Comparative Hybridization Research Articles

Molecular genetic characterization of myeloid neoplasms with idic(X)(q13) and i(X)(q10).

Isodicentric [idic(X)(q13)] and isochromosome [i(X)(q10)] are infrequent aberrations in neoplastic diseases. The former is mainly reported in elderly women with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), whereas the latter is mostly found as a secondary aberration or part of complex karyotypes in various types of neoplasms, including MDS and AML. Here, we present the molecular genetics and clinical features of six patients with myeloid neoplasia and the above-mentioned aberrations. Array comparative genome hybridization (aCGH) and next-generation sequencing (NGS) myeloid panel were used to examine genetic alterations in five bone marrow samples containing neoplastic cells carrying idic(X)(q13) and one sample with i(X)(q10). The breakpoints of idic(X)(q13) were clustered within a 200 kbp region encompassing FAM236B, DMRTC1B, and DMRTC1. The breakpoint of i(X)(q10) was identified within a 112 kbp region on sub-band p11.22 containing SSX2, SSX2B, and SPANXN5. Pathogenic variants of TET2 were identified in four cases, SF3B1 in three cases, ASXL1 and SRSF2 in two cases each, whereas STAG2, RUNX1, U2AF1, and TP53 pathogenic variants were detected in only single cases. The breakpoints of idic(X)(q13) are within a 200kbp. i(X)(q10) in our study turned out to be a cryptic idic(X)(p11) aberration, reported for the first time here. TET2, SF3B1, ASXL1, or SRSF2 were highly prevalent in patients with idic(X)(q13)/i(X)(q10) abnormalities and were often associated with a worse prognosis.

Genetic epilepsy and role of mutation variants in 27 epileptic children: results from a "single tertiary centre" and literature review

<sec><title>Aim and scope</title><p>The wide use of next-generation sequencing has allowed professionals to reach relevant progress in the medical field including diagnoses, prognoses, and genetic counselling and to help in recognizing the pathogenic mechanisms which underlie several epileptic disorders. Genetic epilepsy refers to a disorder in which genetic mutations are recognized as the primary cause of epileptic seizures in the patients. Various types of epilepsy have been highlighted alongside a clear etiologic relationship with genetic mutations; however, for a select number of patients, some doubts remain on the role played by the variant mutations, as well as the adverse pathologic events which may interfere with the clinical manifestations. In this study, twenty-seven children affected by epileptic seizures are reported, with the aim to describe the clinical and neurological involvement presented by the children according to the genes and variant mutations, and to compare the neurological signs observed in these children with those reported in children with similar genetic mutations. Additionally, the roles played by single or associated variants on the clinical expression of the affected children are discussed.</p></sec><sec><title>Material and methods</title><p>A retrospective observational study was conducted on the gene mutations observed in 27 children affected by a clinical history and diagnosis of epilepsy, alongside an electroencephalographic analysis performed with the Pediatric Department of the University-Hospital S. Marco during the years ranging from January 2020 to January 2022. A genetic analysis was performed using an array comparative genomic hybridization (array CGH), a gene panel for epilepsy, Whole Exome Sequencing (WES), and a single nucleotide polymorphism (SNP) array.</p></sec><sec><title>Results</title><p>Nineteen children showed a single nucleotide variant, eight children carried two or more gene mutations in different genes, and one child was not reported. In this study, we identified a total of thirty-six gene mutations, where seventeen were de novo mutations, and twenty-two had either a maternal or paternal inheritance. The genes in which one or more allelic variants were found included <italic>GRIN2A</italic>, <italic>GABRA1</italic>, <italic>SCN2A1,</italic> <italic>KCNT1</italic>, <italic>PCDH19</italic>, <italic>SCN8A</italic>, <italic>KCNK4</italic>, <italic>SLC2A1</italic>, <italic>DNM1</italic>, <italic>ARID1B</italic>, <italic>WWOX</italic>, <italic>GABRG2,</italic> and others.</p></sec><sec><title>Conclusions</title><p>Genetic analyses are recognized to have a central role in the diagnostic process of children with epileptic seizures, alongside their family history, and clinical, neuroradiologic, and neurophysiologic examinations. The gene mutation anomalies reported in this study may be useful to be included in the wide field of factors related to epileptic seizures in children. The roles played by single or associated variant mutations in the clinical expression of the affected epileptic children remain difficult to be defined. Moreover, other factors such as pre-, peri-, and post-natal adverse events may negatively interfere with the severity of the clinical manifestations.</p></sec>

Human Genetics of Ventricular Septal Defect.

