Hemizygous Deletion Research Articles

Genetic alterations that deregulate RB and PDGFRA signaling pathways drive tumor progression in IDH2-mutant astrocytoma

In IDH-mutant astrocytoma, IDH2 mutation is quite rare and biological mechanisms underlying tumor progression in IDH2-mutant astrocytoma remain elusive. Here, we report a unique case of IDH2 mutant astrocytoma, CNS WHO grade 3 that developed tumor progression. We performed a comprehensive genomic and epigenomic analysis for primary and recurrent tumors and found that both tumors harbored recurrent IDH2R172K and TP53R248W mutation with CDKN2A/B hemizygous deletion. We also found amplifications of CDK4 and MDM2 with PDGFRA gain in the recurrent tumor and upregulated protein expressions of these genes. We further developed, for the first time, a xenograft mouse model of IDH2R172K and TP53R248W mutant astrocytoma from the recurrent tumor, but not from the primary tumor. Consistent with parent recurrent tumor cells, amplifications of CDK4 and MDM2 and PDGFRA gain were found, while CDKN2A/B was identified as homozygous deletion in the xenografts, qualifying for integrated diagnosis of astrocytoma, IDH2-mutant, CNS WHO grade 4. Cell viability assay found that CDK4/6 inhibitor and PDGFR inhibitor potently decreased cell viability in recurrent tumor cells, as compared to primary tumor cells. These findings suggest that gene alterations that activate retinoblastoma (RB) signaling pathways and PDGFR may drive tumor progression and xenograft formation in IDH2-mutant astrocytoma, which is equivalent to progressive IDH1-mutant astrocytoma. Also, our findings suggest that these genomic alterations may represent therapeutic targets in IDH2-mutant astrocytoma.

PATH-28. OUTCOME IMPACT OF THE CDKN2A/B HEMI-ZYGOUS DELETION IN IDH-MUTANT ASTROCYTOMA

Abstract Homozygous deletion of CDKN2A/B is a strong adverse prognostic factor in IDH-mutant astrocytomas. However, the impact of hemizygous deletions is unknown. We investigated the impact of CDKN2A/B hemizygous deletions on prognosis. We enrolled 52 adult patients with IDH-mutant astrocytomas between December 2002 and November 2021. CDKN2A/B deletion was evaluated with multiplex ligation-dependent probe amplification (MLPA) with threshold correction using a digital PCR-based IDH-mutant allele frequency. Immunohistochemical analysis of p16/MTAP was performed in CDKN2A/B deletion cases. Of the 52 cases, nine and three were classified as hemizygous and homozygous deletions, respectively. The OS was shorter in the order of homozygous deletion, hemizygous deletion, and copy-neutral groups (median OS:38.5, 59.5, and 93.1 months, respectively). Multivariate analysis revealed hazard ratios of 9.30 (p = 0.0191) and 2.44 (p = 0.0943) for homozygous and hemizygous deletions, respectively. Regarding PFS, in astrocytoma, the time to recurrence tended to be shorter in the order of homozygous deletion, hemizygous deletion, and copy-neutral, with a median PFS of 20.9, 38.9, and 53.0 months, respectively. There was no significant difference in PFS between the hemizygous deletion and copy-neutral groups (p = 0.4030). Malignant transformation was confirmed in 1 of 1 homozygous deletion, 2 of 3 hemizygous deletions, and 5 of 16 copy-neutrals. No significant difference was observed between the hemizygous deletion and copy-neutral groups; however, hemizygous deletion tended to require less time for malignant transformation than copy-neutral group (p = 0.1112). Immunohistochemistry revealed p16-negative and MTAP retention in cases with a hemizygous deletion, whereas homozygous deletions had p16-negative and MTAP loss. In conclusion, CDKN2A/B hemizygous deletions exerted a negative impact on OS in astrocytomas. Immunohistochemistry of p16/MTAP can be utilized to validate hemizygous or homozygous deletions in combination with conventional molecular diagnosis.

