Splice Site Mutation Research Articles

Identification a Novel Molecular Mechanism Underlying the Anti-CD38-Based Treatment Resistance in Multiple Myeloma Patients

Introduction: Anti-CD38 antibody (Daratumumab (DARA)/Isatuximab (ISA))-mediated therapy is now used in combination therapies to treat both newly diagnosed and relapsed/refractory multiple myeloma (MM). Despite a high extent and frequency of response to DARA/ISA containing therapy, MM patient relapse is still common. Several resistance mechanisms to DARA/ISA have been reported including MM cell clonal selection towards reduced CD38 expression; loss of MM cell surface CD38 upon the formation of a CD38-DARA complex leading to immunosuppression; and immunomodulatory effects after the DARA/ISA treatment via the downregulation of intracellular signaling pathways in bone marrow stromal cells. Given the widespread use of anti-CD38-directed treatment in MM, the identification of anti-CD38 based treatment resistance is an urgent clinical need. Methods/results: To understand the underlying molecular mechanism of anti-CD38-based treatment resistance, we assessed post-transcriptional alterations on CD38 transcripts. We performed total and single-cell RNA sequencing of six samples from MM patients treated with DARA/ISA in combination with other MM drugs. Specifically, we examined mRNA splicing aberrations on the CD38 transcripts in 44 MM patient plasma cells (PCs). Four CD38 splice variants (CD38-Va, -Vb, -Vc, and Vd (CD38 SpVs)) were identified in CD138+PCs from DARA-resistant patient samples. These CD38-SpVs are the result of one or more cassette-exon splicing. Some of these splicing events cause in-frame shifts, which lead to the activation of a cryptic stop codon on the CD38 transcript and encode truncated proteins. The cryptic stop codon location at the specific proximity of the exon-intron junction, allowing these transcripts escaping degradation via non-sense mediated decay. In MM PCs, CD38 splice variant transcripts were overexpressed at different levels (2-4 fold) in various combinations with one or more novel splice variants and CD38 full-length (CD38-FL) transcripts. These splice variants were absent in MM PCs from BM aspirates from age-matched healthy donors. Next, we evaluated the expression of the CD38 SpVs and FL transcripts in PCs from MM patients at the time they were sensitive to DARA/ISA treatment and when resistance developed. We identified CD38 splice variant transcripts that were selectively expressed MM PCs at the time of resistance to DARA/ISA treatment. We also identified CD38 splice variant transcripts that are overexpressed in DARA/ISA treatment-sensitive patient samples. Our findings were further validated in DARA resistant patient sample by single-cell RNA-seq analysis, which showed overexpression of CD38Vb in >80% of PCs. We identified splice site mutations that contribute to the expression of CD38 SpVs. To evaluate whether CD38 aberrant splicing is associated with DARA resistance, we generated a KMS11 DARA-resistant cell line (KMS11 DARA-Res) and evaluated the expression of splice variants in these cells. This analysis demonstrated that KMS11DARA-Res over-express CD38 SpVs. To determine whether CD38 splice variants encode functional proteins, we cloned, sequenced, and stably expressed CD38 SpVs transcripts into H929 cells (H929-CD38 SpVs). CD38 SpVs expressions were evaluated by flow cytometry and live cell imaging. These analyses demonstrated that CD38 SpVs encode proteins. Importantly, CD38 splice variant protein folding analyses showed that splicing alterations abrogate DARA/ISA binding sites. Then, to evaluate the functional effects of aberrant splicing of CD38 in MM cells, we carried out DARA/ISA binding assays and evaluated cell killing with ADCC assays in H929- CD38SpV cells. Importantly, these analyses showed that both DARA/ISA binding and ADCC was lost in H929-CD38SpV cells. Conclusions: We identified a novel molecular mechanism that underlies the resistance to anti-CD38-based treatment, both DARA and ISA. By this mechanism, CD38 cognate epitopes are removed from the cell surface of MM cells without an entire disposal of the target protein. The existence of such splicing-based mechanisms of resistance indicates that other antibody-based therapeutics or future CARs should be developed against CD38 epitopes that are created as a result of splicing. This approach may allow for selective targeting of MM PCs while spearing other CD38 expressing normal cells, enhancing cytotoxicity and improving therapeutic index.