Ventricular septal defects (VSDs) are recognized as one of the commonest congenital heart diseases (CHD), accounting for up to 40% of all cardiac malformations, and occur as isolated CHDs as well as together with other cardiac and extracardiac congenital malformations in individual patients and families. The genetic etiology of VSD is complex and extraordinarily heterogeneous. Chromosomal abnormalities such as aneuploidy and structural variations as well as rare point mutations in various genes have been reported to be associated with this cardiac defect. This includes both well-defined syndromes with known genetic cause (e.g., DiGeorge syndrome and Holt-Oram syndrome) and so far undefined syndromic forms characterized by unspecific symptoms. Mutations in genes encoding cardiac transcription factors (e.g., NKX2-5 and GATA4) and signaling molecules (e.g., CFC1) have been most frequently found in VSD cases. Moreover, new high-resolution methods such as comparative genomic hybridization enabled the discovery of a high number of different copynumber variations, leading to gain or loss of chromosomal regions often containing multiple genes, in patients with VSD. In this chapter, we will describe the broad genetic heterogeneity observed in VSD patients considering recent advances in this field.

Array-Based Comparative Genomic Hybridization for the Detection of Copy Number Alterations in Single Cells.

Comprehensive genome-wide analyses of single cells represent an important tool for clinical applications, such as pre-implantation diagnostic and prenatal diagnosis, as well as for cancer research purpose. For the latter, studies of tumor heterogeneity, circulating tumor cells (CTCs), and disseminated cancer cells (DCCs) require the analysis of single-cell genomes. Here we describe a reliable and robust array-based comparative genomic hybridization (aCGH) protocol based on Ampli 1™ whole genome amplification that allows the detection of copy number alterations (CNAs) in single cancer cells as small as 100kb.

Genetic Characterization of Pediatric Mixed Phenotype Acute Leukemia (MPAL).

Mixed phenotype acute leukemia (MPAL) is a rare hematologic malignancy in which the leukemic cells cannot be assigned to any specific lineage. The lack of well-defined, pathogenetically relevant diagnostic criteria makes the clinical handling of MPAL patients challenging. We herein report the genetic findings in bone marrow cells from two pediatric MPAL patients. Bone marrow cells were examined using G-banding, array comparative genomic hybridization, RNA sequencing, reverse transcription polymerase chain reaction, Sanger sequencing, and fluorescence in situ hybridization. In the first patient, the genetic analyses revealed structural aberrations of chromosomal bands 8p11, 10p11, 11q21, and 17p11, the chimeras MLLT10::PICALM and PICALM::MLLT10, and imbalances (gains/losses) on chromosomes 2, 4, 8, 13, and 21. A submicroscopic deletion in 21q was also found including the RUNX1 locus. In the second patient, there were structural aberrations of chromosome bands 1p32, 8p11, 12p13, 20p13, and 20q11, the chimeras ETV6::LEXM and NCOA6::ETV6, and imbalances on chromosomes 2, 8, 11, 12, 16, 19, X, and Y. The leukemic cells from both MPAL patients carried chromosome aberrations resulting in fusion genes as well as genomic imbalances resulting in gain and losses of many gene loci. The detected fusion genes probably represent the main leukemogenic events, although the gains and losses are also likely to play a role in leukemogenesis.

Exploring the intratumoral heterogeneity of DNA ploidy in prostate cancer.

