Splice Acceptor Site Mutation Research Articles

Loss-of-function of activity-dependent neuroprotective protein (ADNP) by a splice-acceptor site mutation causes Helsmoortel-Van der Aa syndrome.

Mutations in ADNP result in Helsmoortel-Van der Aa syndrome. Here, we describe the first de novo intronic deletion, affecting the splice-acceptor site of the first coding ADNP exon in a five-year-old girl with developmental delay and autism. Whereas exome sequencing failed to detect the non-coding deletion, genome-wide CpG methylation analysis revealed an episignature suggestive of a Helsmoortel-Van der Aa syndrome diagnosis. This diagnosis was further supported by PhenoScore, a novel facial recognition software package. Subsequent whole-genome sequencing resolved the three-base pair ADNP deletion c.[-5-1_-4del] with transcriptome sequencing showing this deletion leads to skipping of exon 4. An N-terminal truncated protein could not be detected in transfection experiments with a mutant expression vector in HEK293T cells, strongly suggesting this is a first confirmed diagnosis exclusively due to haploinsufficiency of the ADNP gene. Pathway analysis of the methylome indicated differentially methylated genes involved in brain development, the cytoskeleton, locomotion, behavior, and muscle development. Along the same line, transcriptome analysis identified most of the differentially expressed genes as upregulated, in line with the hypomethylated CpG episignature and confirmed the involvement of the cytoskeleton and muscle development pathways that are also affected in patient cell lines and animal models. In conclusion, this novel mutation for the first time demonstrates that Helsmoortel-Van der Aa syndrome can be caused by a loss-of-function mutation. Moreover, our study elegantly illustrates the use of EpiSignatures, WGS and Phenoscore as novel complementary diagnostic tools in case a of negative WES result.

HLA-C*07:985:01:02Q, a novel allele with an acceptor splice site mutation.

HLA-C*07:985:01:02Q differs from HLA-C*07:985:01 by one nucleotide substitution at the Intron 1 splicing acceptor site.

Investigation of a Fused in Sarcoma Splicing Mutation in a Chinese Amyotrophic Lateral Sclerosis Patient.

Genetic mutations of fused in sarcoma (FUS) causing amyotrophic lateral sclerosis (ALS) may disrupt mRNA splicing events. For example, the FUS c.1394-2delA variant was reported in two western ALS patients, but its molecular mechanism is unclear. In this study, we aim to investigate FUS splice site mutations in Chinese ALS patients. Sanger sequencing was used to identify FUS splicing mutations in Chinese ALS patients. We combined a deep learning tool (SpliceAI), RNA sequencing, and RT-PCR/RT-qPCR to analyze the effect of FUS c.1394-2delA mutation on RNA splicing and expression. AlphaFold was used to predict the protein structure of mutant FUS. In transfected cell lines, we used immunofluorescence to assess cytoplasmic mislocalization of mutant FUS protein. We identified a de novo FUS splice acceptor site mutation (c.1394-2delA, p. Gly466Valfs*14) in one Chinese sporadic ALS patient, which is linked to exon 14 skipping, and upregulated total FUS mRNA expression. The FUS splice site mutation was predicted to be translated into a truncated protein product at C-terminal. In vitro studies revealed that the FUS mutation increased cytoplasmic mislocalization in both HEK293T and SH-SY5Y cells. We identified a de novo FUS splicing mutation (c.1394-2delA, p. Gly466Valfs*14) in 1 out of 233 Chinese ALS patients. It caused abnormal RNA splicing, upregulated gene expression, truncated FUS translation, and cytosolic mislocalization. Our findings suggested that FUS splice site mutation is rare in Chinese ALS patients and extended our knowledge of molecular mechanisms of the FUS c.1394-2delA mutation.

HLA‐DQA1*03:03:01:16Q, a novel allele with an acceptor splice site mutation

DQA1*03:03:01:16Q differs from DQA1*03:03:01:01 by one nucleotide at the Intron 3 splicing acceptor site.

