts where conventional FISH assays are currently being utilised.

Pathogenic sequence variant and microdeletion affecting HMGA2 in Silver-Russell syndrome: case reports and literature review.

Silver-Russell syndrome (SRS) is a representative imprinting disorder characterized by pre- and postnatal growth failure. We encountered two Japanese SRS cases with a de novo pathogenic frameshift variant of HMGA2 (NM_003483.6:c.138_141delinsCT, p.(Lys46Asnfs*16)) and a de novo ~ 3.4 Mb microdeletion at 12q14.2-q15 involving HMGA2, respectively. Furthermore, we compared clinical features in previously reported patients with various genetic conditions leading to compromised IGF2 expression, i.e., HMGA2 aberrations, PLAG1 aberrations, IGF2 aberrations, and H19/IGF2:IG-DMR epimutations (hypomethylations). The results provide further support for HMGA2 being involved in the development of SRS and imply some characteristic features in patients with HMGA2 aberrations.

Extragonadal germ cell tumors: A clinicopathologic study with emphasis on molecular features, clinical outcomes and associated secondary malignancies.

Extragonadal germ cell tumors (EGCTs) are rare, representing <5% of all germ cell tumors (GCTs). Whilst EGCTs share morphological and immunohistochemical features with their gonadal counterparts, they tend to be more aggressive and are frequently associated with secondary somatic malignancies. The aim of our study was to evaluate the clinical, morphological and immunohistochemical features, and to analyze tumors for chromosomal abnormalities of 12p, in addition to any novel genetic alterations, in a series of EGCTs. Seventy-seven EGCTs were included. Anterior mediastinum was the most common anatomic site, followed by central nervous system, retroperitoneum, sacroccygeal area, and neck. Whole genome SNP array identified isochromosome 12p in 26% of tumors. Additional cytogenetic abnormalities included the presence of gain of chr 21 in 37% of tumors. Somatic-type malignancies were identified in 8% of patients. Disease progression (metastasis and/or recurrence) was documented in 8 patients, most of whom died from their relapse. Three patients who died of disease had somatic-type malignancies. Mediastinal seminomas had a significantly better overall survival when compared to mediastinal non-seminomatous GCTs. Our study demonstrates that EGCTs share similar histologic features, but diverse clinical outcomes compared to their gonadal counterparts. Outcomes vary according to anatomic location and histologic subtypes. Our data corroborate that somatic-type malignancies are frequently encountered in mediastinal EGCTs and that their presence portends a poorer prognosis.

MDM2 amplification in rod-shaped chromosomes provides clues to early stages of circularized gene amplification in liposarcoma.

Well-differentiated liposarcoma (WDLS) displays amplification of genes on chromosome 12 (Chr12) in supernumerary ring or giant marker chromosomes. These structures have been suggested to develop through chromothripsis, followed by circularization and breakage-fusion-bridge (BFB) cycles. To test this hypothesis, we compared WDLSs with Chr12 amplification in rod-shaped chromosomes with WDLSs with rings. Both types of amplicons share the same spectrum of structural variants (SVs), show higher SV frequencies in Chr12 than in co-amplified segments, have SVs that fuse the telomeric ends of co-amplified chromosomes, and lack interspersed deletions. Combined with the finding of cells with transient rod-shaped structures in tumors with ring chromosomes, this suggests a stepwise process starting with the gain of Chr12 material that, after remodeling which does not fit with classical chromothripsis, forms a dicentric structure with other chromosomes. Depending on if and when telomeres from other chromosomes are captured, circularized or linear gain of 12q sequences will predominate.

Variant-to-function analysis of the childhood obesity chr12q13 locus implicates rs7132908 as a causal variant within the 3' UTR of FAIM2.

The ch12q13 locus is among the most significant childhood obesity loci identified in genome-wide association studies. This locus resides in a non-coding region within FAIM2; thus, the underlying causal variant(s) presumably influence disease susceptibility via cis-regulation. We implicated rs7132908 as a putative causal variant by leveraging our in-house 3D genomic data and public domain datasets. Using a luciferase reporter assay, we observed allele-specific cis-regulatory activity of the immediate region harboring rs7132908. We generated isogenic human embryonic stem cell lines homozygous for either rs7132908 allele to assess changes in gene expression and chromatin accessibility throughout a differentiation to hypothalamic neurons, a key cell type known to regulate feeding behavior. The rs7132908 obesity risk allele influenced expression of FAIM2 and other genes and decreased the proportion of neurons produced by differentiation. We have functionally validated rs7132908 as a causal obesity variant that temporally regulates nearby effector genes and influences neurodevelopment and survival.