Prostate cancer is morphologically and molecularly heterogeneous. Genomic heterogeneity might be mirrored by variability in DNA ploidy. Aneuploidy is a hallmark of genomic instability and associated with tumor aggressiveness. Little attention has been paid to the biological significance of the diploid tumor cell population that often coexists with aneuploid populations. Here, we investigated the role of DNA ploidy in tumor heterogeneity and clonal evolution. Three radical prostatectomy specimens with intratumoral heterogeneity based on nuclear features on H&E were selected. DNA content of each subpopulation was determined by DNA image cytometry and silver in situ hybridization (SISH). Genomic evolution was inferred from array comparative genomic hybridization (aCGH). Additionally, immunohistochemistry was used to examine the stemness-associated marker ALDH1A1. Nuclear morphology reliably predicted DNA ploidy status in all three cases. In one case, aCGH analysis revealed several shared deletions and one amplification in both the diploid and the aneuploid population, suggesting that these populations could be related. In the other two cases, a statement about relatedness was not possible. Furthermore, ALDH1A1 was expressed in 2/3 cases and exclusively observed in their diploid populations. In this proof-of-concept study, we demonstrate the feasibility to predict the DNA ploidy status of distinct populations within one tumor by H&E morphology. Future studies are needed to further investigate the clonal relationship between the diploid and the aneuploid subpopulation and test the hypothesis that the aneuploid population is derived from the diploid one. Finally, our analyses pointed to an enrichment of the stemness-associated marker ALDH1A1 in diploid populations, which warrants further investigation in future studies.

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.

Angelman syndrome: A genetic challenge for physical and learning disabilities

Angelman syndrome (AS) was first reported in 1965 by Dr. Harry Angelman. It is a condition of neurodevelopment characterized by, a lack of speech, distinctive behavior, seizure, intellectual capacity, and cheerful disposition. The cause of AS is a lack of production by maternal imprinted genes (UBE3A) on the 15q11-q13 chromosome. The complications of AS are strabismus, atrophy of the optical nerve, and blindness, which are rarely reported. There is a possibility of complications in the laboratory diagnostic tests for AS. One method is to evaluate with DNA methylation analysis of AS/Prader-Willi Syndrome (PWS) imprinting center (IC). On cytogenetic analysis, at least 50–60% of patients had a maternally induced de novo mutation of chromosome 15q11-13 with more serious clinical phenotypes such as microcephaly, seizures, language impairment, and motor difficulties. Multiexonic or whole gene deletion is identified by array-comparative genomic hybridisation (CGH) in some cases and laboratory and methodology may vary such deletions. Diagnosis of AS can be suggested by unsteady movements before walking. Based on the patient’s medical history, electroencephalogram (EEG) data, clinical symptoms, and the presence or absence of a chromosome 50 deletion, a diagnosis of AS is made. Incidence estimated for AS is approximately 1 in 12,000–20,000 birth lives, but the epidemiological measures of life expectancy remains unknown. The clinical features of AS phenotype include seizures, sleep disturbance, intellectual disability, and movement disorders such as tremor and ataxia, anxiety, expressive language is limited, behavioral changes, pleasant demeanor, and easily manipulated laughs, EEG abnormalities were discovered in around 100% of the patients. The researcher identified problems with inflammation at the injection site caused by a higher dose of a drug and they monitored proteins in the individual’s blood and cerebrospinal fluid as an additional safety precaution. Genetic counseling for families with one child with AS to address the likelihood of recurrence can be a challenging subject that frequently requires specialist advice.

Assessing the Phenotype of a Homologous Recombination Deficiency Using High Resolution Array-Based Comparative Genome Hybridization in Ovarian Cancer.

Ovarian cancer (OC) cells with homologous recombination deficiency (HRD) accumulate genomic scars (LST, TAI, and LOH) over a value of 42 in sum. PARP inhibitors can treat OC with HRD. The detection of HRD can be done directly by imaging these genomic scars, or indirectly by detecting mutations in the genes involved in HR. We show that HRD detection is also possible using high-resolution aCGH. A total of 30 OCs were analyzed retrospectively with high-resolution arrays as a test set and 19 OCs prospectively as a validation set. Mutation analysis was performed by HBOC TruRisk V2 panel to detect HR-relevant mutations. CNVs were clustered with respect to the involved HR genes versus the OC cases. In prospective validation, the HRD status determined by aCGH was compared with external HRD assessments. Two BRCA mutation carriers did not have HRD. OC could approximately differentiate into two groups with characteristic CNV patterns with different survival rates. Mutation frequencies have a linear regression on the HRD score. Mutations in individual HR-relevant genes do not always indicate HRD. This may depend on the mutation frequency in tumor cells. The aCGH shows the genomic scars of an HRD inexpensively and directly.

