Case Report: Novel SLC9A6 Splicing Variant in a Chinese Boy With Christianson Syndrome With Electrical Status Epilepticus During Sleep

We investigated the existence and potential pathogenicity of a SLC9A6 splicing variant in a Chinese boy with Christianson Syndrome (CS), which was reported for the first time in China. Trio whole-exome sequencing (WES) was performed in the proband and his parents. Multiple computer prediction tools were used to evaluate the pathogenicity of the variant, and reverse transcription-polymerase chain reaction (RT-PCR) analysis and cDNA sequencing were performed to verify the RNA splicing results. The patient presented with characteristic features of CS: global developmental delay, seizures, absent speech, truncal ataxia, microcephaly, ophthalmoplegia, smiling face and hyperkinesis with electrical status epilepticus during sleep (ESES) detected in an electroencephalogram (EEG). A SLC9A6 splicing variant was identified by WES and complete skipping of exon 10 was confirmed by RT-PCR. This resulted in altered gene function and was predicted to be pathogenic. ESES observed early in the disease course is considered to be a significant feature of CS with the SLC9A6 variant. Combined genetic analysis at both the DNA and RNA levels is necessary to confirm the pathogenicity of this variant and its role in the clinical diagnosis of CS.

Novel SCN5A and GPD1L Variants Identified in Two Unrelated Han-Chinese Patients With Clinically Suspected Brugada Syndrome

Brugada syndrome (BrS) is a complexly genetically patterned, rare, malignant, life-threatening arrhythmia disorder. It is autosomal dominant in most cases and characterized by identifiable electrocardiographic patterns, recurrent syncope, nocturnal agonal respiration, and other symptoms, including sudden cardiac death. Over the last 2 decades, a great number of variants have been identified in more than 36 pathogenic or susceptibility genes associated with BrS. The present study used the combined method of whole exome sequencing and Sanger sequencing to identify pathogenic variants in two unrelated Han-Chinese patients with clinically suspected BrS. Minigene splicing assay was used to evaluate the effects of the splicing variant. A novel heterozygous splicing variant c.2437-2A>C in the sodium voltage-gated channel alpha subunit 5 gene (SCN5A) and a novel heterozygous missense variant c.161A>T [p.(Asp54Val)] in the glycerol-3-phosphate dehydrogenase 1 like gene (GPD1L) were identified in these two patients with BrS-1 and possible BrS-2, respectively. Minigene splicing assay indicated the deletion of 15 and 141 nucleotides in exon 16, resulting in critical amino acid deletions. These findings expand the variant spectrum of SCN5A and GPD1L, which can be beneficial to genetic counseling and prenatal diagnosis.

Compound heterozygous c.598_612del and c.1746-20C > G CAPN3 genotype cause autosomal recessive limb-girdle muscular dystrophy-1: a case report

Background Autosomal recessive limb–girdle muscular dystrophy-1 (LGMDR1), also known as calpainopathy, is a genetically heterogeneous disorder characterised by progression of muscle weakness. Homozygous or compound heterozygous variants in the CAPN3 gene are known genetic causes of this condition. The aim of this study was to confirm the molecular consequences of the CAPN3 variant NG_008660.1(NM_000070.3):c.1746-20C > G of an individual with suspected LGMDR1 by extensive complementary DNA (cDNA) analysis. Case presentation In the present study, we report on a male with proximal muscular weakness in his lower limbs. Compound heterozygous NM_000070.3:c.598_612del and NG_008660.1(NM_000070.3):c.1746-20C > G genotype was detected on the CAPN3 gene by targeted next-generation sequencing (NGS). To confirm the pathogenicity of the variant c.1746-20C > G, we conducted genetic analysis based on Sanger sequencing of the proband’s cDNA sample. The results revealed that this splicing variant disrupts the original 3′ splice site on intron 13, thus leading to the skipping of the DNA fragment involving exon 14 and possibly exon 15. However, the lack of exon 15 in the CAPN3 isoforms present in a blood sample was explained by cell-specific alternative splicing rather than an aberrant splicing mechanism. In silico the c.1746-20C > G splicing variant consequently resulted in frameshift and formation of a premature termination codon (NP_000061.1:p.(Glu582Aspfs*62)). Conclusions Based on the results of our study and the literature we reviewed, both c.598_612del and c.1746-20C > G variants are pathogenic and together cause LGMDR1. Therefore, extensive mRNA and/or cDNA analysis of splicing variants is critical to understand the pathogenesis of the disease.