Long-term efficacy of immune checkpoint inhibitors in non-small cell lung cancer patients harboring MET exon 14 skipping mutations.

MET exon 14 skipping mutation, observed in 3-4% of non-small cell lung cancer (NSCLC), is emerging as a targetable alteration. In recent years, immune checkpoint inhibitors (ICI) have been effective in treating several NSCLCs. Our research aimed to investigate the characteristics of patients with NSCLCs harboring MET exon 14 mutations and their response to ICI in Japan. Among the 1954 consecutive NSCLCs diagnosed at Saitama Cancer Center between 2010 and 2019, MET exon 14 skipping mutations were detected in 68 (3.5%) NSCLCs. We evaluated their characteristics such as programmed cell death ligand 1 (PD-L1) expression. Median age of patients with NSCLCs harboring MET exon 14 skipping mutations was 73years. PD-L1 was highly expressed in 17 (70.8%) of the 24 patients examined. Seven patients received ICI monotherapy, and three out of seven had a remarkable treatment response, resulted in objective response rate (ORR) of 42.9% and progression-free survival of 24.7months. Three patients with donor splice-site mutations showed a long-term treatment response, despite the fact that two with acceptor splice-site mutations demonstrated no response and experienced early disease progression with ICI monotherapy. Our results indicated that patients with NSCLCs harboring MET exon 14 mutations presented with a high rate of positive PD-L1 expression. ICI treatment showed a high ORR and long-term efficacy for NSCLCs harboring MET exon 14 mutations. Variants of MET exon 14 splice-site mutations may be associated with ICI response.

Abetalipoproteinemia Due to a Novel Splicing Variant in MTTP in 3 Siblings.

Abetalipoproteinemia (ABL) is a rare recessive condition caused by biallelic loss-of-function mutations in the MTTP gene encoding the microsomal triglyceride transfer protein large subunit. ABL is characterized by absence of apolipoprotein B–containing lipoproteins and deficiencies in fat-soluble vitamins leading to multisystem involvement of which neurological complications are the most serious. We present 3 siblings with ABL who were born to non-consanguineous parents of Filipino and Chinese background. Identical twin boys with long-standing failure to thrive and malabsorption were diagnosed at age 2 years. ABL therapy with vitamins and a specialized diet was initiated, replacing total parenteral nutrition at age 3 years. Their younger sister was diagnosed from a blood sample taken at birth; treatment was instituted shortly thereafter. We observed in the twins reversal and in their sister prevention of ABL systemic features following early implementation of fat restriction and high doses of oral fat-soluble vitamins. A targeted sequencing panel found that each affected sibling is homozygous for a novel MTTP intron 13 -2A>G splice acceptor site mutation, predicted to abolish splicing of intron 13. This variant brings to more than 60 the number of reported pathogenic mutations, which are summarized in this article. The twin boys and their sister are now doing well at 11 and 4 years of age, respectively. This experience underscores the importance of early initiation of targeted specialized dietary and fat-soluble vitamin replacements in ABL.

Mutations in the Human Factor XII Gene

AbstractThe factor XII gene from 31 unrelated factor XII-deficient patients from Germany, Switzerland, and Austria was screened for mutations at the genomic level. Several novel mutations were detected and their absence in a control group of 74 healthy unrelated individuals was checked. Most changes are in the serine protease domain affecting the catalytic triad His-393–Asp-442–Ser-544; two missense mutations, R398Q (arginine 398 to glutamine; gene bank accession no. U71276) and L395M (leucine 395 to methionine; gene bank accession no. U71277), are close to the active site histidine at position 393. Another mutation detected in a cross-reacting material (CRM)-positive female with a history of three abortions affects the active site aspartic acid by changing it to asparagine (D442N; gene bank accession no. U71275). The novel mutation G570R (glycine 570 to arginine; gene bank accession no. U71274) giving rise to a CRM-positive phenotype is located next to Cys571, which forms a vital disulfide bridge. Two mutations are causing reading frame shifts: one single basepair deletion in exon 12 [exon 12: 10590(DelC); gene bank accession no. U71278] and one acceptor splice site mutation [exon 14: 11397(G → A); gene bank accession no. L43615]. The putative regulatory mutation exon 1:−8 (g → c) in the upstream region of the gene is associated with an aberrant Taq I restriction site allele in intron B of the gene (gene bank accession no. X80393).