Outcomes of Patients (pts) with Newly Diagnosed Acute Myeloid Leukemia (AML) and TP53 mutation/Loss Treated on the Phase 2 Study of Venetoclax (Ven) Added to Alternating Cladribine (Clad) Plus Low-Dose Cytarabine (LDAC) and Azacitidine (Aza): A Subgroup Analysis

Background: Despite the introduction of newer drugs and combination therapies, outcomes of patients with AML and TP53mutation (mut) and/or loss continue to remain poor, with a median survival of 6-12 months. The phase 2 study of venetoclax (Ven) added to Clad+ LDAC alternating with azacitidine (Aza) in pts with newly diagnosed (ND) AML demonstrated a composite complete remission (CRc) rate of 93% and median overall survival (OS) not reached (NR) at 22 months (mos) (Kadia et al, JCO, 2022) (NCT03586609) . The trial was amended to increase enrollment, including a prespecified cohort of pts with AML with TP53mut/loss. Methods: This is a subgroup analysis of pts with TP53mut/loss on the phase 2 clinical trial. The treatment protocol has been previously published. Pts with AML with TP53 mut (next generation sequencing, lower limit of detection: 2% variant allele frequency [VAF]) or loss by conventional cytogenetics/fluorescent in situ hybridization and/or array comparative genomic hybridization at AML diagnosis were included. CRc was defined as complete remission (CR) plus CR with incomplete count recovery (CRi). Relapse free survival (RFS) was calculated from response to relapse/death and OS from treatment initiation to death; neither censored for allogeneic hematopoietic stem cell transplantation (HSCT). Results: From Nov 2018-Apr 2023, 124 pts with ND AML were treated on this trial, 14 (11%) of whom had TP53mut/loss. The median age in this subgroup was 66 years (yrs) (range 48-75 yrs) and 8 pts (57%) were male. Two pts (14%) had secondary AML (one treated secondary, post HSCT) and another 2 pts (14%) had therapy-related AML. Twelve pts (86%) had a TP53 mut, 8 of whom had bi-allelic loss (two mut, 1 mut with chromosome 17 loss or 1 mut with VAF≥50%). Two pts (14%) had only TP53 deletion (ie. -17/del17p). CRc was attained in 8 pts (57%) [7 CR (50%) and 1 CRi (7%)] and another pt attained partial remission (PR) with 5% bone marrow blasts as best response. 5/8 pts (63%) pts with CRc attained measurable residual disease (MRD) negativity and 4/8 (50%) also had normalization of their TP53 abnormality . The median therapy cycle to best response was 1. Notably 3 pts (21%) had erythroid leukemia, one of whom attained an MRD+ CR. Nine pts had a complex karyotype, 4 (44%) of whom attained a CRc. 4/8 pts (50%) with bi-allelic TP53 loss attained a CRc (2 became MRD negative by flow cytometry and one cleared both TP53 mutations). Pts received a median of 1 cycle (range, 1-5) of therapy. At a median follow-up of 9 mos from study initiation, the median RFS in the responders was not reached (NR) and 1-yr RFS was 56%; median OS for the entire cohort was NR and 1-yr OS was 56%. The 1 yr-OS in responders was 75%. 4/8 pts with CRc and the pt with PR underwent HSCT at a median of 4.5 mos (range 3.9-5.9 mos) from study therapy initiation; all are alive and in remission at a median of 6 mos (range 1-12.3 mos) from HSCT. Conclusion: The phase 2 trial of Ven added to alternating Clad+LDAC and Aza showed promising remission rates of 57% in pts with ND AML having TP53 mut/loss and high rates of MRD negativity in responders; median RFS and OS was not reached at 9 mos follow-up. Five pts (36% overall, and 50% of the responders) could be consolidated with HSCT, with none of them relapsing after HSCT. The trial continues to accrue pts.