Retroperitoneal leiomyosarcoma in a female patient with a germline splicing variant RAD51D c.904-2A > T: a case report

Background RAD51D (RAD51 paralog D) is an intermediate cancer susceptibility gene for primary ovarian cancer, including fallopian tube and peritoneal carcinomas and breast cancer. Although gynecological non-epithelial tumors such as uterine sarcomas are associated with genomic instability, including BRCA impairment, there is no clear evidence of the relationship between RAD51D variants and the risk of sarcoma development. Case presentation A Japanese woman in her 50s underwent multiple surgical resections and several regimens of chemotherapy for tumors that originated in the retroperitoneum and recurred in the peritoneum over a clinical course of approximately 4 years. The peritoneal tumor was histologically diagnosed as a leiomyosarcoma and was genetically identified to show a splice variant of RAD51D c.904-2A > T [NM_002878] through tumor profiling performed as a part of cancer precision medicine. The confirmatory genetic test performed after genetic counseling revealed that the RAD51D splicing variant detected in her tumor was of germline origin. In silico analyses supported the possible pathogenicity of the detected splice variant of RAD51D with a predicted attenuation in mRNA transcription and truncated protein production due to frameshifting, which was attributed to a single-nucleotide alteration in the splicing acceptor site at the 3′-end of intron 9 of RAD51D. Considering her unfavorable clinical outcome, which showed a highly aggressive phenotype of leiomyosarcoma with altered RAD51D, this case provided novel evidence for the relationship of a RAD51D splicing variant with malignant tumor development or progression. We report the findings of this rare case with possible involvement of the germline variant of RAD51D c.904-2A > T as a potential predisposing factor for malignant tumors, including leiomyosarcoma. Conclusions We present the findings of a case of leiomyosarcoma in the peritoneum of a female patient with a novel germline splicing variant of RAD51D as potential evidence for the pathogenicity of the variant and its involvement in the risk of sarcoma etiology and/or development. To the best of our knowledge, this is the first case report describing a leiomyosarcoma carrying a germline RAD51D splicing variant and elucidating its pathogenicity on the basis of computational prediction of the impairment of normal transcription and the presumed loss of functional protein production.

ASPP2 k, a Dominant-Negative Splicing Variant of the Apoptosis-Stimulating Protein of p53-2 (ASPP2), Modulates Treatment Response Towards BCL-Signaling Inhibitors in Acute Myeloid Leukemia

In recent years, it has become increasingly apparent that BCL-2 inhibition in AML is a clinically highly effective approach - and the first BCL-2 inhibitor, venetoclax, has gained FDA and EMA approval. However, drug resistance frequently occurs and mechanisms or biological markers to predict response to BCL-2 inhibition are urgently needed. ASPP2 plays a central role to orchestrate induction of apoptosis via binding of p53 - but also via binding of antiapoptotic BCL-2, allowing release of proapoptotic proteins. We recently described an alternatively spliced oncogenic ASPP2 isoform (ASPP2k) (Schittenhelm et al., 2019), which is characterized by truncation of the C-terminus allocating the BCL2- as well as the p53 binding sites (in analogy to major TP53 mutations lacking the ASPP2 binding motifs). We therefore hypothesized that expression of ASPP2k attenuates efficacy of pro-apoptotic compounds: AML cell line models, including MOLM13/14 (FLT3 ITD+), OCI-AML3 (NPM1 A+) and HL60, as well as freshly isolated native leukemia blasts (n=40) are used in dose-dilution assays to assess for pro-apoptotic efficacy in annexin V-based assays. Bone marrow donors served as a control population. A lentiviral approach was used for isoform-specific ASPP2k-shRNA transduction. A HisMax vector was used to forcely express ASPP2k. Several compounds targeting BCL-2 signaling, which are under clinical investigation, were tested (BCL-2: venetoclax, BCL-2/Xl: AZD4320, MCL-1: AZD5991 and CDK9: AZD4573). To summarize, leukemia cells demonstrated variable and preferential sensitivity profiles towards the tested compounds: MOLM cells were sensitive towards all tested compounds. In contrast, OCI-AML3 proofed reduced sensitivity towards BCL-2/Xl inhibition - whereas MCL-1 and CDK9 inhibition (indirectly targeting MCL-1 signaling) showed IC50s in the nanomolar range (AZD5991: 307nM, AZD4573: 16nM). Furthermore, HL60 were relatively resistant towards both BCL-2/Xl and MCL-1 inhibition - however remained high sensitivity towards CDK9 inhibition (IC50 25nM). Priming with a hypomethylating agent (HMA, decitabine) resulted in additive (CI close to 1) to synergistic (CI 0.25 - 0.7) proapoptotic effects in isobologram analysis and led to a release of drug resistance in primary resistant cell lines. Exposure of native leukemia cells towards all inhibitors confirmed (individually differing) sensitivity in the nanomolar ranges - whereas bone marrow donor controls were relatively resistant towards the tested compounds, which argues for a therapeutic clinical window. In a last step, we addressed, whether ASPP2k functionally impedes the proapoptotic efficacy observed for the tested compounds. Indeed, we demonstrate that isoform-specific ASPP2k interference similarly results in a significant increase of pro-apoptotic capacities of all tested BCL-2, BCL-2/XL, MCL-1 and CDK9 inhibitors, (resp. attenuation thereof after forcely expressing the dominant-negative splicing variant). To summarize, we show that inhibition of BCL-2 signaling is a promising approach to target acute leukemia - as a monoagent as well as in combination with HMA: Further, we provide a path for exploration of ASPP2k as a predictive tool as well as a therapeutic sensitizer of pro-apoptotic drugs, which will be addressed in future studies. Disclosures Schittenhelm: BMS: Other: advisory board; Astellas: Other: advisory board; Takeda: Other: advisory board; University of Tuebingen: Patents & Royalties: patent for ASPP2k. Kampa-Schittenhelm: University of Tuebingen: Patents & Royalties: patent related to ASPP2k.