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.

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.

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.

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.

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.

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.

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.

SIN3-HDAC complex-associated factor, a chromatin remodelling gene located in the 12p amplicon, is a potential germ cell tumour-specific oncogene.

A genetic hallmark of malignant germ cell tumours (GCTs) is isochromosome 12p, but oncogenes located in 12p that are specifically expressed in GCT have not yet been identified. SIN3-HDAC complex-associated factor (SINHCAF) is a subunit of the Sin3/histone deacetylase (HDAC) complex, and it defines a Sin3a-Hdac complex variant that is required for the self-renewal of mouse embryonic stem cells. This study demonstrated that SINHCAF is expressed in a vast majority of malignant GCTs and is rarely expressed in somatic malignancy. Fluorescence in situ hybridisation revealed SINHCAF amplification in malignant GCTs. SINHCAF silencing using shRNA reduced anchorage-dependent cell proliferation and tumoursphere formation and inhibited tumour cell migration and invasion in GCT cell lines. Moreover, in the GCT cell line NTERA2/D1, SINHCAF silencing inhibited the expression of genes associated with embryonic stem cells and induced the expression of genes associated with neuronal and white fat cell differentiation. Compared with somatic cell lines, GCT cell lines were more susceptible to HDAC inhibitor treatment. Thus, we identified SINHCAF to be a potential oncogene located in the amplicon of chromosome 12p and showed that SINHCAF was specifically expressed in malignant GCTs. HDAC inhibitor treatment may counteract the oncogenic activity of SINHCAF and is a promising therapeutic approach for GCTs. © 2022 The Pathological Society of Great Britain and Ireland.

Histopathological Correlation of Chromosome 12 Polysomy by Fluorescence in Situ Hybridization in Adipocytic Neoplasms.

Background: Identification of MDM2 amplification by fluorescence in situ hybridization is an important diagnostic tool for evaluation of adipocytic neoplasms. Rarely, neoplasms can show increased copies of MDM2 and CEP12 probes (polysomy) without amplification (MDM2/CEP12 ratio <2.0). While noted in the literature, this finding has not been the focus of any study to date. Methods: Consecutive cases were retrospectively screened for increased copies of MDM2 and CEP12 and were classified as: high polysomy (ratio<2.0, CEP12≥10.0), low polysomy (ratio<2.0, but >0.5, CEP12≥4.0 but <9.9), and CEP12 amplification (ratio≤0.5, CEP12 > 4.0). H&E slides were classified by a pathologist into diagnostic categories based on morphology without knowledge of MDM2 amplification result. Correlations between chromosome 12 polysomy and histological features in the same region of the tumor were investigated. Results: There were 19 (0.7%) high polysomy, 52 (2.0%) low polysomy and 3 (0.1%) CEP12 amplification cases identified in the 2541 cases screened. While low polysomy was seen across benign and malignant adipocytic tumors and other sarcomas, high level polysomy was primarily seen in liposarcomas, both atypical lipomatous tumor/well-differentiated liposarcoma (ALT/WDLPS) and dedifferentiated liposarcoma (DDLPS). No lipomas were high polysomy. Conclusion: Polysomy is an uncommon, but distinct, finding in adipocytic neoplasms found across the spectrum of benign to malignant with little insight into the pathophysiology or prognosis. While low polysomy is also observed in benign adipocytic neoplasms, high polysomy is almost always seen in malignant adipocytic neoplasms and is uncommon in benign adipocytic neoplasms.

Posterior Amorphous Corneal Dystrophy: New Chromosomal Breakpoints in the Small Leucine-Rich Proteoglycan-Coding Region.

PURPOSE: The purpose of this study was to report the clinical features and describe the results obtained by multimodal corneal imaging of a patient with novel chromosomal breakpoints of the 12q21.33 locus. METHODS: This study was a case report and literature review. RESULTS: A 12-year-old girl presented with visual loss whose examination revealed a best-corrected visual acuity of 20/50 in her right eye and 20/35 in her left eye and corneal flattening and gray sheet-like opacities deep in the stroma. Anterior segment optical coherence tomography and ultrabiomicroscopy showed an evenly distributed hyperreflective line in the posterior stroma. Confocal microscopy revealed enlarged keratocytes and the presence of small reflective deposits from the pre-Descemet line to the endothelium. In addition, a 447-kb deletion that included the small leucine-rich proteoglycan-coding region in locus 12q21.33 was found. She was, therefore, diagnosed with PACD. CONCLUSIONS: PACD is a rare genetic disorder of the cornea characterized by gray sheet-like opacification of the posterior stroma in combination with corneal flattening. Confocal microscopy provides histologic segmentation of each corneal layer and shows the degree to which they are affected. New chromosomal breakpoints of a deletion in the small leucine-rich proteoglycan-coding region are hereby reported. PACD may be a contiguous gene syndrome, and further tests are required to identify the exact position responsible for the phenotypic variation.

Utilizing next-generation sequencing to characterize a case of acute myeloid leukemia with t(4;12)(q12;p13) in the absence of ETV6/CHIC2 and ETV6/PDGFRA gene fusions.

The t(4;12)(q12;p13) has been rarely reported in both myeloid/lymphoid neoplasms with eosinophilia (ETV6/PDGFRA gene fusion) and acute myeloid leukemia (AML) (ETV6/CHIC2 gene fusion). The ability to accurately characterize t(4;12) is critical as myeloid neoplasms with PDGFRA rearrangements may be amenable to tyrosine kinase inhibitor (TKI) therapy. Herein, we describe a 60-year-old male with newly diagnosed AML and t(4;12)(q12;p13) by conventional chromosome studies. While the ETV6 break-apart fluorescence in situ hybridization (FISH) probe set demonstrated a balanced ETV6 gene rearrangement, the FIP1L1/CHIC2/PDGFRA tri-color and PDGFRA break-apart FISH probe sets could not resolve the ETV6 gene fusion partner. Mate-pair sequencing (MPseq), a next-generation sequencing assay, was subsequently performed and identified an ETV6 gene rearrangement (at 12p13) that involved an intergenic chromosomal region at 4q12, located between the CHIC2 and PDGFRA gene regions. Having excluded involvement by the PDGFRA gene region, the patient will not be considered for TKI therapy at any point during his medical management. The accurate characterization of structural rearrangements by NGS-based technologies, as demonstrated in this case, highlights the clinical relevance and potential impact on patient medical management of modern cytogenetic techniques.