Conventional Cytogenetic Analysis and Array CGH+SNP Identify Essential Thrombocythemia (ET)/Prefibrotic Primary Myelofibrosis (pre-PMF) Patients Who Are at Risk for Disease Progression

ET and pre-PMF are myeloproliferative neoplasms (MPNs) that are each characterized by thrombocytosis but are difficult to distinguish by clinical and histopathological criteria. Moreover, each has the propensity to progress at variable rates to more advanced forms of the disease. The cause of progression to more overt forms of myelofibrosis (MF) or accelerated /blast phase (AP/BP) has yet to be fully understood, making prognostication difficult. To identify patients (pts) who are at risk for disease progression, we retrospectively reviewed the karyotypes from conventional cytogenetics (CC) and array Comparative Genomic Hybridization + Single Nucleotide Polymorphism (aCGH+SNP) of 158 pts with a diagnosis of ET or pre-PMF from 2017 to 2022 at our institution. Among these pts, 58% were female (92 pts) and 42% were male (66 pts), and the median age was 61 years (range 13-92). Mutational analysis was performed in 155 pts, and JAK2 mutations were present in 63% of cases, CALR in 19%, and MPL in 7% of cases. The remaining 11% of pts were triple negative. An abnormal karyotype was observed in 15% (24/158), and the remaining 85% (134/158) were either normal or had non-diagnostic results due to failed or limited peripheral blood chromosome analysis. Of the 111 pts that had aCGH+SNP testing, 6% (7/111) had prior abnormalities as detected by conventional cytogenetic analyses, while abnormalities were found in 30% (33/111) with aCGH+SNP exclusively. In this cohort, 29% (46/158) of pts underwent disease progression. Among these cases, 65% (30/46) had progression to either MF or chronic myeloid leukemia or myelodysplasia, and 22% (10/46) progressed to AP/BP. In addition, 13% (6/46) evolved to polycythemia vera. Disease progression, irrespective of its form, was associated with genomic alterations detected by aCGH+SNP, cytogenetic analysis, or both at the time of first encounter. Of the 46 pts undergoing progression during their clinical course, 65% (30/46) had an abnormal genomic finding by either technology compared to 35% (16/46) who were normal (p<0.001). Strikingly, among 24 pts who were abnormal by classical cytogenetics, 79% (19/24) progressed to either AP/BP or MF compared to only 20% (26/125) who were cytogenetically normal (p<0.001). As shown in Figure 1, there was a shorter progression-free survival among pts who were cytogenetically abnormal as compared to normal pts (p<0.001). Among 134 pts with ET/pre-PMF who were normal or had non-diagnostic results by classical cytogenetic studies, aCGH+SNP testing was performed in 104 cases and additional genomic aberrations were detected in 32% (33/104). Within this group, 30% (10/33) progressed to MF with a trend towards a shorter progression-free survival. In those patients that progressed to MF, the most frequent abnormality was 9p CNLOH occurring in 6 pts. In the remaining 71 pts that were cytogenetically and aCGH+SNP normal, only 11% (8/71) had disease progression. Overall, the combination of both conventional cytogenetics and aCGH+SNP increased the sensitivity of detecting genomic abnormalities from 15% (cytogenetics alone) to 37% when incorporating aCGH+SNP. These data indicate that genomic alterations detected by conventional cytogenetic analyses or CGH+SNP array identify a subset of ET/pre-PMF pts that are at a higher risk of disease progression.

Cytogenetics of the Hybridogenetic Frog Pelophylax grafi and Its Parental Species Pelophylax perezi.

Hybrid taxa from the genus Pelophylax can propagate themselves in a modified way of sexual reproduction called hybridogenesis ensuring the formation of clonal gametes containing the genome of only one parental (host) species. Pelophylax grafi from South-Western Europe is a hybrid composed of P. ridibundus and P. perezi genomes and it lives with a host species P. perezi (P-G system). Yet it is unknown, whether non-Mendelian inheritance is fully maintained in such populations. In this study, we characterize P. perezi and P. grafi somatic karyotypes by using comparative genomic hybridization, genomic in situ hybridization, fluorescent in situ hybridization, and actinomycin D-DAPI. Here, we show the homeology of P. perezi and P. grafi somatic karyotypes to other Pelophylax taxa with 2n = 26 and equal contribution of ridibundus and perezi chromosomes in P. grafi which supports F1 hybrid genome constitution as well as a hemiclonal genome inheritance. We show that ridibundus chromosomes have larger regions of interstitial (TTAGGG)n repeats flanking the nucleolus organizing region on chromosome no. 10 and a high quantity of AT pairs in the centromeric regions. In P. perezi, we found species-specific sequences in metaphase chromosomes and marker structures in lampbrush chromosomes. Pericentromeric RrS1 repeat sequence was present in perezi and ridibundus chromosomes, but the blocks were stronger in ridibundus. Various cytogenetic techniques applied to the P-G system provide genome discrimination between ridibundus and perezi chromosomal sets. They could be used in studies of germ-line cells to explain patterns of clonal gametogenesis in P. grafi and broaden the knowledge about reproductive strategies in hybrid animals.