SURG-36. CLINICO-RADIOLOGICAL PARAMETERS, MOLECULAR AND CHROMOSOMAL ALTERATIONS AFFECTING SURGICAL OUTCOME OF PETROCLIVAL MENINGIOMAS

Abstract Petroclival meningiomas were reviewed for correlation of extent of resection with clinico-radiological parameters and molecular/chromosomal alterations. 53 surgical procedures in 50 patients of petroclival meningioma were performed. Cavernous sinus and sellar extension were present in 37(69.8%) and 5(9.4%) respectively; contralateral extension in 10(20%). Modified anterior transpetrous rhomboid-MATRA (44=83.0%) was the most commonly used approach, followed by retrosigmoid (2=3.8%), and combined (7=13.2%) approaches. Hypointense tumors on T2-weighted MRI (OR:5.85;95%CI:1.70-20.41) and presence of brainstem edema (OR:4.53;95%CI:1.36-15.12) were found to be significant factors increasing the likelihood of subtotal resection-STR (p = 0.004 & p = 0.011 respectively). In the presence of both tumor T2-hypointensity and brainstem edema, there was significant increase in the likelihood of STR (p = 0.001;OR:25;95%CI:3.52-177.48). The most common cranial nerve to suffer new-onset deficit postoperatively was found to be VI nerve(19=35.8%) followed by V(9=16.9%) and VII(8=15.8%) nerves. On long-term follow-up, 38 patients had good outcome. Out of the 16 cases for which molecular analysis was performed, no specimen was found to have pTERT-C228T,C250T, AKT-1, E17K and SMO-L412F,W535L mutations. Eight (50.0%) cases showed H3K27me3 loss while 5 (31.25%) had hemizygous CDKN2A deletion. All(100%) patients harboring H3K27me3 loss and/or hemizygous CDKN2A deletion had cavernous sinus extension compared to 62.5% patients without H3K27me3 loss and 72.7% with hemizygous CDKN2A retention. Similarly, hemizygous CDKN2A deletion and H3K27me3 loss are associated with increase in the rate of brainstem edema from 27.3% to 60% and 25% to 50% respectively. Cases (n = 4) with both these alterations have 100% cavernous sinus extension and 75% chances of brainstem edema and lack of arachnoid plane. The MATRA is a versatile approach for petroclival meningiomas with good surgical outcome and acceptable complication rates. T2 hypointense tumor and brainstem edema on pre-operative imaging are significant predictors of STR. H3K27me3 loss and hemizygous CDKN2A deletion may be associated with cavernous sinus extension suggesting their role in tumor spread.

STS and PUDP Deletion Identified by Targeted Panel Sequencing with CNV Analysis in X-Linked Ichthyosis: A Case Report and Literature Review.

X-linked recessive ichthyosis (XLI) is clinically characterized by dark brown, widespread dryness with polygonal scales. We describe the identification of STS and PUDP deletions using targeted panel sequencing combined with copy-number variation (CNV) analysis in XLI. A 9-month-old infant was admitted for genetic counseling. Since the second day after birth, the infant's skin tended to be dry and polygonal scales had accumulated over the abdomen and upper extremities. The infant's maternal uncle and brother (who had also exhibited similar skin symptoms from birth) presented with polygonal scales on their trunks. CNV analysis revealed a hemizygous deletion spanning 719.3 Kb on chromosome Xp22 (chrX:7,108,996-7,828,312), which included a segment of the STS gene and exhibited a Z ratio of -2 in the proband. Multiplex ligation-dependent probe amplification (MLPA) confirmed this interstitial Xp22.31 deletion. Our report underscores the importance of implementing CNV screening techniques, including sequencing data analysis and gene dosage assays such as MLPA, to detect substantial deletions that encompass the STS gene region of Xq22 in individuals suspected of having XLI.

CRISPR deletion of a SINE-VNTR-Alu (SVA_67) retrotransposon demonstrates its ability to differentially modulate gene expression at the MAPT locus.

SINE-VNTR-Alu (SVA) retrotransposons are hominid-specific elements which have been shown to play important roles in processes such as chromatin structure remodelling and regulation of gene expression demonstrating that these repetitive elements exert regulatory functions. We have previously shown that the presence or absence of a specific SVA element, termed SVA_67, was associated with differential expression of several genes at the MAPT locus, a locus associated with Parkinson's Disease (PD) and frontotemporal dementia. However, we were not able to demonstrate that causation of differential gene expression was directed by the SVA due to lack of functional validation. We performed CRISPR to delete SVA_67 in the HEK293 cell line. Quantification of target gene expression was performed using qPCR to assess the effects on expression in response to the deletion of SVA_67. Differences between CRISPR edit and control cell lines were analysed using two-tailed t-test with a minimum 95% confidence interval to determine statistical significance. In this study, we provide data highlighting the SVA-specific effect on differential gene expression. We demonstrate that the hemizygous deletion of the endogenous SVA_67 in CRISPR edited cell lines was associated with differential expression of several genes at the MAPT locus associated with neurodegenerative diseases including KANSL1, MAPT and LRRC37A. This data is consistent with our previous bioinformatic work of differential gene expression analysis using transcriptomic data from the Parkinson's Progression Markers Initiative (PPMI) cohort. As SVAs have regulatory influences on gene expression, and insertion polymorphisms contribute to interpersonal differences in expression patterns, these results highlight the potential contribution of these elements to complex diseases with potentially many genetic components, such as PD.