Targeting a Putative Intronic Splicing Silencer Salvages Expression from the Recurrent SBDS C.258+2T>C Mutant Allele in Shwachman-Diamond Syndrome Patient Cells and Mouse Model

Shwachman-Diamond syndrome (SDS) is an autosomal recessive inherited bone marrow failure syndrome and leukemia predisposition disorder due to biallelic mutations of SBDS, whose gene product positively regulates 80S ribosome assembly. In addition to predisposition to pancytopenia, myelodysplasia and acute myeloid leukemia, patients frequently experience exocrine pancreatic insufficiency, skeletal dysplasia, cardiac abnormalities, and immune dysfunction. Faithful animal models are lacking to study hematopoiesis and clonal evolution in SDS. Almost all patients carry at least one copy of the SBDS c.258+2T>C mutation that disrupts the intron 2 5' splice site (SS). We hypothesize that this recurrent mutant allele allows for sufficient residual SBDS expression to promote SBDS cellular function and permit organismal development, and thus residual expression could be potentiated through novel therapeutic approaches. Here we present RNA-seq analysis that shows residual expression of full-length SBDS originating from the non-canonical SS of the SBDS c.258+2T>C allele. Most transcripts were the product of aberrant splicing from a cryptic exon 2 SS and exon 2 skipping, both of which result in nonsense transcripts. Prior animal models of SDS have relied on complete or tissue-specific Sbds knock-out and thus fail to replicate the splicing defect and residual SBDS expression. To develop a mouse model to test novel sequence-specific therapeutic strategies, we generated, by CRISPR/Cas9-mediated HDR, mice with a humanized intron 2 5' SS containing the c.258+2T>C mutation at the endogenous mouse Sbds gene (c.199+2T>C). Homozygous Sbds SS mutant knock-in mouse embryos were found to be viable until at least mid-gestation (E14.5) though live births were not observed. In contrast, constitutive Sbds knock-out mice are reported to succumb to early embryonic lethality by E8.51. Using isolated MEFs, we demonstrate that the humanized mouse Sbds mutant allele recapitulates low-level residual expression, aberrant splicing from a cryptic exon 2 SS, and exon 2 skipping, although exon skipping appears to be greater and residual expression lower as compared to the human mutant allele. Consistent with the characteristic SDS molecular defect, homozygous Sbds c.199+2T>C MEFs show impaired 80S ribosome assembly, accumulation of EIF6 on the 60S ribosomal subunit, and induction of a p53-dependent stress response. To explore strategies to further increase residual wild type SBDS transcript expression from the SBDS c.258+2T>C allele, we designed anti-sense oligonucleotides (ASOs) targeting potential splice regulatory sequences. We evaluated 38 antisense oligos, identifying several that block a putative intronic splicing silencer (ISS) that suppresses normal splicing from the non-canonical c.258+2T>C SS, with the most potent ASO resulting in 4.0-fold increase in SBDS expression (p<0.01) in fibroblasts from 4 different SDS patients. Disruption of sequences within this putative ISS by Cas12a nuclease-mediated indels result in 1.4 and 2.4 fold increased SBDS/Sbds expression from SDS patient fibroblasts and homozygous MEFs respectively (p<0.05). Together these studies present a novel mouse model for SDS recapitulating the recurrent SS mutation with residual intact splicing and indicate that salvaging residual SBDS expression by modulating aberrant splicing is a promising strategy for SDS therapeutics.