Application of array comparative genomic hybridization in clinical diagnostics of intellectual disability/developmental delay in children

Background: This study was designed to analyze and evaluate the potential pathogenic genomic imbalance in children with unexplained intellectual disability (ID) and/or developmental delay (DD) and its association with phenotypes, and to investigate the value of array-based comparative genomic hybridization (array-CGH). Methods: A total of 72 Children with ID/DD were evaluated by array-CGH for detection of genomic copy number variations (CNVs). Results: The results of the array-CGH revealed that 10(14%) of the 72 patients had pathogenic CNVs, in that six cases had pathogenic CNV in a single chromosome, 2 cases had multiple microdeletions and 2 cases had combined microdeletion and microduplication, 2 cases had pathogenic CNVs in chromosome 1p36 and Xq28 region. One case had variation of unknown significance in chromosome region 15q11.2. Large bands of copy neutral loss of heterozygosity were detected in 2 cases comprising more than 10% of genome. Conclusions: Array-CGH being a high-throughput and rapid tool, allows for the etiological diagnosis in some of the children with unexplained ID/DD.

Titin copy number variations associated with dominant inherited phenotypes

BackgroundTitinopathies are caused by mutations in the titin gene (TTN). Titin is the largest known human protein; its gene has the longest coding phase with 364 exons. Titinopathies are very...

Association between copy number variations in parkin (PRKN) and schizophrenia and autism spectrum disorder: A case-control study.

The present study aimed to examine the association between copy number variations (CNVs) in parkin (PRKN) and schizophrenia (SCZ) and autism spectrum disorder (ASD) in a large case-control sample. Array comparative genomic hybridization was performed on 3111 cases with SCZ, 1236 cases with ASD, and 2713 controls. We systematically prioritized likely pathogenic CNVs (LP-CNVs) in PRKN and examined their association with SCZ and ASD. In total, 3014 SCZ cases (96.9%), 1205 ASD cases (97.5%), and 2671 controls (98.5%) passed quality control. We found that monoallelic carriers of LP-CNVs in PRKN were common (70/6890, 1.02%) and were not at higher risk of SCZ (p = 0.29) or ASD (p = 0.72). We observed that the distribution pattern of LP-CNVs in the Japanese population was consistent with those in other populations. We also identified a patient diagnosed with SCZ and early-onset Parkinson's disease carrying biallelic pathogenic CNVs in PRKN. The absence of Parkinson's symptoms in 10 other monoallelic carriers of the same pathogenic CNV further reflects the lack of effect of monoallelic pathogenic variants in PRKN in the absence of a second hit. The present findings suggest that monoallelic CNVs in PRKN do not confer a significant risk for SCZ or ASD. However, further studies to investigate the association between biallelic CNVs in PRKN and SCZ and ASD are warranted.

Single-Exon Deletions of ZNRF3 Exon 2 Cause Congenital Adrenal Hypoplasia.

Primary adrenal insufficiency (PAI) is a life-threatening condition characterized by the inability of the adrenal cortex to produce sufficient steroid hormones. E3 ubiquitin protein ligase zinc and ring finger 3 (ZNRF3) is a negative regulator of Wnt/β-catenin signaling. R-spondin 1 (RSPO1) enhances Wnt/β-catenin signaling via binding and removal of ZNRF3 from the cell surface. This work aimed to explore a novel genetic form of PAI. We analyzed 9 patients with childhood-onset PAI of biochemically and genetically unknown etiology using array comparative genomic hybridization. To examine the functionality of the identified single-exon deletions of ZNRF3 exon 2, we performed three-dimensional (3D) structure modeling and in vitro functional studies. We identified various-sized single-exon deletions encompassing ZNRF3 exon 2 in 3 patients who showed neonatal-onset adrenal hypoplasia with glucocorticoid and mineralocorticoid deficiencies. Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis showed that the 3 distinct single-exon deletions were commonly transcribed into a 126-nucleotide deleted mRNA and translated into 42-amino acid deleted protein (ΔEx2-ZNRF3). Based on 3D structure modeling, we predicted that interaction between ZNRF3 and RSPO1 would be disturbed in ΔEx2-ZNRF3, suggesting loss of RSPO1-dependent activation of Wnt/β-catenin signaling. Cell-based functional assays with the TCF-LEF reporter showed that RSPO1-dependent activation of Wnt/β-catenin signaling was attenuated in cells expressing ΔEx2-ZNRF3 as compared with those expressing wild-type ZNRF3. We provided genetic evidence linking deletions encompassing ZNRF3 exon 2 and congenital adrenal hypoplasia, which might be related to constitutive inactivation of Wnt/β-catenin signaling by ΔEx2-ZNRF3.