JS04.5.A HEMIZYGOUSCDKN2A DELETION CONFERS WORSE SURVIVAL OUTCOMES IN IDH-MUTANT GLIOMAS

Abstract BACKGROUND The tumor suppressor gene CDKN2A is frequently deactivated in cancer. Homozygous CDKN2A deletion is a Grade 4 defining criterion for IDH-mutant gliomas. However, the prognostic value of hemizygous CDKN2A deletions is unclear. We assessed CNV profiles of 1238 initial and 479 recurrent IDHmut gliomas to evaluate the association of CDKN2A hemizygous deletions with overall survival (OS) outcomes. MATERIAL AND METHODS The GLASS primary/recurrent glioma dataset was used to infer CDKN2A status with DNAseq (n=210) and DNA methylation array (n=100) data. Validation was conducted using DFCI, MSKCC, TCGA and DKFZ cohorts. RESULTS Longitudinal analyses of IDH-mutant gliomas without 1p-/19q-codeletion showed significant increases in CDKN2A homozygous deletion from n=6 to n=23 (5% to 20%, P=0.0001, Fisher’s-exact test) and hemizygous deletion from n=19 to n=34 (17% to 30%, P=0.002) at recurrence. CDKN2A hemizygous deletions were significantly associated with poor OS at initial diagnosis (P<0.0001, log-rank test) and recurrence (P<0.0001), remaining significant in multivariable cox regression model that accounted for age, treatment with radiotherapy and/or alkylating agents, and 1p/19q-codeletion status: HR 2.40 (95%-CI: 1.31-4.37, P=0.004). Validation in independent datasets confirmed increased frequencies of CDKN2A hemizygous deletions in recurrent cases as well as the association between CDKN2A hemizygous deletion and worse OS in IDHmut gliomas. Using the DKFZ DNA methylation profiling dataset we further confirmed that CDKN2A status can be accurately assessed through DNAseq and DNA methylation array profiling. CONCLUSION CDKN2A hemizygous deletion is associated with significantly worse OS in initial and recurrent IDHmut gliomas. Similar to CDKN2A homozygous deletion, CDKN2A hemizygous deletion is enriched in post-treatment, recurrent IDHmut gliomas, confirming the value of CDKN2A status (re-)assessment in recurrences. Our results highlight the importance of incorporating CDKN2A status in diagnostic workup to inform prognosis and treatment strategies, revealing the previously unrecognized prognostic value of hemizygous CDKN2A deletions.

Dorsal visual stream and LIMK1: hemideletion, haplotype, and enduring effects in children with Williams syndrome

BackgroundWilliams syndrome (WS), a rare neurodevelopmental disorder caused by hemizygous deletion of ~ 25 genes from chromosomal band 7q11.23, affords an exceptional opportunity to study associations between a well-delineated genetic abnormality and a well-characterized neurobehavioral profile. Clinically, WS is typified by increased social drive (often termed “hypersociability”) and severe visuospatial construction deficits. Previous studies have linked visuospatial problems in WS with alterations in the dorsal visual processing stream. We investigated the impacts of hemideletion and haplotype variation of LIMK1, a gene hemideleted in WS and linked to neuronal maturation and migration, on the structure and function of the dorsal stream, specifically the intraparietal sulcus (IPS), a region known to be altered in adults with WS.MethodsWe tested for IPS structural and functional changes using longitudinal MRI in a developing cohort of children with WS (76 visits from 33 participants, compared to 280 visits from 94 typically developing age- and sex-matched participants) over the age range of 5–22. We also performed MRI studies of 12 individuals with rare, shorter hemideletions at 7q11.23, all of which included LIMK1. Finally, we tested for effects of LIMK1 variation on IPS structure and imputed LIMK1 expression in two independent cohorts of healthy individuals from the general population.ResultsIPS structural (p < 10−4 FDR corrected) and functional (p < .05 FDR corrected) anomalies previously reported in adults were confirmed in children with WS, and, consistent with an enduring genetic mechanism, were stable from early childhood into adulthood. In the short hemideletion cohort, IPS deficits similar to those in WS were found, although effect sizes were smaller than those found in WS for both structural and functional findings. Finally, in each of the two general population cohorts stratified by LIMK1 haplotype, IPS gray matter volume (pdiscovery < 0.05 SVC, preplication = 0.0015) and imputed LIMK1 expression (pdiscovery = 10−15, preplication = 10−23) varied according to LIMK1 haplotype.ConclusionsThis work offers insight into neurobiological and genetic mechanisms responsible for the WS phenotype and also more generally provides a striking example of the mechanisms by which genetic variation, acting by means of molecular effects on a neural intermediary, can influence human cognition and, in some cases, lead to neurocognitive disorders.