Pineocytoma in a child with Pallister-Killian syndrome: a case report and review of the literature.

Pallister-Killian syndrome (PKS; OMIM #601803) is a rare genetic disorder typically characterized by developmental delay, seizures, sparse temporal hair, and facial dysmorphisms. PKS is most frequently caused by mosaic supernumerary isochromosome 12p. Here, we report a 27-month-old girl with a prenatal diagnosis of PKS and a histopathological diagnosis of pineocytoma.

Structural brain abnormalities in Pallister-Killian syndrome: a neuroimaging study of 31 children.

BACKGROUND: Pallister-Killian syndrome (PKS) is a rare genetic disorder caused by mosaic tetrasomy of 12p with wide neurological involvement. Intellectual disability, developmental delay, behavioral problems, epilepsy, sleep disturbances, and brain malformations have been described in most individuals, with a broad phenotypic spectrum. This observational study, conducted through brain MRI scan analysis on a cohort of patients with genetically confirmed PKS, aims to systematically investigate the neuroradiological features of this syndrome and identify the possible existence of a typical pattern. Moreover, a literature review differentiating the different types of neuroimaging data was conducted for comparison with our population. RESULTS: Thirty-one individuals were enrolled (17 females/14 males; age range 0.1-17.5 years old at first MRI). An experienced pediatric neuroradiologist reviewed brain MRIs, blindly to clinical data. Brain abnormalities were observed in all but one individual (compared to the 34% frequency found in the literature review). Corpus callosum abnormalities were found in 20/30 (67%) patients: 6 had callosal hypoplasia; 8 had global hypoplasia with hypoplastic splenium; 4 had only hypoplastic splenium; and 2 had a thin corpus callosum. Cerebral hypoplasia/atrophy was found in 23/31 (74%) and ventriculomegaly in 20/31 (65%). Other frequent features were the enlargement of the cisterna magna in 15/30 (50%) and polymicrogyria in 14/29 (48%). Conversely, the frequency of the latter was found to be 4% from the literature review. Notably, in our population, polymicrogyria was in the perisylvian area in all 14 cases, and it was bilateral in 10/14. CONCLUSIONS: Brain abnormalities are very common in PKS and occur much more frequently than previously reported. Bilateral perisylvian polymicrogyria was a main aspect of our population. Our findings provide an additional tool for early diagnosis.Further studies to investigate the possible correlations with both genotype and phenotype may help to define the etiopathogenesis of the neurologic phenotype of this syndrome.

Trisomy 12 compromises the mesendodermal differentiation propensity of human pluripotent stem cells.

Trisomy 12 is one of the most frequent chromosomal abnormalities in cultured human pluripotent stem cells (hPSCs). Although potential oncogenic properties and augmented cell cycle caused by trisomy 12 have been reported, the consequences of trisomy 12 in terms of cell differentiation, which is the basis for regenerative medicine, drug development, and developmental biology studies, have not yet been investigated. Here, we report that trisomy 12 compromises the mesendodermal differentiation of hPSCs. We identified sublines of hPSCs carrying trisomy 12 after their prolonged culture. Transcriptome analysis revealed that these hPSC sublines carried abnormal gene expression patterns in specific signaling pathways in addition to cancer-related cell cycle pathways. These hPSC sublines showed a lower propensity for mesendodermal differentiation in embryoid bodies cultured in a serum-free medium. BMP4-induced exit from the self-renewal state was impaired in the trisomy 12 hPSC sublines, with less upregulation of key transcription factor gene expression. As a consequence, the differentiation efficiency of hematopoietic and hepatic lineages was also impaired in the trisomy 12 hPSC sublines. We reveal that trisomy 12 disrupts the genome-wide expression patterns that are required for proper mesendodermal differentiation.

Genomic analysis of spermatocytic tumors demonstrates recurrent molecular alterations in cases with malignant clinical behavior.

