Intragenic Deletion Research Articles

Prenatal and postnatal diagnosis of Phelan-McDermid syndrome: A report of 21 cases from a medical center and review of the literature.

Background: Phelan–McDermid syndrome (PMS), caused by deletions at 22q13.3 and pathogenic variants in the SHANK3 gene, is a rare developmental disorder characterized by hypotonia, developmental delay (DD), intellectual disability (ID), autism spectrum disorder (ASD), dysmorphic features, absence of or delayed language, and other features. Methods: Conventional karyotyping, chromosomal microarray analysis (CMA), and whole exome sequencing (WES) have been used to detect genetic defects causing PMS. We summarized the genetic and clinical findings from prenatal to postnatal stages of detected cases of PMS and mapped potential candidate haploinsufficient genes for deletions of 22q13. This study aimed to summarize the laboratory findings, genetic defects, and genotype–phenotype correlations for Chinese patients with PMS. Results: Seven prenatal cases and fourteen postnatal cases were diagnosed with PMS in our center. Thirteen cases had a deletion ranging in size from 69 to 9.06 Mb at 22q13.2-q13.33, and five cases had a pathogenic variant or an intragenic deletion in the SHANK3 gene. Three familial cases with a parental carrier of a balanced translocation were noted. A review of the literature noted another case series of 29 cases and a report of five cases of PMS in China. Genotype–phenotype correlations confirmed haploinsufficiency of the SHANK3 gene for PMS and suggested other candidate haploinsufficient genes TNFRSFI3C and NFAM1 genes for immunological features and TCF20, SULT4A1, PARVB, SCO2, and UPK3A genes for intellectual impairment and behavioral abnormality, neurological features, macrocephaly/hypotonia, oculopathy, and renal adysplasia, respectively. Conclusion: Indications for prenatal diagnosis of PMS are not specific, and approximately 85% prenatally diagnosed PMS elected termination of pregnancies after genetic counseling. For postnatal cases, 62.5% were caused by a deletion at 22q13 and 37.5% were caused by a pathogenic variant or an intragenic deletion in the SHANK3 gene. Approximately 6.7% of cases with a deletion were familial, and almost all pathogenic variants were de novo. Combined karyotype, CMA, and WES should be performed to increase the diagnostic yield. The identification of other candidate haploinsufficient genes in deletions of 22q13.2-q13.33 could relate to more severe dysmorphic features, neurologic defects, and immune deficiency. These results provided evidence for diagnostic interpretation, genetic counseling, and clinical management for the Chinese cases of PMS.

HIDEA syndrome is caused by biallelic, pathogenic, rare or founder P4HTM variants impacting the active site or the overall stability of the P4H-TM protein.

HIDEA syndrome is caused by biallelic pathogenic variants in P4HTM. The phenotype is characterized by muscular and central hypotonia, hypoventilation including obstructive and central sleep apneas, intellectual disability, dysautonomia, epilepsy, eye abnormalities, and an increased tendency to develop respiratory distress during pneumonia. Here, we report six new patients with HIDEA syndrome caused by five different biallelic P4HTM variants, including three novel variants. We describe two Finnish enriched pathogenic P4HTM variants and demonstrate that these variants are embedded within founder haplotypes. We review the clinical data from all previously published patients with HIDEA and characterize all reported P4HTM pathogenic variants associated with HIDEA in silico. All known pathogenic variants in P4HTM result in either premature stop codons, an intragenic deletion, or amino acid changes that impact the active site or the overall stability of P4H-TM protein. In all cases, normal P4H-TM enzyme function is expected to be lost or severely decreased. This report expands knowledge of the genotypic and phenotypic spectrum of the disease.