Comparative genomic hybridisation and transcriptome microarray analysis in triple negative breast cancer: An INDIAN study.

Triple-negative breast cancer (TNBC), which accounts for approximately 15–20% of all breast cancers, defined as lack of expression of estrogen receptor, progesterone receptor and Her-2 neu receptors. TNBC has two subtypes basal like and non-basal like, the former characterised by aggressive biology with limited therapeutic options. This study explored molecular markers involved in pathogenesis of TNBC and investigated novel potential diagnostic and therapeutic targets by CGH array and transcriptome array. aCGH analysis in TNBC demonstrated genes amplified were 3888, number of pathway hits was 1554 and major pathways amplified was found to be WNT signalling pathway and Cadherin signalling pathway. Among all metastatic sites and remission, activation of WNT signalling pathway is commonly observed. TNBC exhibited 1486 copy number variations (CNVR) which is approximately 250 times higher than controls. More than 20 CNVR was observed in all chromosomes and more than 80 CNVR was observed in Chr 1 to Chr 4, Chr 7, Chr 11 and Chr X. Common CNVR associated with amplified regions in Chr 22, Chr 14, Chr 8 and Chr 2 was observed in TNBC and CNVR associated with Chr 22q11.22–23, Chr 6p21.32–33, Chr11q12.2, Chr14q32.22–23, Chr 8p11.22–23, was observed in metastatic disease. In transcriptome array analysis a total of 11,359 differentially expressed genes with fold change 2.0 were in observed in TNBC comprise of 7639 upregulated genes and 3720 downregulated genes. Further, with fold change 10, 1526 upregulated genes and 839 down regulated genes were identified. Panther pathway analysis identified the main pathways of upregulated genes were Wnt signalling pathway, Integrin signalling pathway and Cadherin signalling pathway. The main pathways of down regulated genes were Inflammation mediated by chemokine and cytokine signalling pathways. PPI network shows that COL12A1, COL6A3, FN1, MMP3, WNT5A were key upregulated genes and ITGB7, PTPRC, ITGA4, LCK and CD247 were key down regulated genes. Cytoscape analysis followed by multiple list comparator tool identified top 5 significant hub genes were FN1, MMP3, COLL11A1, COL12A1 and COL3A1. The significant pathway genes obtained by CGH array and transcriptome array when compared, exhibited 5 common genes COL4A1, FN1, COL6A3, COL5A2 and PCDH7. These genes were not overexpressed in Controls and therefore involved in pathogenesis of TNBC. Expressions of these genes were validated by studying protein expression by immunohistochemistry. FN1 and COL6A3 protein over expression predicted worse DFS in TNBC and can be considered as therapeutic targets at diagnosis to reduce the disease metastases. These findings provide new insights into the pathogenesis of TNBC and guide for selection of targets related to diagnosis, prognosis and prediction of treatment in TNBC.

Neuroblastoma Patients' Outcome and Chromosomal Instability.

Chromosomal instability (CIN) induces a high rate of losses or gains of whole chromosomes or parts of chromosomes. It is a hallmark of most human cancers and one of the causes of aneuploidy and intra-tumor heterogeneity. The present study aimed to evaluate the potential prognostic role of CIN in NB patients at diagnosis. We performed array comparative genomic hybridization analyses on 451 primary NB patients at the onset of the disease. To assess global chromosomal instability with high precision, we focused on the total number of DNA breakpoints of gains or losses of chromosome arms. For each tumor, an array-CGH-based breakpoint instability index (BPI) was assigned which defined the total number of chromosomal breakpoints per genome. This approach allowed us to quantify CIN related to whole genome disruption in all NB cases analyzed. We found differences in chromosomal breakages among the NB clinical risk groups. High BPI values are negatively associated with survival of NB patients. This association remains significant when correcting for stage, age, and MYCN status in the Cox model. Stratified analysis confirms the prognostic effect of BPI index in low-risk NB patients with non-amplified MYCN and with segmental chromosome aberrations.