Spermatocytic tumor (ST) is a rare type of germ cell tumor that occurs exclusively in the postpubertal testis and typically affects elderly men. Most STs are benign, but rare cases exhibit aggressive clinical behavior, often in association with transition to sarcomatoid histology. Limited molecular analyses have been performed on STs; therefore, their genomic and epigenomic features remain incompletely described. Twenty-seven samples from 25 individual patients were analyzed with a combination of DNA sequencing panels, genomic methylation profiling, SNP array, isochromosome (12p) [i(12p)] FISH, and immunohistochemistry. The series included five metastasizing tumors (three with sarcomatoid transformation, one anaplastic, and one conventional) and 20 non-metastasizing tumors (14 anaplastic and six conventional). Anaplastic tumors comprised a monomorphic population of intermediate-sized neoplastic cells, as previously described. Multiomic analyses demonstrated that there were two genomic subgroups of STs: one with diploid genomes and hotspot RAS/RAF variants and the other with global ploidy shift and absence of recurrent mutations. Relative gain of chromosome 9 was a consistent finding in both subgroups. A comparison of metastasizing and non-metastasizing cases demonstrated that aggressive behavior was associated with the acquisition of pathogenic TP53 mutations and/or relative gains of 12p/i(12p). In cases with sarcomatoid transformation, TP53 mutations seem to underlie the transition to sarcomatoid histology. Genomic methylation analysis demonstrated that aggressive cases with gains of 12p cluster closer to pure seminomas than to STs without gains of 12p. In conclusion, STs include two genomic subgroups, characterized by global ploidy shifts without recurrent mutations and diploid genomes with RAS/RAF hotspot mutations, respectively. Biologic progression was associated with relative gains of 12p and TP53 mutations. The findings in STs with relative gains of 12p suggest that they may exhibit biologic characteristics akin to those seen in germ cell neoplasia in situ-related germ cell tumors rather than non-germ cell neoplasia in situ-derived STs. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

A novel genotype-phenotype between persistent-cloaca-related VACTERL and mutations of 8p23 and 12q23.1.

The mechanism underlying anorectal malformations (ARMs)-related VACTERL (vertebral defects, anal atresia, cardiac defects, tracheo-esophageal fistula, and renal and limb abnormalities) remains unclear. Copy number variation (CNV) contributed to VACTERL pathogenicity. Here, we report a novel CNV in 8p23 and 12q23.1 identified in a case of ARMs-related VACTERL association. This 12-year-old girl presented a cloaca (urethra, vagina, and rectum opening together and sharing a single tube length), an isolated kidney, and a perpetuation of the left superior vena cava at birth. Her intelligence, growth, and development were slightly lower than those of normal children of the same age. Array comparative genomic hybridization revealed a 9.6-Mb deletion in 8p23.1-23.3 and a 0.52-Mb duplication in 12q23.1 in her genome. Furthermore, we reviewed the cases involving CNVs in patients with VACTERL, 8p23 deletion, and 12q23.1 duplication, and our case was the first displaying ARMs-related VACTERL association with CNV in 8p23 and 12q23.1. These findings enriched our understanding between VACTERL association and the mutations of 8p23 deletion and 12q23.1 duplication. IMPACT: This is a novel case of a Chinese girl with anorectal malformations (ARMs)-related VACTERL with an 8p23.1-23.3 deletion and 12q23.1 duplication. Cloaca malformation is presented with novel copy number variation in 8p23.1-23.3 deletion and 12q23.1 duplication.

Genetic correlation for alcohol consumption between Europeans and East Asians.

Genome-wide association studies (GWAS) have identified many genetic variants associated with alcohol consumption in Europeans and East Asians, as well as other populations. However, the genetic homogeneity and heterogeneity between these populations have not been thoroughly investigated, despite evidence of varying effect sizes of variants between ethnicities and the presence of population-specific strong signals of selection on loci associated with alcohol consumption. In order to better understand the relationship between Europeans and East Asians in the genetic architecture of alcohol consumption, we compared their heritability and evaluated their genetic correlation using GWAS results from UK Biobank (UKB) and Biobank Japan (BBJ). We found that these two populations have low genetic correlation due to the large difference on chromosome 12. After excluding this chromosome, the genetic correlation was moderately high ([Formula: see text] = 0.544, p = 1.12e-4) and 44.31% of the genome-wide causal variants were inferred to be shared between Europeans and East Asians. Given those observations, we conducted a meta-analysis on UKB and BBJ and identified new signals, including the CADM2 gene on chromosome 3, which has been associated with various behavioral and metabolic traits. Overall, our findings suggest that the genetic architecture of alcohol consumption is largely shared between Europeans and East Asians, but there are exceptions such as the enrichment of heritability on chromosome 12 in East Asians.