Molecular Challenges in the Diagnosis of X-Linked Chronic Granulomatous Disease: CNVs, Intronic Variants, Skewed X-Chromosome Inactivation, and Gonosomal Mosaicism.

Chronic granulomatous disease (CGD) is a prototypical inborn error of immunity affecting phagocytes, in which these cells are unable to produce reactive oxygen species. CGD is caused by defects in genes encoding subunits of the NADPH oxidase enzyme complex (CYBA, CYBB, CYBC1, NCF1, NCF2, NCF4); inflammatory responses are dysregulated, and patients are highly susceptible to recurrent severe bacterial and fungal infections. X-linked CGD (XL-CGD), caused by mutations in the CYBB gene, is the most common and severe form of CGD. In this study, we describe the analytical processes undertaken in 3 families affected with XL-CGD to illustrate several molecular challenges in the genetic diagnosis of this condition: in family 1, a girl with a heterozygous deletion of CYBB exon 13 and skewed X-chromosome inactivation (XCI); in family 2, a boy with a hemizygous deletion of CYBB exon 7, defining its consequences at the mRNA level; and in family 3, 2 boys with the same novel intronic variant in CYBB (c.1151 + 6T > A). The variant affected the splicing process, although a small fraction of wild-type mRNA was produced. Their mother was a heterozygous carrier, while their maternal grandmother was a carrier in form of gonosomal mosaicism. In summary, using a variety of techniques, including an NGS-based targeted gene panel and deep amplicon sequencing, copy number variation calling strategies, microarray-based comparative genomic hybridization, and cDNA analysis to define splicing defects and skewed XCI, we show how to face and solve some uncommon genetic mechanisms in the diagnosis of XL-CGD.

Exploiting nanopore sequencing for characterization and grading of IDH-mutant gliomas.

The 2021 WHO Classification of Central Nervous System Tumors recommended evaluation of cyclin-dependent kinase inhibitor 2A/B (CDKN2A/B) deletion in addition to codeletion of 1p/19q to characterize IDH-mutant gliomas. Here, we demonstrated the use of a nanopore-based copy-number variation sequencing (nCNV-seq) approach to simultaneously identify deletions of CDKN2A/B and 1p/19q. The nCNV-seq approach was initially evaluated on three distinct glioma cell lines and then applied to 19 IDH-mutant gliomas (8 astrocytomas and 11 oligodendrogliomas) from patients. The whole-arm 1p/19q codeletion was detected in all oligodendrogliomas with high concordance among nCNV-seq, FISH, DNA methylation profiling, and whole-genome sequencing. For the CDKN2A/B deletion, nCNV-seq detected the loss in both astrocytoma and oligodendroglioma, with strong correlation with the CNV profiles derived from whole-genome sequencing (Pearson correlation (r) = 0.95, P < 2.2 × 10-16 to r = 0.99, P < 2.2 × 10-16 ) and methylome profiling. Furthermore, nCNV-seq can differentiate between homozygous and hemizygous deletions of CDKN2A/B. Taken together, nCNV-seq holds promise as a new, alternative approach for a rapid and simultaneous detection of the molecular signatures of IDH-mutant gliomas without capital expenditure for a sequencer.

Dört Aileden Pelizaeus-Merzbacher Sendromlu Altı Hastanın Klinik ve Moleküler Sitogenetik Analizleri

Objective: Pelizaeus-Merzbacher Disease is a rare X-linked recessive leukodystrophy caused by a mutation in the proteolipid protein (PLP) gene on chromosome Xq22. PMD is an early-onset neurological disorder characterized by nystagmus, spastic quadriplegia, ataxia, and developmental delay. Genetic analysis has identified Xq22 microduplications (60-70%), point mutations (10–25%), and deletions (5-10%) within the coding region of the PLP genes in Pelizaeus-Merzbacher Disease. This study evaluated six patients with PLP1 deletion and duplication in four Turkish families. Material and Methods: To detect the duplication and deletion of PLP1, chromosomal microarray analysis, and multiplex ligation-related probe amplification assays were performed. Results: In these four families, two brothers had a hemizygous deletion in the PLP1 gene, their carrier mother had a deletion in the PLP1 gene, and another two unrelated boys and one girl had duplication of the PLP1. Also, we identified the rare case of two brother patients who were found to have a hemizygous deletion in the PLP1 gene. Their carrier mother had unexplained dementia. Conclusion: Genotype-phenotype correlations of the PLP1 mutation in these families were identified in this study while trying to elucidate the genetic etiology of six individuals from four different families.