Clinical, Biochemical, and Molecular Characterization of Metachromatic Leukodystrophy Among Egyptian Pediatric Patients: Expansion of the ARSA Mutational Spectrum.

Metachromatic leukodystrophy (MLD) is a neurodegenerative disorder characterized by progressive demyelination due to deficiency of the enzyme arylsulfatase A (ARSA) in leukocytes, and consequently leads to impaired degradation and accumulation of cerebroside-3-sulfate (sulfatide). This study aimed to sequence the ARSA gene in a total of 43 patients with metachromatic leukodystrophy descendant from 40 Egyptian families. In addition, four carrier parents from two families with children who had died from MLD came to the clinic for genetic analysis. Prenatal diagnosis was performed for four families with molecularly diagnosed MLD sibs. Different mutations were characterized in our cohort, including missense, nonsense, splice, and deletion. Overall, 21 different mutations in the ARSA gene were detected, with 12 novel mutations, i.e. p.Arg60Pro, p.Tyr65*, p.Val112Asp, p.Arg116*, p.Gly124Asp, p.Pro193Ser, p.Gln238*, p.Gln456*, p.Thr276Lys, and p.Gly311Arg, in addition to two new acceptor splice-site mutations 685-1G > A and c.954_956 delCTT. The amniotic fluid samples revealed two carrier fetuses with heterozygous monoallelic mutations, and two affected fetuses had the homozygous biallelic mutations. In conclusion, the current study sheds light on the underlying ARSA gene defect, with an expansion of the mutation spectrum. To our knowledge, this is the first molecular study of MLD among the Egyptian population.

Generation of two patient-derived iPSC lines from siblings (LIBUCi001-A and LIBUCi002-A) and a genetically modified iPSC line (JMUi001-A-1) to mimic dilated cardiomyopathy with ataxia (DCMA) caused by a homozygous DNAJC19 mutation.

Dilated cardiomyopathy with ataxia (DCMA) is an autosomal recessive disorder arising from mutations in DNAJC19. Two patient-derived dermal fibroblast cell lines of siblings with the same homozygous splice acceptor site mutation in DNAJC19 (NM_145261.4):c.130-1G>C were reprogrammed into induced pluripotent stem cell (iPSC) lines (LIBUCi001-A and LIBUCi002-A) using non-integrative Sendai virus. Additionally, a third DNAJC19tv (truncation variant) iPSC line (JMUi001-A-1) was generated by CRISPR/Cas9 in healthy control iPSCs (JMUi001-A). All three DCMA iPSC lines present normal karyotypes, high expression of pluripotency markers and the capacity to differentiate into cells of all three germ layers.

Insights into Chinese prostate cancer germline gene mutation profile: HOXB13 G84E mutation is unsuitable for genetic testing.