Genome-Wide Exploration and Expression Analysis of the CNGC Gene Family in Eggplant (Solanum melongena L.) under Cold Stress, with Functional Characterization of SmCNGC1a.

Eggplant (Solanum melongena L.) is an important economic crop, and to date, there has been no genome-wide identification and analysis of the cyclic nucleotide-gated channel (CNGC) gene family in eggplant. In this study, we identified the CNGC gene family in eggplant, and the results showed that 29 SmCNGC genes were classified into five groups, unevenly distributed across the 12 chromosomes of eggplant. The gene structure and motif analysis indicated that the SmCNGC family proteins may exhibit apparent preferences during evolution. Furthermore, our study revealed the presence of numerous light-responsive elements, hormone-responsive elements, and transcription factor binding sites in the promoter regions of SmCNGC genes, suggesting their significant role in environmental adaptability regulation. Finally, we analyzed the expression patterns of all SmCNGC genes under cold stress and found that SmCNGC1a was significantly upregulated under cold stress. Subcellular localization experiments indicated that this gene is located on the plasma membrane. Subsequently, its importance in the low-temperature response of eggplant was validated through virus-induced gene silencing (VIGS), and its protein interactome was predicted. In summary, our study provides a comprehensive understanding of the function and regulatory mechanisms of the CNGC gene family in eggplant, laying an important foundation for further research on cold adaptation in eggplant.

Replication stress causes delayed mitotic entry and chromosome 12 fragility at the ANKS1B large neuronal gene in human induced pluripotent stem cells.

Substantial background level of replication stress is a feature of embryonic and induced pluripotent stem cells (iPSCs), which can predispose to numerical and structural chromosomal instability, including recurrent aberrations of chromosome 12. In differentiated cells, replication stress-sensitive genomic regions, including common fragile sites, are widely mapped through mitotic chromosome break induction by mild aphidicolin treatment, an inhibitor of replicative polymerases. IPSCs exhibit lower apoptotic threshold and higher repair capacity hindering fragile site mapping. Caffeine potentiates genotoxic effects and abrogates G2/M checkpoint delay induced by chemical and physical mutagens. Using 5-ethynyl-2'-deoxyuridine (EdU) for replication labeling, we characterized the mitotic entry dynamics of asynchronous iPSCs exposed to aphidicolin and/or caffeine. Under the adjusted timing of replication stress exposure accounting revealed cell cycle delay, higher metaphase chromosome breakage rate was observed in iPSCs compared to primary lymphocytes. Using differential chromosome staining and subsequent locus-specific fluorescent in situ hybridization, we mapped the FRA12L fragile site spanning the large neuronal ANKS1B gene at 12q23.1, which may contribute to recurrent chromosome 12 missegregation and rearrangements in iPSCs. Publicly available data on the ANKS1B genetic alterations and their possible functional impact are reviewed. Our study provides the first evidence of common fragile site induction in iPSCs and reveals potential somatic instability of a clinically relevant gene during early human development and in vitro cell expansion.

Prenatal diagnosis of a case with complete and uniform tetrasomy 12p by the utility of noninvasive prenatal testing.

PURPOSE: To report a rare type of Pallister-Killian syndrome (PKS) diagnosed prenatally by the utility of non-invasive prenatal testing (NIPT). METHODS: NIPT was performed in the first trimester. Conventional karyotyping and chromosomal microarray analysis (CMA) were performed on the amniotic samples in the second trimester. Copy number variation sequencing (CNV-seq) was used for the validation of fetal skin and the placental tissue after pregnancy termination. RESULTS: NIPT results showed increased signal from chromosome 12p. Subsequent prenatal diagnostic testing by karyotype revealed 47, XY, +i (12p), and CMA displayed four copies of 12p: 12p13.33-12p11.1(173786_34835641) × 4. The CNV-seq results of the fetal skin and the fetal side of placenta showed four copies of 12p13.33-p11 and an estimated chimeric duplication of 34.08 Mb (chimerism ratio: 10%) in 12 p13.33-p11, respectively. However, no abnormality was detected by CNV-seq at the maternal side of placenta. CONCLUSIONS: Our findings suggest that a positive signal from chromosome 12p on NIPT should raise suspicion for PKS. With the wide application of NIPT, the true positive of incidental finding is expected to increase.