PD13 Severe cutaneous manifestations in an Indian child with X-linked dominant erythropoietic protoporphyria

Abstract Erythropoietic protoporphyria (EPP) is usually caused by autosomal dominant mutations in FECH: raised protoporphyrin levels cause itching, burning and erythema after sun exposure, with facial scarring and sometimes liver damage. We report a child with the much rarer X-linked dominant form (XLDPP) and consider reasons for his unusually severe features. A 4-year-old boy living in India presented with long-standing erosions and scarring on the face and sides of the feet, and marked distal onycholysis with subungual hyperkeratosis affecting all nails, suggestive of pachyonychia congenita (PC). There was no relevant family history or parental consanguinity. He was anaemic (haemoglobin 7.2 g dL−1). Genetic testing revealed no variants in PC genes, but there was a hemizygous deletion in ALAS2, c.1706_1709del (p.Glu569GlyfsT er24) reported recurrently in XLDPP. Further review revealed a history of photosensitivity, low vitamin D3 and raised liver enzymes. Measurement of erythrocyte protoporphyrin was not available, but XLDPP was diagnosed on the basis of photosensitivity, liver abnormalities, probable photo-onycholysis and genetic testing. ALAS2 encodes 5-aminolaevulinate synthase, the first enzyme in the haem synthetic pathway. Most ALAS2 mutations block haemoglobin production resulting in sideroblastic anaemia. However, specific deletions, including this one, activate the pathway, increasing protoporphyrin to levels higher than what can be used in haematopoiesis. The manifestations of XLDPP therefore resemble EPP: protoporphyrin accumulation is caused in the former by increased production and in the latter by a block in its conversion to haem. Iron deficiency, common in Indian children, reduces haem production and consequently protoporphyrin utilization, potentially exacerbating the protoporphyrin accumulation caused by XLDPP. In a previous patient with XLDPP and anaemia due to gastrointestinal bleeding, iron therapy successfully lowered red cell protoporphyrin levels (Whatley SD, Ducamp S, Gouya L et al. C-terminal deletions in the ALAS2 gene lead to gain of function and cause X-linked dominant protoporphyria without anemia or iron overload. Am J Hum Genet 2008; 83:408–14). Nail dystrophy has not previously been reported in XLDPP. The appearance here is consistent with photo-onycholysis in a child whose feet are always exposed in sandals. The delay in diagnosing photosensitivity and severity of his skin changes can be attributed to the difficulty in interpreting symptoms in young children and to local conditions where sun exposure is unavoidable. Management here involves iron therapy, photoprotection, monitoring of liver function and genetic counselling; female carriers may also have symptoms. We are grateful to national experts for helpful discussion of this case.

CREATION OF ISOGENIC CELL LINE MODELS OF 17P DELETION TO STUDY CLONAL EVOLUTION AND MECHANISMS OF THERAPY RESISTANCE IN CLL AND DLBCL

Introduction: A deletion of chromosome 17p (del17p), on which the tumour suppressor gene TP53 is located, is a prevalent genetic aberration in many cancer types. In chronic lymphocytic leukaemia (CLL), diffuse large B-cell lymphoma (DLBCL), and other B cell malignancies this chromosomal mutation is associated with high fitness advantage in the setting of therapy, and the most adverse clinical outcome. Whilst resistance to the chemoimmunotherapies often used in the treatment of CLL and DLBCL can be explained by the functional loss of p53, such deactivation does not explain the increase in aggressive behaviour of the malignant cells. To study this, we aimed to develop in-vitro models of 17p deletion in CLL and DLBCL by creating TP53 (15.6 kbp) and 17p (20.5 Mbp) deletions by means of CRISPR-Cas9 genome editing. Methods: The efficiency of the gene editing machineries were firstly assessed in the adherent human colorectal cancer cell line HCT116 prior to their application on the B-cell lines, which are less amenable to genetic manipulation. TP53 gene removal (15.6 kbp) was performed by transient transfection of a vector encoding Cas9 as well as single guide (sg) RNAs targeting upstream and downstream of the TP53 coding region. For deletion of 17p (20.5 Mbp) sgRNAs targeting exon 2 of GEMIN4 (telomeric) and intron 5 of MAP2K3 (centromeric) were designed. PCR was used to confirm deletions. Results: Isogenic HCT116 cell lines with hemizygous and homozygous TP53 deletion, as well as hemizygous 17p deletion were obtained (Figure 1). HG-3 cells, a CLL cell line, were successfully transduced with inducible-Cas9, and subsequently with the sgRNAs for removal of TP53 and 17p. Keyword: Tumor Biology and Heterogeneity No conflicts of interests pertinent to the abstract.