e17515 Background: Philadelphia Prostate Cancer Consensus Conference 2017 developed an expert consensus-driven working framework for comprehensive genetic evaluation of inherited PCA. However, the consensus was mainly based on data from Caucasian populations. Genetic differences in the PC molecular landscape between Asian and Caucasian men have been established. Whether Caucasian-based genetic information can be used to guide clinical practice in Chinese population needs further evidence. Methods: 1123 patients with confirmed prostate cancer admitted from March 2018 to August 2019 from 18 Chinese centers were included in this retrospective study. We sequenced the association of 50 gene with prostate cancer risk, clinical treatment implications and predisposition, focusing on DNA repair gene and genetic susceptibility genes pathogenic mutations in Philadelphia Prostate Cancer Consensus Conference. Only protein-truncating alterations (nonsense/stop-gains, frameshift insertions and deletions, and donor and acceptor splice-site mutations) were coded as pathogenic or likely pathogenic protein-truncating alterations. Results: Among all cases, DNA repair gene pathogenic mutations were found in 133 men (11.84%), including BRCA2 (62 [5.5%]), ATM (15 [1.3%]), PALB2 (11 [0.97%]), GEN1 (7 [0.62%]), BRCA1 (6 [0.53%]), MLH3 (4 [0.35%]), MUTYH (4 [0.35%]), PMS1 (4 [0.35%]), RAD50 (4 [0.35%]), ATR (3 [0.27%]), CHEK2 (3 [0.27%]), FANCD2 (3 [0.27%]) etc. Similar germline DRG mutation frequencies were observed between our cohort and Caucasian men. Noticeably, G84E mutation in HOXB13, a rare but recurrent missense mutation associated with a significantly increased risk of hereditary prostate cancer was not found in our cohort of patients. Furthermore, the frequency of HSD3B1 (1245C) allele in the Chinese patients is much lower than that in the Caucasian populations (11.7% vs 63%). Conclusions: Our study retrospectively analyzed germline mutation profile of prostate cancer in Chinese population. The frequency of HSD3B1 (1245C) allele varies across ethnic populations in spite of the similar DNA repair gene mutations rates. Considering the gene list should be tested based on clinical/familial scenarios from the Philadelphia Prostate Cancer Consensus Conference, a prevalence of certain mutations, G84E mutation in HOXB13 was unnecessary for genetic testing in Chinese population.

Mutated RAP1GDS1 causes a new syndrome of dysmorphic feature, intellectual disability & speech delay.

BackgroundRAP1GDS1 (RAP1, GTP‐GDP dissociation stimulator 1), also known as SmgGDS, is a guanine nucleotide exchange factor (GEF) that regulates small GTPases, including, RHOA, RAC1, and KRAS. RAP1GDS1 was shown to be highly expressed in different tissue types including the brain. However, mutations in the RAP1GDS1 gene associated with human diseases have not previously been reported.MethodsWe report on four affected individuals, presenting intellectual disability, global developmental delay (GDD), and hypotonia. The probands’ DNA was subjected to whole‐genome sequencing, revealing a homozygous splice acceptor site mutation in the RAP1GDS1 gene (1444‐1G > A). Sanger sequencing was performed to confirm the segregation of the variant in two Saudi families. The possible aberrant splicing in the patients’ RNA was investigated using RT‐PCR and changes in mRNA expression of the patients were confirmed using qRT‐PCR.ResultsThe identified splice variant was found to segregate within the two families. RT‐PCR showed that the mutation affected RAP1GDS1 gene splicing, resulting in the production of aberrant transcripts in the affected individuals. Quantitative gene expression analysis demonstrated that the RAP1GDS1 mRNA expression in all the probands was significantly decreased compared to that of the control, and Sanger sequencing of the probands’ cDNA revealed skipping of exon 13, further strengthening the pathogenicity of this variant.ConclusionWe are the first to report the mutation of the RAP1GDS1 gene as a potential cause of GDD and hypotonia. However, further investigations into the molecular mechanisms involved are required to confirm the role of RAP1GDS1 gene in causing GDD and hypotonia.