PATH-39. A 44-YEAR-OLD FEMALE WITH DICER1-MUTANT PRIMARY INTRACRANIAL SARCOMA WITH IMAGING NEAR END OF LIFE

Abstract DICER1-mutant intracranial sarcomas were recently defined by WHO in the 2021 CNS tumor classifications. Currently there is a lack of literature describing the clinical nature of this disease. Here we present a case of primary intracranial sarcoma with DICER1 mutation and unknown etiology. A 44-year-old female presented with aphasia and right-sided weakness. Clinical imaging confirmed the presence of a hemorrhagic mass in the left frontal lobe and superior falx. The patient underwent two surgical resections, but a clear diagnosis was difficult to make due to heterogenous histological features and inconclusive genomic testing. The surgical samples were sent to the National Institute of Health where genetic testing indicated a DICER1 E1813D mutation with high allele frequency (97%), a TP53 mutation with high allele frequency (95%), and a high variant allele frequency NF1 splice site mutation (96%). This resulted in a diagnosis of DICER1-mutant primary intracranial sarcoma. The patient was treated with temozolomide, bevacizumab, and CPT-11. 382 days after the mass was originally identified, the patient died. A detailed analysis of the radiographic images was performed by creating T1-weighted post-contrast enhancement maps (T1C) and T2-weighted fluid attenuated inversion recovery (FLAIR) hyper-intensity maps for each timepoint. We plotted these volumes over time alongside the patient’s treatment history. Large spikes in T1C and FLAIR volume were seen 4 days prior to initiation of temozolomide and 31 days after CPT-11 treatment was ended. At the final clinical MRI, both volumes were about 4 times greater than that of the initial scan. Despite this disease’s aggressive nature, it can be difficult to accurately diagnose, due in part to very few studies investigating the clinical features of this rare tumor. With the addition of this diagnosis to WHO CNS5, future studies should examine the behavior of these tumors, aiding in earlier diagnosis and improvement of treatment guidance.

Compound heterozygous loss-of-function variants in BRAT1 cause lethal neonatal rigidity and multifocal seizure syndrome.

Lethal neonatal rigidity and multifocal seizure syndrome (RMFSL, OMIM 614498) is a rare autosomal recessive disease characterized by the onset of rigidity and intractable seizures at or soon after birth. The BRAT1 has been identified to be the disease-causing gene for RMFSL. This study aimed to determine the underlying pathogenic mutations of a Chinese family with RMFSL and to confirm the effect of the splice-site mutation by reverse transcription analysis. Detailed family history and clinical data were recorded, and peripheral blood samples were collected from all available family members. Whole exome sequencing (WES), Sanger sequencing, and bioinformatics analysis were performed to investigate the causative variants. The impact of the intronic variant on splicing was subsequently analyzed by RT-PCR analysis. We identified two compound heterozygous variants in the BRAT1, c.431-2A>G in intron 3 and c.1359_1361del(p.Leu454del) in exon 9 in the proband, one inherited from each parent. Furthermore, the 3'-splice site acceptor (c.431-2A>G) variant was found to activate a cryptic acceptor splice site, which resulted in the loss of 29 nucleotides and generation of a premature stop codon at code 180, producing a truncated BRAT1 (c.432_460del; p.Ala145Argfs*36). This research identified two mutations in the BRAT1 of one Chinese family with RMFSL. These data can aid in developing clinical diagnoses as well as providing genetic counseling and prenatal interventions to the family. These findings also expand our knowledge of the spectrum of BRAT1 pathogenic variants in RMFSL syndrome.

A splice site mutation in the FvePHP gene is associated with leaf development and flowering time in woodland strawberry.