LeishIF3d is a non-canonical cap-binding protein in Leishmania.

Translation of most cellular mRNAs in eukaryotes proceeds through a cap-dependent pathway, whereby the cap-binding complex, eIF4F, anchors the pre-initiation complex at the 5' end of mRNAs driving translation initiation. The genome of Leishmania encodes a large repertoire of cap-binding complexes that fulfill a variety of functions possibly involved in survival along the life cycle. However, most of these complexes function in the promastigote life form that resides in the sand fly vector and decrease their activity in amastigotes, the mammalian life form. Here we examined the possibility that LeishIF3d drives translation in Leishmania using alternative pathways. We describe a non-canonical cap-binding activity of LeishIF3d and examine its potential role in driving translation. LeishIF3d is required for translation, as reducing its expression by a hemizygous deletion reduces the translation activity of the LeishIF3d(+/-) mutant cells. Proteomic analysis of the mutant cells highlights the reduced expression of flagellar and cytoskeletal proteins, as reflected in the morphological changes observed in the mutant cells. Targeted mutations in two predicted alpha helices diminish the cap-binding activity of LeishIF3d. Overall, LeishIF3d could serve as a driving force for alternative translation pathways, although it does not seem to offer an alternative pathway for translation in amastigotes.

Nance-Horan Syndrome: characterization of dental, clinical and molecular features in three new families

BackgroundNance–Horan syndrome (NHS; MIM 302,350) is an extremely rare X-linked dominant disease characterized by ocular and dental anomalies, intellectual disability, and facial dysmorphic features.Case presentationWe report on five affected males and three carrier females from three unrelated NHS families. In Family 1, index (P1) showing bilateral cataracts, iris heterochromia, microcornea, mild intellectual disability, and dental findings including Hutchinson incisors, supernumerary teeth, bud-shaped molars received clinical diagnosis of NHS and targeted NHS gene sequencing revealed a novel pathogenic variant, c.2416 C > T; p.(Gln806*). In Family 2, index (P2) presenting with global developmental delay, microphthalmia, cataracts, and ventricular septal defect underwent SNP array testing and a novel deletion encompassing 22 genes including the NHS gene was detected. In Family 3, two half-brothers (P3 and P4) and maternal uncle (P5) had congenital cataracts and mild to moderate intellectual deficiency. P3 also had autistic and psychobehavioral features. Dental findings included notched incisors, bud-shaped permanent molars, and supernumerary molars. Duo-WES analysis on half-brothers showed a hemizygous novel deletion, c.1867delC; p.(Gln623ArgfsTer26).ConclusionsDental professionals can be the first-line specialists involved in the diagnosis of NHS due to its distinct dental findings. Our findings broaden the spectrum of genetic etiopathogenesis associated with NHS and aim to raise awareness among dental professionals.

Social Behavioral Impairments in SYNGAP1-related Intellectual Disability (P11-9.003)