A novel CREBBP mutation and its phenotype in a case of Rubinstein–Taybi syndrome

BackgroundThis study was to report a novel CREBBP mutation and phenotype in a child with Rubinstein–Taybi syndrome.MethodsCase report of a 9-year-old boy.ResultsWe described the patient’s clinical manifestations in detail, and found that in addition to the typical systemic manifestations of the syndrome, the outstanding manifestation of the child was severe intellectual deficiency and prominent ocular abnormalities. Whole-exome sequencing and sanger sequencing were performed on the patient and his parents, a large intragenic deletion, covering the exon 1 region and part of the intron 1 region of the TRAP1 gene, and the entire region from intron 27 to exon 30 of the CREBBP gene (chr16:3745393-3783894) was identified on the patient. This mutation affected the CREBBP histone acetyltransferase (HAT) domain.ConclusionsThis findings in our patient add to the spectrum of genetic variants described in Rubinstein–Taybi syndrome and present a RSTS patient with various ocular anomalies including early onset glaucoma.

Genomic testing for copy number and single nucleotide variants in spermatogenic failure.

To identify clinically significant genomic copy number (CNV) and single nucleotide variants (SNV) in males with unexplained spermatogenic failure (SPGF). Peripheral blood DNA from 97/102 study participants diagnosed with oligozoospermia, severe oligozoospermia, or non-obstructive azoospermia (NOA) was analyzed for CNVs via array comparative genomic hybridization (aCGH) and SNVs using whole-exome sequencing (WES). Of the 2544 CNVs identified in individuals with SPGF, > 90% were small, ranging from 0.6 to 75kb. Thirty, clinically relevant genomic aberrations, were detected in 28 patients (~ 29%). These included likely diagnostic CNVs in 3/41 NOA patients (~ 7%): 1 hemizygous, intragenicTEX11 deletion, 1 hemizygous DDX53 full genedeletion, and 1 homozygous, intragenic STK11 deletion. High-level mosaicism for X chromosome disomy (~ 10% 46,XY and ~ 90% 47,XXY) was also identified in 3 of 41 NOA patients who previously tested normal with conventional karyotyping. The remaining 24 CNVs detected were heterozygous, autosomal recessive carrier variants. Follow-up WES analysis confirmed 8 of 27 (30%) CNVs (X chromosome disomy excluded). WES analysis additionally identified 13 significant SNVs and/or indels in 9 patients (~ 9%) including X-linked AR, KAL1, and NR0B1 variants. Using a combined genome-wide aCGH/WES approach, we identified pathogenic and likely pathogenic SNVs and CNVs in 15 patients (15%) with unexplained SPGF. This value equals the detection rate of conventional testing for aneuploidies and is considerably higher than the prevalence of Y chromosome microdeletions. Our results underscore the importance of comprehensive genomic analysis in emerging diagnostic testing of complex conditions like male infertility.

A unique Smith-Magenis patient with a de novo intragenic deletion on the maternally inherited overexpressed RAI1 allele

RAI1 is a dosage-sensitive gene whose decreased or increased expression by recurrent and non-recurrent 17p11.2 deletions or duplications causes Smith-Magenis (SMS) or Potocki-Lupski syndromes (PTLS), respectively. Here we report on a 21-year-old female patient showing SMS phenotype who was found to carry a 3.4 kb de novo intragenic RAI1 deletion. Interestingly, a significant increase in RAI1 transcript levels was identified in the patient’s, brother’s and mother’s peripheral blood cells. Allele-specific dosage analysis revealed that the patient’s maternally inherited overexpressed RAI1 allele harbors the intragenic deletion, confirming the SMS diagnosis due to the presence of a single wild-type RAI1 functional allele. The mother and brother do not present any PTLS neurologic/behavioral clinical features. Extensive sequencing of RAI1 promoter and predicted regulatory regions showed no potential causative variants accounting for gene overexpression. However, the mother and both children share a novel private missense variant in RAI1 exon 3, currently classified as a VUS (uncertain significance), though predicted by two bioinformatic tools to disrupt the binding site of one specific transcription factor. The reported familial case, the second showing RAI1 overexpression in the absence of RAI1 duplication, may help to understand the regulation of RAI1 dosage sensitivity although its phenotypic effect remains to be determined.