Leaves and flowers are crucial for the growth and development of higher plants. In this study we identified a mutant with narrow leaflets and early flowering (nlef) in an ethyl methanesulfonate-mutagenized population of woodland strawberry (Fragaria vesca) and aimed to identify the candidate gene. Genetic analysis revealed that a single recessive gene, nlef, controlled the mutant phenotype. We found that FvH4_1g25470, which encodes a putative DNA polymerase α with a polymerase and histidinol phosphatase domain (PHP), might be the candidate gene, using bulked segregant analysis with whole-genome sequencing, molecular markers, and cloning analyses. A splice donor site mutation (C to T) at the 5' end of the second intron led to an erroneous splice event that reduced the expression level of the full-length transcript of FvePHP in mutant plants. FvePHP was localized in the nucleus and was highly expressed in leaves. Silencing of FvePHP using the virus-induced gene silencing method resulted in partial developmental defects in strawberry leaves. Overexpression of the FvePHP gene can largely restore the mutant phenotype. The expression levels of FveSEP1, FveSEP3, FveAP1, FveFUL, and FveFT were higher in the mutants than those in 'Yellow Wonder' plants, probably contributing to the early flowering phenotype in mutant plants. Our results indicate that mutation in FvePHP is associated with multiple developmental pathways. These results aid in understanding the role of DNA polymerase in strawberry development.

ATYPICAL PRESENTATION OF X-LINKED IL-2 RECEPTOR GAMMA MUTATION CHARACTERIZED BY RECURRENT CUTANEOUS AND GENITAL WARTS

<h3>Introduction</h3> Combined immunodeficiency syndromes constitute a group of inherited immune disorders characterized by diminished lymphocyte number and function. Late onset combined immunodeficiency can be difficult to diagnose as patients initially have a less severe phenotype compared to their SCID counterparts. Here we describe a case of an atypical presentation of late onset combined immunodeficiency due to a hemizygous IL-2 receptor gamma (IL-2Rg) mutation. <h3>Case</h3> The patient is a 41-year-old male with a 25-year history of recurrent cutaneous warts of the hands and feet. At the age of 36 he developed perianal condylomata acuminata that later developed into squamous cell carcinoma. He reported no history of recurrent sinopulmonary infection, atopic disease, or autoimmunity. Immune phenotyping demonstrated normal B- and NK cell counts but CD4+ T-cell lymphopenia with poor proliferation to antigenic stimulation. Total immunoglobulins were normal but without vaccine responses. Genetic testing revealed a hemizygous variant of unknown significance in IL2Rg gene. NK cell cytotoxicity assay showed suboptimal cytotoxicity and CD8+ T-cells exhibited impaired pSTAT5 confirming the likely pathogenicity of this variant. <h3>Discussion</h3> IL-2 plays a critical role in the activation among a host of T-cells and NK cells. Deleterious mutations of the IL-2Rg gene are associated with X-linked SCID displaying a T−B+NK− phenotype. Milder phenotypes have been reported with hypomorphic mutations of IL-2Rg, with a recent report describing recurrent cutaneous warts and respiratory infections in two brothers with splice site mutations. This patient's chronic HPV infection resulted in malignant sequalae, highlighting the importance for awareness of the phenotypic heterogeneity that can occur in X-linked SCID.

Candidate genes and sequence variants for susceptibility to mycobacterial infection identified by whole-exome sequencing.

Inborn errors of immunity are known to influence susceptibility to mycobacterial infections. The aim of this study was to characterize the genetic profile of nine patients with mycobacterial infections (eight with BCGitis and one with disseminated tuberculosis) from the Republic of Moldova using whole-exome sequencing. In total, 12 variants in eight genes known to be associated with Mendelian Susceptibility to Mycobacterial Disease (MSMD) were detected in six out of nine patients examined. In particular, a novel splice site mutation c.373-2A>C in STAT1 gene was found and functionally confirmed in a patient with disseminated tuberculosis. Trio analysis was possible for seven out of nine patients, and resulted in 23 candidate variants in 15 novel genes. Four of these genes - GBP2, HEATR3, PPP1R9B and KDM6A were further prioritized, considering their elevated expression in immune-related tissues. Compound heterozygosity was found in GBP2 in a single patient, comprising a maternally inherited missense variant c.412G>A/p.(Ala138Thr) predicted to be deleterious and a paternally inherited intronic mutation c.1149+14T>C. Functional studies demonstrated that the intronic mutation affects splicing and the level of transcript. Finally, we analyzed pathogenicity of variant combinations in gene pairs and identified five patients with putative oligogenic inheritance. In summary, our study expands the spectrum of genetic variation contributing to susceptibility to mycobacterial infections in children and provides insight into the complex/oligogenic disease-causing mode.