<h3>Objective:</h3> The goal of this study is to characterize the prevalence and severity of social impairments for children with <i>SYNGAP1</i>-related intellectual disability (<i>SYGNAP1</i>-ID), and to compare the severity of social impairment between patients with <i>SYNGAP1</i>-ID and, the phenotypically similar, Phelan-McDermid Syndrome (PMD). <h3>Background:</h3> Synaptopathies are neurodevelopmental disorders caused by genetic mutations disrupting the development and function of neuronal synapses. <i>SYNGAP1</i>-related intellectual disability is due to either de novo loss-of-function single nucleotide variants in <i>SYNGAP1</i> or a hemizygous deletion of the chromosome 6p21.3. Phenotypic presentations of <i>SYNGAP1</i>-ID can include epilepsy, intellectual disability, autism, speech impairments, sleep abnormalities, global developmental delays, and behavioral issues. <h3>Design/Methods:</h3> We utilized the validated Social Responsiveness Scale, Second Edition (SRS-2) to investigate the phenotypic presentation of social-behavioral impairments for two synaptopathies—<i>SYNGAP1</i>-ID (n=32) and PMD (n=19). The short form SRS-2, derived for populations with severe intellectual disability by Sturm et al., was also utilized. The short form SRS-2 limits the influence of age, expressive language, behavioral problems, and nonverbal IQ on survey scores, while maintaining a unidimensional factor structure. <h3>Results:</h3> For the full form analysis, both <i>SYNGAP1</i>-ID and PMD had significantly elevated total and subcategory T-scores as compared to controls, with no significant difference between <i>SYNGAP1</i>-ID and PMD. Similarly, the short form analysis showed significantly elevated total scores for both <i>SYNGAP1</i>-ID and PMD as compared to healthy controls, with no significant difference between the two synaptopathies. <h3>Conclusions:</h3> The survey data showed 87.5% of <i>SYNGAP1</i>-ID and 100% of PMD individuals met the criteria for mild to severe deficiencies in reciprocal social behavior—with majority falling within the “Severe” category (68.8%, <i>SYNGAP1</i>-ID; 73.7%, PMD). Survey item completion data suggests improved utility of the Sturm et al. short form assessment for this patient population. <b>Disclosure:</b> Ms. Naveed has nothing to disclose. Maria McCormack has nothing to disclose. Dr. Holder has received personal compensation in the range of $0-$499 for serving as a Consultant with Stoke Pharmaceutical.

Atypical meningiomas with an immunohistochemical profile consistent with hypermetabolic or proliferative molecular groups show high mitotic index, chromosomal instability, and higher recurrence risk

The use of adjuvant radiotherapy is controversial for atypical meningiomas undergoing gross total resection. It has recently been proposed that meningiomas may be classified into four molecular groups (MG): immunogenic (MG1), benign NF2-wildtype (MG2), hypermetabolic (MG3), and proliferative (MG4). The two latter have the worst prognosis, and it has been suggested that they can be identified using ACADL and MCM2 immunostainings. We studied 55 primary atypical meningiomas, treated with gross total resection and no adjuvant therapies, to assess whether ACADL and MCM2 immuno-expression may identify patients at higher recurrence risk, thus requiring adjuvant treatments. Twelve cases resulted ACADL-/MCM2-, 9 ACADL + /MCM2-, 17 ACADL + /MCM2 + , and 17 ACADL-/MCM2 + . MCM2 + meningiomas displayed more frequent atypical features (prominent nucleoli, small cells with high nuclear-to-cytoplasmic ratio) and CDKN2A hemizygous deletion (HeDe) (P = 0.011). The immunoexpression of ACADL and/or MCM2 was significantly associated with higher mitotic index, 1p and 18q deletions, increased recurrence rate (P = 0.0006), and shorter recurrence-free survival (RFS) (P = 0.032). At multivariate analysis, carried out including ACADL/MCM2 immuno-expression, mitotic index, and CDKN2A HeDe as covariates, this latter resulted a significant and independent prognosticator of shorter RFS (P = 0.0003).

KaryoCreate: A CRISPR-based technology to study chromosome-specific aneuploidy by targeting human centromeres

Aneuploidy, the presence of chromosome gains or losses, is a hallmark of cancer. Here, we describe KaryoCreate (karyotype CRISPR-engineered aneuploidy technology), a system that enables the generation of chromosome-specific aneuploidies by co-expression of an sgRNA targeting chromosome-specific CENPA-binding ɑ-satellite repeats together with dCas9 fused to mutant KNL1. We design unique and highly specific sgRNAs for 19 of the 24 chromosomes. Expression of these constructs leads to missegregation and induction of gains or losses of the targeted chromosome in cellular progeny, with an average efficiency of 8% for gains and 12% for losses (up to 20%) validated across 10 chromosomes. Using KaryoCreate in colon epithelial cells, we show that chromosome 18q loss, frequent in gastrointestinal cancers, promotes resistance to TGF-β, likely due to synergistic hemizygous deletion of multiple genes. Altogether, we describe an innovative technology to create and study chromosome missegregation and aneuploidy in the context of cancer and beyond.