Molecular Genetic Analysis of Patients with Duchenne/Becker Muscular Dystrophy by Multiplex Ligation-Dependent Probe Amplification and Next-Generation Sequencing Techniques

Background: Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are neuromuscular genetic disorders, which are characterized by mutations in the dystrophin gene. Large deletions or duplications in the dystrophin gene are found in approximately 60 to 70% of cases, and the remaining have point mutations, small deletions, or insertions. Objectives: The aim of this study was to assess mutations in the dystrophin gene in clinically suspicious subjects of DMD/ BMD by multiplex ligation-dependent probe amplification (MLPA) and next-generation sequencing (NGS) techniques. Methods: This study consisted of 13 clinically suspected DMD/BMD patients. MLPA, NGS, and Sanger sequencing were used to determine mutations in the dystrophin gene. In order to minimize the time and cost, MLPA (SALSA P034/P035 DMD test kit) was performed as the first step to detect large deletions or duplications, followed by NGS and Sanger sequencing for MLPA-negative cases. Results: This study included nine males and four females with clinical suspicion of DMD or BMD. The MLPA was performed for all cases, among which ten cases were definitely diagnosed with DMD/BMD. NGS and Sanger sequencing were performed for the three remaining subjects to find out possible point mutations and small deletions, which could not be detected by MLPA. Nine intragenic deletions were identified, mostly in exons 46 to 47 and 4 to 7. Furthermore, an intragenic triplication was detected in exon 71 by MLPA. Conclusions: In agreement with other reports, our findings indicated that exonic deletions of the dystrophin gene are more frequent than duplications and point mutations in Duchenne/ Becker muscular dystrophy. Most deletions were demonstrated to occur in exons 46 to 47 and 4 to 7.

What do we know about the molecular and biological features of EGFR in glioblastomas and non-small cell lung cancer?

The EGFR mutation is one of the most common mutations in malignant neoplasms. The epidermal growth factor receptor (EGFR) is a growth factor receptor that induces cell differentiation and proliferation when activated by binding one of its ligands. The receptor is located on the cell surface, where ligand binding activates a tyrosine kinase in the intracellular region of the receptor. The tyrosine kinase phosphorylates a number of intracellular substrates and further activates pathways leading to cell growth, DNA synthesis and oncogene expression. Gene amplification is a process characterized by an increase in the copy number of a restricted region in the chromosome shoulder, which is associated with overexpression of the corresponding amplified gene. Amplification of the EGFR gene is detected in about 40% of glioblastoma cases. It should be noted that EGFR gene amplification is accompanied by the acquisition of many mutations, which include intragenic deletions and point mutations. The most common EGFR mutation in glioblastomas of the brain is a deletion in exon 2-7 (EGFRvIII) frame, which occurs in 50% of all cases of EGFR-amplified glioblastoma. Despite great advances in molecular biology and targeted therapies, patients with non-small cell lung cancer (NSCLC) and glioblastoma still lead in mortality. Most of them have “classical” EGFR mutations (deletions in exon 19 and 21), but 15-20% of patients have rare mutations, which most often include point mutations, deletions and insertions in exon 18 and 25. Thus, rare EGFR mutations are a promising diagnostic and therapeutic target in cancer. This review summarizes data on the role of EGFR in the carcinogenesis of NMPL and glioblastoma. The literature search was performed using the Pubmed database.

Feasibility and clinical utility of comprehensive genomic profiling of hematological malignancies.

Identification of genetic alterations through next‐generation sequencing (NGS) can guide treatment decision‐making by providing information on diagnosis, therapy selection, and prognostic stratification in patients with hematological malignancies. Although the utility of NGS‐based genomic profiling assays was investigated in hematological malignancies, no assays sufficiently cover driver mutations, including recently discovered ones, as well as fusions and/or pathogenic germline variants. To address these issues, here we have devised an integrated DNA/RNA profiling assay to detect various types of somatic alterations and germline variants at once. Particularly, our assay can successfully identify copy number alterations and structural variations, including immunoglobulin heavy chain translocations, IKZF1 intragenic deletions, and rare fusions. Using this assay, we conducted a prospective study to investigate the feasibility and clinical usefulness of comprehensive genomic profiling for 452 recurrently altered genes in hematological malignancies. In total, 176 patients (with 188 specimens) were analyzed, in which at least one alteration was detected in 171 (97%) patients, with a median number of total alterations of 7 (0–55). Among them, 145 (82%), 86 (49%), and 102 (58%) patients harbored at least one clinically relevant alteration for diagnosis, treatment, and prognosis, respectively. The proportion of patients with clinically relevant alterations was the highest in acute myeloid leukemia, whereas this assay was less informative in T/natural killer‐cell lymphoma. These results suggest the clinical utility of NGS‐based genomic profiling, particularly for their diagnosis and prognostic prediction, thereby highlighting the promise of precision medicine in hematological malignancies.