Impact of RB1 gene mutation type in retinoblastoma patients on clinical presentation and management outcome

Objective/backgroundRetinoblastoma (RB), the most common intraocular malignancy in children, is caused by biallelic inactivation of the human retinoblastoma susceptibility gene (RB1). We are evaluating the impact of the type of RB1 gene mutation on clinical presentation and management outcome. MethodsA retrospective case series of 50 patients with RB. Main outcomes were clinical and pathologic features and types of RB1 gene mutations detected using quantitative multiplex polymerase chain reaction (PCR), allele-specific PCR, next-generation sequencing analysis, and Sanger sequencing. ResultsTwenty (40%) patients had unilateral RB and 30 (60%) had bilateral RB. Overall, 36 (72%) patients had germline disease, 17 (47%) of whom inherited the disease. Of these 17 inherited cases, paternal origin of the RB1 mutation was seen in 15 (88%). The overall eye salvage rate was 74% (n = 49/66; 100% for Groups A + B + C, and 79% for Group D eyes). The most frequent type of mutation was a nonsense mutation generating a stop codon (15/36, 42%). Other mutations that result in a premature stop codon due to deletions or insertions with donor splice site or receptor splice site mutations were detected in 7/36 (19%), 10/36 (28%), and 2/26 (6%) patients, respectively. The remaining two (6%) patients had frameshift mutation. Patients with deletion, acceptor splice site, and frameshift mutations presented with more advanced ICRB (International Classification of Retinoblastoma) stage (75% diagnosed with Group D or E), even though there was no significant difference in eye salvage rate or tumor invasiveness between patients with different types of mutations. ConclusionDespite the heterogeneous nature of RB1 gene mutations, tumor stage remains the most important predictive factor for clinical presentation and outcome. Furthermore, acceptor splice site and frameshift mutations are associated with more advanced tumor stage at diagnosis.

Functional Studies on Novel RET Mutations and Their Implications for Genetic Counseling for Hirschsprung Disease.

Hirschsprung disease (HSCR) is a genetic disorder characterized by the absence of ganglion cells in the gut. RET is considered to be the main susceptibility gene. In our previous screening of 83 HSCR patients, targeted exome sequencing identified nine rare variants of RET, most of which were new discoveries. Here, we performed in vitro arrays with functional studies to investigate their effects. Two variants (p.R77C and p.R67insL) were demonstrated to disrupt the glycosylation of RET and affect its subcellular localization. Three nonsense mutations (p.W85X, p.E252X, and p.Y263X) could not produce detectable RET full-length protein, and the other three mutations (p.R770X, p.Q860X, and p.V778Afs*1) were translated into truncated proteins of predicted sizes. One canonical splice acceptor site mutation (c.2802-2 A > G) was verified to affect gene regulation through aberrant splicing. In addition, we explored the effects of read-through reagents on RET nonsense mutations and showed that G418 significantly increased the full-length RET protein expression of p.Y263X in a dose-dependent manner, together with a mild recovery of p-ERK and p-STAT3. Our data provide a functional analysis of novel RET mutations and suggest that all of the rare variants detected from patients with clinically severe HSCR are indeed pathogenic. Thus, our findings have implications for proper genetic counseling.

Three novel point mutations in the keratinocyte transglutaminase (TGK) gene in lamellar ichthyosis: significance for mutant transcript level, TGK immunodetection and activity.

We have investigated 8 patients from 7 unrelated families with lamellar ichthyosis (LI) for defects in the keratinocyte transglutaminase (TGK) gene. We have characterized three novel homozygous mutations and a previously reported splice acceptor site mutation. One patient showed a C-to-T change in the binding site for the transcription factor Sp1 within the promoter region. Another patient had a Gly 143-to-Glu mutation in exon 3 and a third patient, affected with a particular form of LI sparing the four limbs, demonstrated a Val382-to-Met mutation within exon 7. These three patients exhibited drastically reduced transglutaminase activity and an absence of detectable TGK polypeptide, as assessed by immunofluorescence and immunoblotting. Northern blot analysis showed that the Sp1 site mutation was associated with profound reduction of TGK transcript levels whereas normal transcript levels were observed for the two missense mutations. We hypothesize that the Sp1 site mutation impairs transcription of the TGK gene, whereas the two missense mutations induce structural changes leading to protein instability. Linkage to TGK was excluded in another family and no evidence for TGK defect was found in 3 other patients. These results further support the involvement of TGK in some patients with LI. They identify a TGK mutation as a cause for non-generalized LI and further delineate the molecular mechanisms underlying TGK deficiency in LI.