Nemaline Myopathy in Brazilian Patients: Molecular and Clinical Characterization.

Nemaline myopathy (NM), a structural congenital myopathy, presents a significant clinical and genetic heterogeneity. Here, we compiled molecular and clinical data of 30 Brazilian patients from 25 unrelated families. Next-generation sequencing was able to genetically classify all patients: sixteen families (64%) with mutation in NEB, five (20%) in ACTA1, two (8%) in KLHL40, and one in TPM2 (4%) and TPM3 (4%). In the NEB-related families, 25 different variants, 11 of them novel, were identified; splice site (10/25) and frame shift (9/25) mutations were the most common. Mutation c.24579 G>C was recurrent in three unrelated patients from the same region, suggesting a common ancestor. Clinically, the “typical” form was the more frequent and caused by mutations in the different NM genes. Phenotypic heterogeneity was observed among patients with mutations in the same gene. Respiratory involvement was very common and often out of proportion with limb weakness. Muscle MRI patterns showed variability within the forms and genes, which was related to the severity of the weakness. Considering the high frequency of NEB mutations and the complexity of this gene, NGS tools should be combined with CNV identification, especially in patients with a likely non-identified second mutation.

Correlation Analysis of FⅧGene Mutation and the Production of FⅧ Inhibitor with Severe Hemophilia A Patients in a Single Medical Center

To investigate the relationship between the type of FⅧgene mutation and the development of FⅧ inhibitors in patients with severe haemophilia A (HA). The medical records of 172 patients with severe hemophilia A from January 2009 to September 2020 were reviewed. The types of FⅧgene mutations and the production of factor Ⅷ inhibitors were collected and divided into high-risk mutation group ( intron 1 inversions, large deletions, nonsense mutations), low-risk mutation group (missense mutations, small deletions and insertions, splice site mutations) and intron 22 inversions group. The correlation of FⅧgenotype and the production of FⅧ inhibitors in patients with HA were analyzed. Among 172 patients with severe HA, 21 cases(12.21%) developed FⅧ inhibitors. The cumulative incidence of FⅧ inhibitor development was 32%(10/31) in high risk group (75% patients with large deletions, 43% patients with intron 1 inversions, 20% patients with nonsense mutations) and 5%(2/43) in low risk group(6% patients with missense mutations, 5% patients with small deletions or insertions and 0% patient with a splice site mutation) and 9%(9/98) in intron 22 inversions group. Compared with the risk of FⅧ inhibitor development in intron 22 inversions group, the risk of FⅧ inhibitor development in high risk group was higher (OR＝4.7, 95% CI： 1.7-13.0), the risk of FⅧ inhibitor development in low risk group was equal (OR＝0.5, 95% CI： 0.1-2.3). Compared with the risk of inhibitor development in low risk group, the risk of FⅧ inhibitor development in high risk group was higher (OR＝9.8, 95% CI： 2.0-48.7). Gene mutations of patients with severe HA in high-risk group which include intron 1 inversions, large deletions, nonsense mutations are a risk factor for FⅧ inhibitor production.