Abstract 798: Comprehensive genomic profiling of tissue and liquid biopsies reveals the landscape of reversion mutations

Abstract Loss of function mutations in homologous recombination (HR) repair genes predispose individuals to cancer due to the genomic instability brought about by the inability to properly repair DNA. These alterations may also identify patients eligible for treatment with poly (ADP-ribose) polymerase (PARP) inhibitors and platinum drugs that exploit synthetic lethality; however, resistance eventually occurs. This resistance is largely the result of reversion mutations, which restore the DNA repair function of mutated HR genes. We employed a computational approach to identify patients with breast, ovarian, pancreas, or prostate cancer who underwent comprehensive genomic profiling (CGP; Foundation Medicine, Inc.) and had reversion mutations in the BRCA1, BRCA2, PALB2, RAD51C, or RAD51D genes. Reversion mutations were identified in six different categories: 1) missense mutations that occur at the same amino acid position as a deleterious alteration, 2) non-frameshift deletion mutations that encompass a deleterious alteration, 3) frameshift mutations that restore the open reading frame of another frameshift mutation, 4) splice-site mutations that occur at the same exon as a deleterious alteration, 5) copy number losses that remove a deleterious alteration, and 6) intragenic deletions that lead to loss of deleterious mutation. Using our computational approach, we identified 483 patients who 1,112 reversion mutation pairs in both solid tissue- and liquid biopsy-derived samples. The most frequently observed category was a non-frameshift deletion of a deleterious mutation. Overall, a greater number of reversion mutations were found in liquid biopsy samples. BRCA2 was the most frequently reverted gene followed by BRCA1, PALB2, RAD51D, and RAD51C. BRCA1-mutated samples had up to six reversion mutations, and for BRCA2, we observed as many as 228 putative reversion pairs in one sample. PALB2, RAD51C, and RAD51D had no more than two reversion mutations. We further examined co-occurring alterations and found correlations with genes in multiple signaling pathways including the MYC signaling pathway, which contained more mutations in reversion-positive samples. Lastly, we explored real-world longitudinal data from reversion-positive samples and found that the presence of a reversion mutation was significantly associated with lower overall survival for breast, ovarian, and prostate cancer patients. In summary, we characterized the landscape of reversion mutations in patients who underwent tissue or liquid biopsy-based CGP during clinical cancer treatment. Our data provide a better perspective for the identification and treatment of cancers involving a reversion mechanism. Citation Format: Shannon T. Bailey, Meijuan Li, James Thornton, Lei Yang, Chenming Cui, Mark Hartman, Garrett Frampton, Brennan Decker, Christine Vietz. Comprehensive genomic profiling of tissue and liquid biopsies reveals the landscape of reversion mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 798.

Abstract 2158: Preclinical evaluation of CFT1946 as a selective degrader of mutant BRAF for the treatment of BRAF driven cancers