A homozygous canonical splice acceptor site mutation in PRUNE1 is responsible for a rare childhood neurodegenerative disease in Manitoba Cree families.

Autosomal recessive PRUNE1 mutations are reported to cause a severe neurodevelopmental disorder with microcephaly, hypotonia, and brain malformations. We describe clinical and neuropathological features in a cohort of nine individuals of Cree descent who, because of a founder effect, are homozygous for the same PRUNE1 mutation. They follow the course of a combined neuromuscular and neurodegenerative disease, rather than a pure failure of normal development. This cohort presented in infancy with features of lower motor neuron disease, such as hypotonia, contractures, tongue fasciculations, and feeding difficulties in the absence of congenital brain anomalies and microcephaly. A neurodegenerative course followed with onset of seizures, spasticity, and respiratory insufficiency. Muscle biopsies showed denervation/reinnervation features, nonspecific atrophy and end-stage atrophy. Autopsy findings in two patients are also described, suggesting length dependent central motor axon degeneration, peripheral motor axon degeneration, possible spinal motor neuron degeneration, and accumulation of beta amyloid precursor protein inclusions in select brainstem nuclei. Exome sequencing and homozygosity mapping identified a homozygous PRUNE1 mutation in a canonical splice site, which produces two abnormal PRUNE1 mRNA products. Based on our studies and the histopathology and phenotypic data, we provide further evidence that this disorder leads to a neurodegenerative disease affecting both the peripheral and central nervous systems and suggest that the pathogenic c.521-2A>G mutation could lead to an altered effect on tubulin dynamics.

Microglandular adenosis is an advanced precursor breast lesion with evidence of molecular progression to matrix-producing metaplastic carcinoma

Microglandular adenosis (MGA) is a rare breast lesion reported to be associated with invasive carcinoma in up to 20% to 30% of cases and has been proposed as a nonobligate precursor to basal-like breast cancers. We identified a case of matrix-producing metaplastic carcinoma with morphologic and immunohistochemical evidence of progression from MGA to atypical MGA, carcinoma in situ, and invasive carcinoma. We performed whole-exome sequencing of each component (MGA, atypical MGA, carcinoma in situ, and cancer) to characterize the mutational landscape of these foci. There was a significant copy number overlap between all foci, including a segmental amplification of the CCND1 locus (partial chromosome 11 trisomy) and MYC (8q24.12-13). Using a bioinformatics approach, we were able to identify 3 putative mutational clusters and recurrent, stop-gain nonsynonymous mutations in both ZNF862 and TP53 that were shared across all foci. Finally, we identified a novel deleterious splice-acceptor site mutation of chr5:5186164 G>T (chromosome 5p15) encoding the gene, ADAMTS16, in the invasive component.

Complete androgen insensitivity syndrome caused by c.1769-1G > C mutation and activation of a cryptic splice acceptor site in the androgen receptor gene

Androgen insensitivity syndrome (AIS) is the most common cause of 46,XY disorders of sex development (46,XY DSD). This syndrome is an X-linked recessive genetic disease characterized by resistance to the actions of androgens in an individual with a male karyotype and it is caused by mutations in the androgen receptor (AR) gene. We evaluated two siblings with primary amenorrhea, normal secondary sex characteristics, absence of uterus and ovaries, intra-abdominal testis, and elevated testosterone levels. Sequence analysis of the AR gene revealed a splice acceptor site mutation in intron 2 (c.1769-1G > C). The analysis of mRNA showed that this mutation resulted in the activation of a cryptic splice acceptor site located in intron 2 and in the synthesis of an aberrant mRNA transcript with 69 nucleotides insertion between exon 2 and exon 3, leading to an insertion of 23 amino acids in the AR protein instead of generating a premature termination codon. The additional 23 amino acids insertion affects AR intracellular trafficking by impairing its translocation from the cytoplasm to the nucleus after hormone stimulation. The c.1769-1G > C mutation provides new insights into the molecular mechanism involved in splicing defects and expands the spectrum of mutations associated with the androgen insensitivity syndrome.