Pan-cancer genomic analysis shows hemizygous PTEN loss tumors are associated with immune evasion and poor outcome

In tumors, somatic mutations of the PTEN suppressor gene are associated with advanced disease, chemotherapy resistance, and poor survival. PTEN loss of function may occur by inactivating mutation, by deletion, either affecting one copy (hemizygous loss) leading to reduced gene expression or loss of both copies (homozygous) with expression absent. Various murine models have shown that minor reductions in PTEN protein levels strongly influence tumorigenesis. Most PTEN biomarker assays dichotomize PTEN (i.e. presence vs. absence) ignoring the role of one copy loss. We performed a PTEN copy number analysis of 9793 TCGA cases from 30 different tumor types. There were 419 (4.28%) homozygous and 2484 (25.37%) hemizygous PTEN losses. Hemizygous deletions led to reduced PTEN gene expression, accompanied by increased levels of instability and aneuploidy across tumor genomes. Outcome analysis of the pan-cancer cohort showed that losing one copy of PTEN reduced survival to comparable levels as complete loss, and was associated with transcriptomic changes controlling immune response and the tumor microenvironment. Immune cell abundances were significantly altered for PTEN loss, with changes in head and neck, cervix, stomach, prostate, brain, and colon more evident in hemizygous loss tumors. These data suggest that reduced expression of PTEN in tumors with hemizygous loss leads to tumor progression and influences anticancer immune response pathways.

Haemophilia B, severe childhood obesity and other extra-haematological features associated with similar 4Mb-deletions on Xq27: Clinical findings, molecular insights and literature update.

Haemophilia B (HB) is associated with pathogenic variants in F9. Hemizygous deletions encompassing the entire F9 and proximate genes may express extra-haematological clinical phenotypes. To analyse the genotype/phenotype correlations in two unrelated boys with severe early childhood obesity (SCO), global developmental delay (GDD) and similar bleeding phenotype associated with comparable Xq27 deletions spanning the entire F9 and proximate genes, and characterise the pathogenic events estimating the most likely mutational mechanism involved. Entire F9-deletions were detected in three hemizygous unrelated probands with HB: two cases, C#1/C#2, presented SCO and GDD and a control patient (Co), who only had severe bleeding symptoms. Dense SNP-array and case-specific STS walking scan allowed characterisation of the deletion breakpoints. Extensive use of bioinformatics, statistics and clinical databases allowed the investigation of genotype-phenotype associations. Patients C#1/C#2 and Co resulted in a complete F9 and additional gene deletions of variable extensions on Xq26.3-Xq27.2 (C#1/C#2/Co: 4.3Mb/3.9Mb/160Kb). C#1/C#2 common deleted gene SOX3 is directly associated with SCO, GDD and pituitary hypothyroidism (PH) whilst C#2 extra-deleted gene MAGEC2 indirectly relates to anal atresia (AA). Breakpoint analysis revealed the involvement of the mechanisms of Alu/Alu recombination for the first time in HB and non-homologous or alternative end-joining. Our results represent the first report of unrelated patients with HB, SCO and GDD. This study and the literature update expand the spectrum of clinical findings and molecular insights observed in patients with HB caused by complete F9 and nearby SOX3 and MAGEC2 gene deletions, which may configure a contiguous gene syndrome.

High-Grade Endometrial Stromal Sarcomas With YWHAE::NUTM2 Gene Fusion Exhibit Recurrent CDKN2A Alterations and Absence of p16 Staining is a Poor Prognostic Marker

High-grade endometrial stromal sarcomas (HGESSs) are aggressive uterine tumors harboring oncogenic fusion proteins. We performed a molecular study of 36 HGESSs with YWHAE::NUTM2 gene fusion, assessing co-occurring genetic events, and showed that these tumors frequently harbor recurrent events involving the CDKN2A locus on chromosome 9p. Using array-based copy number profiling and CDKN2A fluorescence in situ hybridization, we identified homozygous and hemizygous deletions of CDKN2A in 18% and 14% of tumors (n = 22 analyzed), respectively. While all YWHAE-rearranged HGESSs with retained disomy for CDKN2A were immunohistochemically positive for p16INK4 (p16), all tumors with homozygous deletion of CDKN2A showed complete absence of p16 staining. Of the 2 tumors with a hemizygous deletion of CDKN2A, 1 showed diffuse and strong p16 positivity, whereas the other showed complete absence of staining. In the p16-negative case, we did not find intragenic mutations or DNA promoter methylation to explain the p16 protein loss, implicating other mechanisms in the regulation of protein expression. In our cohort, subclonal or complete absence of p16 staining was associated with worse overall survival compared with positive p16 staining (1-year overall survival: 28.6% vs 90.7%, respectively; n = 32; P < .001), with all 7 patients in the p16-negative group having succumbed to their disease within 2 years of diagnosis. Our results suggested CDKN2A alterations as a cooperative driver of tumorigenesis in a subset of HGESSs with the YWHAE::NUTM2 gene fusion and showed p16 to be a potential prognostic marker.