Abstract The BRAF kinase is a critical node in the MAPK signaling pathway and is mutated in approximately 8% of human cancers including melanoma (~60%), thyroid (~60%), and lung adenocarcinoma (~10%). The most common mutation in BRAF is V600E (Class I), occurring in half of malignant melanomas. This mutation hyperactivates ERK and signals as a RAF inhibitor-sensitive monomer. BRAF inhibitors including vemurafenib, dabrafenib and encorafenib have produced impressive responses in V600X patients, however resistance usually emerges within a year, including RAS mutation, BRAFV600E amplification, and BRAFV600E intragenic deletion or splice variants. These inhibitors are also ineffective against non-V600 BRAF mutants (Class II & III). To address some of these limitations we have developed CFT1946, a bifunctional degradation activating compound (BiDAC™) degrader comprising a BRAF kinase domain targeting ligand linked to a cereblon ligand. CFT1946 is capable of degrading BRAF V600E (Class I), G469A (Class II), G466V (Class III) mutations, and the p61-BRAFV600E splice variant while maintaining exquisite selectivity against the proteome including WT BRAF and CRAF. In A375 cells, CFT1946 potently degraded BRAFV600E (Emax = 26%; DC50 = 14nM at 24hr) and, inhibited ERK phosphorylation (IC50 = 11nM at 24hr) and cell growth (GI50 = 94nM at 96hr) while having no effect in the mutant KRAS driven cell line HCT116. In A375 xenografts, oral delivery of CFT1946 resulted in deeper tumor regressions when dosed at 10 mg/kg PO BID and compared favorably to a clinically relevant dose of encorafenib. We further evaluated CFT1946 in an engineered A375-BRAFV600E/NRASQ61K double mutant model of BRAF inhibitor resistance. CFT1946 was able to degrade BRAFV600E in these cells and was much more effective than encorafenib at inhibiting viability in vitro. In this model, in vivo dosing of single agent CFT1946 caused robust tumor growth inhibition and combination with the MEK inhibitor, trametinib, resulted in tumor regressions. The combination of encorafenib and trametinib showed no activity in the same model. Next, we demonstrated that CFT1946 was able to degrade additional BRAF mutant proteins including G469A (Class II), G466V (Class III), and the p61-BRAFV600E splice variant using heterologous expression in HEK293T cells. Additionally, we also showed that CFT1946, but not encorafenib, inhibited proliferation of the BRAFG466V heterozygous lung tumor cell line H1666. Based on its activity in preclinical models, including models of BRAF inhibitor resistance, and its drug-like properties we are progressing CFT1946 as a candidate for clinical development in patients with solid tumors bearing BRAF V600X mutations. Further, given CFT1946’s activity on non-V600 BRAF mutations, we are continuing to explore CFT1946 and related BiDAC degraders as therapeutic options for patients bearing Class II or Class III BRAF mutations. Citation Format: Mathew E. Sowa, Bridget Kreger, Joelle Baddour, Yanke Liang, Jeffrey R. Simard, Laura Poling, Ping Li, Robert Yu, Ashley Hart, Roman V. Agafonov, Grace Sarkissian, Joe Sahil Patel, Richard Deibler, Kyle S. Cole, Scott Eron, David Cocozziello, Fazlur Rahman, Moses Moustakim, Christopher G. Nasveschuk, Katrina L. Jackson, Mark Fitzgerald, Victoria Garza, Morgan O’Shea, Gesine Veits, Jeremy L. Yap, Andrew J. Phillips, Elizabeth Norton, Adam S. Crystal, Stewart L. Fisher, Roy M. Pollock. Preclinical evaluation of CFT1946 as a selective degrader of mutant BRAF for the treatment of BRAF driven cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2158.

ETMR-06. Molecular and clinical characteristics of CNS tumors withBCOR(L1) fusion/internal tandem duplication

Central nervous system (CNS) tumor with BCOR internal tandem duplication (BCOR-ITD) have recently been introduced in the 5th edition of the WHO classification of CNS tumors, however, their molecular makeup and clinical characteristics remain widely enigmatic. This is further complicated by the recent discovery of tumors characterized by gene fusions involving BCOR or its homologue BCORL1. We identified a cohort of 206 BCOR altered CNS tumors via DNA methylation profiling and conducted in-depth molecular and clinical characterization in an international effort. By performing t-SNE clustering analysis we found that BCOR-fusion tumors form a distinct cluster (n=61), adjacent to BCOR-ITD cases (n=145). The identified fusion partners of BCOR(L1) included EP300 (n=20), CREBBP (n=5), and NUTM2HP (n=1). Notably, three cases within the BCOR-ITD cluster harbored a c-terminal intragenic deletion within BCOR. With respect to clinical characteristics gender ratio was balanced in BCOR-fusion cases (m/f, 1.1), whereas predominance of male patients was observed in the BCOR-ITD group (m/f, 1.5). Moreover, age at diagnosis of BCOR-fusion patients was higher as compared to BCOR-ITD cases (15 vs 4.5 years). Interestingly, BCOR-fusion tumors were exclusively found in the supratentorial region being originally diagnosed as ependymomas or gliomas whereas BCOR-ITD emerged across the entire CNS with diverse original diagnoses. 8% of BCOR-ITD and none of BCOR-fusion cases were disseminated at diagnosis. In line with this observation, 40% of first relapses within the BCOR-ITD group were metastatic which was less frequent in BCOR-fusion tumors. Survival estimates demonstrated no differences, generally showing short median PFS (BCOR-fusion, 2 years, n=15; BCOR-ITD, 1.8 years, n=55) and intermediate OS rates (BCOR-fusion, 6.8 years, n=18; BCOR-ITD 6.3 years, n=60). Further molecular and clinical characterization is ongoing potentially revealing first therapeutic leads for these highly aggressive CNS tumor types.