A genotype-phenotype correlation in split-hand/foot malformation type 1: further refinement of the phenotypic subregions within the 7q21.3 locus.

Background: Split-hand/foot malformation type 1 (SHFM1) refers to the group of rare congenital limb disorders defined by the absence or hypoplasia of the central rays of the autopods with or without accompanying anomalies, such as hearing loss, craniofacial malformation, and ectodermal dysplasia. Consequently, the condition is characterized by clinical variability that hinders diagnostic and counseling procedures. SHFM1 is caused by pathogenic variants affecting the DLX5/6 genes and/or their tissue-specific enhancers at the 7q21.3 locus. Herein, we report on seven patients from five unrelated Polish families affected by variable symptoms of the SHFM1 spectrum, all harboring 7q21.3 or 7q21.2-q21.3 rearrangements, and provide a genotype-phenotype correlation in the studied cohort. Methods: We applied GTG banding, array-based comparative genomic hybridization (aCGH), and whole-genome sequencing (WGS) in order to identify the causative aberrations in all affected patients. Results: The identified pathogenic structural variants included deletions and/or translocations involving the 7q21.3 locus, i.e., t(7;10)(q21.3;q22.2) and t(7;12)(q21.3;q21.2) in all affected individuals. Interestingly, a sporadic carrier of the latter aberration presented the SHFM1 phenotype with additional features overlapping with Baker-Gordon syndrome (BAGOS), which resulted from the translocation breakpoint at chromosome 12 within the SYT1 gene. Conclusion: Clinical variability of the studied cohort reflects the composition of the DLX5/6 regulatory elements that were dislocated from their target genes by chromosomal rearrangements. The correlation of our data with the previously published observations enabled us to update the phenotypic subregions and regulatory units within the SHFM1 locus. In addition, we present the first case of SHFM1 and BAGOS-like phenotype that resulted from translocation breakpoints at chromosomes 7 and 12, both of which were pathogenic, and consequently, we show the first evidence that BAGOS can also result from the regulatory loss-of-function SYT1 mutations. In this paper, we emphasize the utility of sequence-based approaches in molecular diagnostics of disorders caused by regulatory structural variants.

Newly discovered genomic mutation patterns in radiation-induced small intestinal tumors of ApcMin/+ mice.

Among the small intestinal tumors that occur in irradiated mice of the established mouse model B6/B6-Chr18MSM-F1 ApcMin/+, loss of heterozygosity analysis can be utilized to estimate whether a deletion in the wild-type allele containing the Adenomatous polyposis coli (Apc) region (hereafter referred to as Deletion), a duplication in the mutant allele with a nonsense mutation at codon 850 of Apc (Duplication), or no aberration (Unidentified) has occurred. Previous research has revealed that the number of Unidentified tumors tends to increase with the radiation dose. In the present study, we investigated the molecular mechanisms underlying the development of an Unidentified tumor type in response to radiation exposure. The mRNA expression levels of Apc were significantly lower in Unidentified tumors than in normal tissues. We focused on epigenetic suppression as the mechanism underlying this decreased expression; however, hypermethylation of the Apc promoter region was not observed. To investigate whether deletions occur that cannot be captured by loss of heterozygosity analysis, we analyzed chromosome 18 using a customized array comparative genomic hybridization approach designed to detect copy-number changes in chromosome 18. However, the copy number of the Apc region was not altered in Unidentified tumors. Finally, gene mutation analysis of the Apc region using next-generation sequencing suggested the existence of a small deletion (approximately 3.5 kbp) in an Unidentified tumor from a mouse in the irradiated group. Furthermore, nonsense and frameshift mutations in Apc were found in approximately 30% of the Unidentified tumors analyzed. These results suggest that radiation-induced Unidentified tumors arise mainly due to decreased Apc expression of an unknown regulatory mechanism that does not depend on promoter hypermethylation, and that some tumors may result from nonsense mutations which are as-yet undefined point mutations.