Splicing defect in FKBP10 gene causes autosomal recessive osteogenesis imperfecta disease: a case report

BackgroundOsteogenesis imperfecta (OI) is a group of connective tissue disorder caused by mutations of genes involved in the production of collagen and its supporting proteins. Although the majority of reported OI variants are in COL1A1 and COL1A2 genes, recent reports have shown problems in other non-collagenous genes involved in the post translational modifications, folding and transport, transcription and proliferation of osteoblasts, bone mineralization, and cell signaling. Up to now, 17 types of OI have been reported in which types I to IV are the most frequent cases with autosomal dominant pattern of inheritance.Case PresentationHere we report an 8- year- old boy with OI who has had multiple fractures since birth and now he is wheelchair-dependent. To identify genetic cause of OI in our patient, whole exome sequencing (WES) was carried out and it revealed a novel deleterious homozygote splice acceptor site mutation (c.1257-2A > G, IVS7-2A > G) in FKBP10 gene in the patient. Then, the identified mutation was confirmed using Sanger sequencing in the proband as homozygous and in his parents as heterozygous, indicating its autosomal recessive pattern of inheritance. In addition, we performed RT-PCR on RNA transcripts originated from skin fibroblast of the proband to analyze the functional effect of the mutation on splicing pattern of FKBP10 gene and it showed skipping of the exon 8 of this gene. Moreover, Real-Time PCR was carried out to quantify the expression level of FKBP10 in the proband and his family members in which it revealed nearly the full decrease in the level of FKBP10 expression in the proband and around 75% decrease in its level in the carriers of the mutation, strongly suggesting the pathogenicity of the mutation.ConclusionsOur study identified, for the first time, a private pathogenic splice site mutation in FKBP10 gene and further prove the involvement of this gene in the rare cases of autosomal recessive OI type XI with distinguished clinical manifestations.

Clinical and genetic analysis of two unrelated patients with Angelman syndrome and novel UBE3A mutations

To explore the genetic cause for two familial Angelman syndrome cases and correlation between the clinical phenotypes and their genetic basis. Karyotyping analysis and microarray assay were carried out to exclude chromosome anomalies and uniparental disomy. The UBE3A gene was analyzed for potential point mutations, deletions, insertions and splice site mutations. Reverse transcription PCR was used to evaluate splicing mutation of the RNA transcripts. DNA sequencing showed the proband of family 1 has carried a novel maternal UBE3A splice acceptor site mutation, resulting in a guanine-to-cytosine transversion (IVS15-1G>C). Reverse transcription PCR revealed the proband and his mother both carried heterozygous mutant transcripts with loss of 54 and 59 nucleotides in exon 16, respectively. The proband displayed severe mental retardation, ataxia, seizures and inappropriate laughter. The siblings of family 2 has carried a novel maternal missense mutation in exon 16 of the UBE3A gene (c.2540C>T). She also presented with mental retardation, absent speech, mild ataxia and inappropriate laughter. The novel IVS15-1G>C and c.2540 C>T mutations of the UBE3A gene probably underlie the AS in the two families. Compared with small-scale mutations, larger fragments mutations can produce more severe phenotypes.