Exome sequencing reveals the first intragenic deletion in ABCA5 underlying autosomal recessive hypertrichosis.

Hereditary hypertrichosis (HH) is characterized by excessive hair growth on various body areas, which is independent of the individual's age. This rare hair disorder has been classified by its origin (genetic or acquired), age of onset, breadth of hair distribution (universal or localized) and the affected body areas. HH is often linked to several additional congenital abnormalities involving teeth, heart and bones. Human HH is associated with heterozygous genomic duplications and deletions in the chromosomal region 17q24.2-q24.3, containing genes such as ABCA5, ABCA6, ABCA10 and MAP2K6. Recently, a homozygous splice-site variant in ABCA5 has been reported to cause autosomal recessive congenital generalized hypertrichosis terminalis (CGHT; OMIM 135400). To investigate the clinical and genetic basis of autosomal recessive hypertrichosis in a large consanguineous Pakistani family. In the present study, we characterized a family of Pakistani origin segregating CGHT in an autosomal recessive pattern, using whole exome sequencing followed by Sanger sequencing RESULTS: We identified a novel 2-bp intragenic deletion [NM_172232.4(ABCA5);c.977_978delAT] causing a frameshift variant (p.His326ArgfsTer5) in ABCA5. To our knowledge, this is the first intragenic deletion in ABCA5 underlying CGHT. The findings further validate the involvement of ABCA5 in hair development. The study will facilitate genetic counselling of families carrying CGHT-related features in Pakistani and other populations.

19. Phase determination and demonstration of parental mosaicism of intragenic PRKN deletions initially identified by chromosomal microarray analysis

Biallelic variants in PRKN are associated with autosomal recessive (AR) juvenile Parkinson disease. Various PRKN mutation types cause disease, however deletions are frequent due to its large size (∼1.4Mb) and its localization within a common fragile site. Here we present a case with biallelic intragenic PRKN deletions identified using chromosomal microarray (CMA) in a newborn. CMA was performed on a six-day old female with a family history of a 17q12 duplication. This maternal 1.5 Mb duplication was observed, along with two non-overlapping intragenic deletions encompassing exons 2 (227 Kb) and 7 (140 Kb) of PRKN.

Familial platelet disorder due to germline exonic deletions in RUNX1: a diagnostic challenge with distinct alterations of the transcript isoform equilibrium

Germline pathogenic variants in RUNX1 are associated with familial platelet disorder with predisposition to myeloid malignancies (FPD/MM) with intragenic deletions in RUNX1 accounting for almost 7% of all reported variants. We present two new pedigrees with FPD/MM carrying two different germline RUNX1 intragenic deletions. The aforementioned deletions encompass exons 1–2 and 9–10 respectively, with the exon 9–10 deletion being previously unreported. RNA sequencing of patients carrying the exon 9–10 deletion revealed a fusion with LINC00160 resulting in a change in the 3′ sequence of RUNX1. Expression analysis of the transcript isoform demonstrated altered RUNX1a/b/c ratios in carriers from both families compared to controls. Our data provide evidence on the impact of intragenic RUNX1 deletions on transcript isoform expression and highlight the importance of routinely performing copy number variant analysis in patients with suspected MM with germline predisposition.

Familial intragenic X-linked OPHN1 gene deletion in a newborn male infant with low birth weight and distinctive facial appearance that diagnosed by advanced microarray-CGH method

The oligophrenin-1 (OPHN1) gene is localized in the Xq12 region and it encodes the rho-GTPase-activating protein which spans 500 kb in size and consists of 25 exons. Gene plays crucial role in synaptic function and dendritic morphogenesis. Here we report a 391 kb deletion in OPHN1 gene in a mother and her newborn male child with recognizable pattern of clinical and neuroradiological hallmarks. Mother has short stature, and her son has distinctive facial appearance, bilateral choroid plexus cysts and low birth weight (1600 g). After clinical evaluation, the current large intragenic gene deletion was identified by microarray-CGH and confirmed by MLPA techniques. The P106 MRX probemix kit (MRC Holland C1- 0416, Amsterdam) and Coffalyser software were used for MLPA and Agilent sure print G3 HUMAN CGH 60k Microarray platform and Agilent cytogenomics 4.0.2.21 software (Singapore) were used for advance chromosomal genotyping for mother and his son in the presented results. Presented results showed that mother with X chromosome deletion has a great risk to have a son with mental retardation due to deleted X chromosome transmission in 50% possibility. If the son has clinical findings, the genotype should be screened by using the advanced genetic methodology. Results also showed that once these cases are first diagnosed correctly, they may be candidate to IVF for preimplantation genetic diagnosis by giving appropriate genetic counseling. It is also comment that pregnant women who have the history of having X-linked mental retarded child or a mentally retarded brother need to be tested genetically for prenatal diagnosis.

Unscrambling cancer genomes via integrated analysis of structural variation and copy number

Complex somatic genomic rearrangements and copy number alterations are hallmarks of nearly all cancers. We have developed an algorithm, LINX, to aid interpretation of structural variant and copy number data derived from short-read, whole-genome sequencing. LINX classifies raw structural variant calls into distinct events and predicts their effect on the local structure of the derivative chromosome and the functional impact on affected genes. Visualizations facilitate further investigation of complex rearrangements. LINX allows insights into a diverse range of structural variation events and can reliably detect pathogenic rearrangements, including gene fusions, immunoglobulin enhancer rearrangements, intragenic deletions, and duplications. Uniquely, LINX also predicts chained fusions that we demonstrate account for 13% of clinically relevant oncogenic fusions. LINX also reports a class of inactivation events that we term homozygous disruptions that may be a driver mutation in up to 9% of tumors and may frequently affect PTEN, TP53, and RB1.

EP380: Sequencing of entire 2.2 MB DMD gene facilitates diagnostic testing and aids selection of patients for therapeutic intervention

Duchenne Muscular Dystrophy (DMD) is an X-linked inherited neuromuscular disorder caused by pathogenic variants in the DMD gene. The variant spectrum of DMD is unique including 60% intragenic deletions, 5% duplications and 35% sequence variants. The clinical presentation of DMD includes progressive weakness of the skeletal muscles that leads to loss of ambulation by age 10-12 years. Affected individuals also have significantly elevated creatine kinase (CK) levels in blood. Present mean age of diagnosis of DMD is about 5 years; however, it can be diagnosed as early as immediately after birth through newborn screening (NBS) by measuring serum CK levels followed by confirmatory molecular testing.

Activity of KIN-3248, a next-generation pan-FGFR inhibitor, against acquired FGFR-gatekeeper and molecular-brake drug resistance mutations.