Functional Analyses of BTK Mutations in Follicular Lymphoma

Introduction: Follicular lymphoma (FL) constitutes the second most common non-Hodgkin's lymphoma in the Western world. Recently, novel mutations in the Bruton's Tyrosine Kinase (BTK) have been identified in FL (Krysiak et al, Blood 2017). Additional mutations in other components of BCR signaling pathways in FL have been reported as well but functional consequences have not been fully explored.Methods: Using a combination of WES on N=82 FLand BTK resequencing in an extended FL panel we uncovered novel BTK mutations in FL. These BTK mutations are all distinct from the previously identified mutations suggesting that a full complement of BTK mutations has not yet been identified. Using crispr-Cas9-mediated BTK gene targeting in multiple lymphoma cell lines, we generated novel BTK-/- null lines. We introduced all BTK mutations by site-directed mutagenesis into a reference BTK cDNA. Subsequently, we re-introduced HA-tagged wild type and mutated BTK constructs using lentiviral transduction into the BTK-/- cell lines and also into BTK-/- DT40 cells. We measured signal transduction pathway components following BCR cross linking. The location of BTK mutations was modeled onto a 3D-model assembled from various published BTK substructures.Results: Mutations in BTK are distributed along the entire length of the gene and are targeting the PH-, SH2- and catalytic domain, with the catalytic domain carrying the majority of BTK mutations. Interestingly, occasional frame shift mutations as reported and a splice acceptor site mutation by genetic criteria suggest gene inactivation. The 3D modeling data show that all BTK mutations are on the surface of the BTK protein within the various functional domains. The quantitation of BTK protein after reconstitution into various engineered BTK-/- cell lines uncovered lower steady state levels of various mutants especially for mutations targeting the catalytic domain. PLCγ2 remains the best studied BTK substrate and various sites have been reported to be phosphorylated by BTK in vivo (PLCγ2 amino acids 753, 759 and 1217). Following BCR crosslinking in engineered BTK-/- lymphoma cell lines we found only a modest reduction in the phosphorylation of PLCγ2 residue 759 or 1217 demonstrating that these sites are under the control of various tyrosine kinases and not exclusively targeted by BTK. After BCR crosslinking in the BTK-/- cells lines we detected a substantial increase in PLCy2 residues 759 or 1217 phosphorylation over baseline and this increase was abolished by ibrutinib pre-treatment. Therefore, PLCγ2 residue 759 or 1217 phosphorylation is due to multiple BCR stimulated and ibrutinib sensitive tyrosine kinases. In engineered BTK-/- lymphoma cell lines reconstituted with HA-tagged wild type and mutated BTK we did not detect major effects by the BTK mutants on the phosphorylation state of PLCγ2 residue 759 or 1217. In addition, phosphorylation of BTK at residue 223 (a major autophosphorylation site) was reduced or absent in multiple BTK mutants. Summarizing these findings, FL-associated BTK mutations do not activate the BTK kinase; instead, multiple BTK mutants are inactivating.Focusing on BCR-regulated signaling pathways, we measured ERK and AKT phosphorylation in the engineered BTK-/- lymphoma cell lines reconstituted with HA-tagged wild type and mutated BTK. Following BCR crosslinking, we detected increased p-AKT-473 phosphorylation in most BTK mutants in a total of 4 separate cell lines. In contrast, p-ERK was largely unaffected. Recent reports implicated AKT activation as a major factor in tonic BCR signaling in germinal center type DLBCL. We did not detect elevated AKT phosphorylation prior to BCR stimulation in our BTK reconstituted cell systems but an exaggerated response following BCR engagement. The effects of BTK mutations on protein-protein interactions within the BTK signalosome as well as effects on sensitivity to ibrutinib will be updated at the meeting.Conclusion : Our initial efforts at functional characterization of FL-associated BTK mutations uncovered that despite reduced stability and impaired catalytic activity most BTK mutations resulted in enhanced AKT activation following BCR crosslinking. Given the established pro-growth and pro-survival function of AKT, the data provide a clear reason why FL would select for such mutations. DisclosuresPhillips:Incyte: Other: Travel/Expenses; Pharmacyclics: Consultancy; KITE: Consultancy; Seattle Genetics: Consultancy. Malek:Abbvie: Equity Ownership; Janssen R&D: Honoraria, Research Funding; Pharmacyclics: Honoraria, Research Funding.