461 Background: Oncogenic FGFR (FGFR1, FGFR2, FGFR3, and FGFR4) gene alterations are observed in approximately 7% of all human cancers and present as point mutations, small intragenic deletions, genomic amplifications, or chromosomal rearrangements/ gene fusions. FGFR2 gene fusions and FGFR3 activating alterations are predicted oncogenic drivers in 10-20% of cholangiocarcinoma and 20-35% of urothelial cancers, respectively. While currently approved FGFR inhibitors (e.g., erdafitinib, pemigatinib, infigratinib) and those in development (e.g., futibatinib) provide clinical benefit in these cancer indications, disease progression typically occurs within 6-8 months of starting treatment and is often associated with the emergence of on-target resistance mutations within the FGFR kinase domain. KIN-3248 is a next-generation, irreversible, small molecule, pan-FGFR inhibitor designed to target clinically relevant primary FGFR driver alterations and secondary resistance mutations, including FGFR2 and FGFR3 gatekeeper, molecular brake, and activation loop mutations. Methods: KIN-3248 was evaluated across wild-type FGFR family members and clinically relevant fusions and kinase domain resistance mutations in vitro. In addition, KIN-3248 activity was assessed in FGFR-driven and FGFR inhibitor-resistant human gastrointestinal xenograft tumor models. Results: KIN-3248 exhibited low nanomolar biochemical potency against wild-type FGFR family members as well as mutants associated with resistance to FGFR inhibitors (IC50 3.9 – 24.1 nM). Consistently, KIN-3248 was active in human FGFR2-PHGDH fusion-positive CCLP-1 and FGFR2-OPTN fusion-positive ICC13-7 cholangiocarcinoma cell lines engineered to express wild-type or clinically relevant gatekeeper, molecular brake, and activation loop mutant alleles (EC50 2.4 – 9.9 nM). Lastly, KIN-3248 led to dose-dependent tumor growth inhibition and regressions in FGFR inhibitor-resistant, patient-derived gastric cancer and cholangiocarcinoma models harboring secondary FGFR2 kinase domain mutations. This efficacy was accompanied by both pharmacodynamic biomarker modulation, downstream pathway inhibition, and apoptotic cell death across models in vitro and in vivo. Conclusions: KIN-3248 is a next-generation, irreversible, orally available, small molecule pan-FGFR inhibitor that overcomes clinically observed secondary mutations in FGFR2 and FGFR3 known to drive resistance to both reversible and irreversible FGFR inhibitors and associated with disease progression. Its highly-selective, potent and broad-spectrum activity against mutations in both the FGFR2 and FGFR3 kinase domains – including gatekeeper, molecular brake, and activation loop alterations – is unique among FGFR inhibitors and has the potential to extend the clinical response of cancer patients with FGFR-altered tumors.

A Clinical and Integrated Genetic Study of Isolated and Combined Dystonia in Taiwan

Dystonia is a clinically and genetically heterogeneous movement disorder. However, genetic causes of dystonia remain largely unknown in Asian subjects. To address this, we applied an integrated two-step approach that included gene dosage analysis and a next-generation sequencing panel containing 72 known genes causative for dystonia and related movement disorders to 318 Taiwanese patients with isolated or combined dystonia. Whole-genome sequencing was performed for one multiplex family with no known causative variant. The panel confirmed the genetic diagnosis in 40 probands (12.6%). A genetic diagnosis was more likely with juvenile onset compared with adult onset (24.2% vs 10.8%; P=0.03) and those with combined features, especially with myoclonus, compared with isolated dystonia (35.3% vs 10.5%; P=0.004). The most common causative genes were SGCE followed by GCH1, TH, CACNA1B, PRRT2, MR1, CIZ1, PLA2G6, and PRKN. Genetic causes were identified from single cases in TOR1A, TUBB4A, THAP1, ATP1A3, ANO3, GNAL, KMT2B, SLC6A3, ADCY5, CYP27A1, PANK2, C19orf12, and SPG11. The whole-genome sequencing analysis identified a novel intragenic deletion in OPHN1 in a multiplex family with X-linked dystonia and intellectual delay. Our findings delineate the genetic architecture and clinical spectrum of dystonia-causing pathogenic variants in an Asian population.

Highly penetrant AUTS2 syndrome phenotype in a boy with AUTS2 C-terminal intragenic deletion

Introduction Syndromic autism spectrum disorders represent a group of childhood neurological conditions, that has been distinguished from nonsyndromic or idiopathic autism based on the presence or absence of other additional morphological signs or clinical symptoms [1]. Syndromic autism is typically associated with chromosomal abnormalities or mutations in a single gene. According to