IBCL-365 Variety of Mutations In Russian B-Cell Lymphoma Patients

TP53 plays a key role in almost all cellular processes. Mutations in this gene are found in most types of neoplasms. Among hematological malignancies, TP53 lesions are the most actively studied in CLL, although available data for other lymphomas remains limited. To study the frequency and diversity of TP53 mutations in Russian patients with B-cell lymphomas. The study included 145 DNA samples from patients with B-cell lymphomas: 53 patients with DLBCL, 47 patients with MCL, and 45 patients with FL, 16 of whom experienced transformation to DLBCL. Mutations in TP53 (exons 4-10) in most samples were detected using NGS (138 patients) or Sanger sequencing (7 patients). The clinical significance of detected variants was assessed using the Seshat online tool based on the UMD TP53 database. TP53 mutations were detected in 24 DLBCL patients (45.4%), 21 of which were missense mutations, 2 were splicing site mutations, and 1 was an insertion. Sixteen variants were identified as (probably) pathogenic, and 8 mutations were of unclear significance. Del17p was simultaneously detected in 7 patients with TP53 mutations (of 11 examined). In MCL patients, mutations were found in 19 cases (40%). Eighteen were missense mutations, and 1 was a splicing mutation. Sixteen variants were identified as (probably) pathogenic, and 3 were variants of unclear significance. Chromosome 17 abnormalities were found in 7 MCL patients with TP53 mutations of 11 studied. In FL patients, TP53 mutations were detected in 12 cases (26.7%). Eight were missense mutations, 2 were nonsense mutations, 1 was a splicing mutation, and 1 was a deletion. Nine variants were recognized as (probably) pathogenic, and 2 were variants of unclear significance. Only 1 patient of 6 examined had del17p. The frequency of TP53 mutations in our cohort of patients with B-cell lymphomas was significantly higher than the frequency reported for other cohorts. This bias may be due to differences in patient selection. We are planning further studies examining the type of mutations, allelic loads, and combinations with other chromosome 17 lesions in association with the disease course.

Invasive lobular carcinoma with extracellular mucin (ILCEM): clinicopathologic and molecular characterization of a rare entity

AbstractInvasive lobular carcinoma with extracellular mucin (ILCEM) is a rare histologic subtype of breast cancer. Little is known about the pathologic or genomic signatures that distinguish ILCEM from classic invasive lobular carcinoma (ILC) or mucinous carcinoma. We studied 17 breast cancers with lobular morphology and extracellular mucin. Thirteen tumors with sufficient tissue for DNA extraction were analyzed by a next generation sequencing (NGS) assay that interrogates 447 genes for mutations and copy number variations (CNVs). Median patient age was 66 yrs (range: 31–77 yrs). Sixteen patients presented with masses, 7 of which were >2 cm. Seven patients had lymph node metastases. The cases of ILCEM were moderately (n = 13) or poorly differentiated (n = 4), frequently exhibiting variant morphology that has not been previously described or emphasized, including grade 3 nuclei (n = 11), diffuse signet ring cells (n = 10), solid growth (n = 4), tumor necrosis (n = 3) or apocrine features (n = 2). All tumors showed absent or reduced membranous E-cadherin expression. Concurrent lobular carcinoma in situ (LCIS) was seen in 11/17 cases, 1 of which was a striking example of signet ring cell LCIS with extracellular mucin. Receptor profiles were ER+/HER2− (n = 15) and ER+/HER2+ (n = 2). With a median follow-up of 83.5 months (range: 3–171 months) in 12 patients with available information, 8 patients had recurrences resulting in 4 cancer-related deaths. The most common CNVs were 16q loss (n = 11) and 1q gain (n = 9). CDH1 gene-level alterations were detected in all but one case, including frameshift (n = 7), nonsense (n = 2), and donor splice site (n = 1) mutations and indels (n = 2). Recurrent mutations were also seen in PIK3CA (n = 3), POLQ (n = 3), TP53 (n = 3), ERBB3 (n = 3), ERBB2 (n = 2), and RUNX1 (n = 2). Genes with recurrent amplifications included GATA3 (n = 4), FOXA1 (n = 3), CCND1 (n = 2). Our data highlights ILCEM as a distinct variant of ILC that often presents with higher-grade and variant morphologic features and is associated with an aggressive clinical course. NGS data support an overall lobular-type molecular profile and reveal potentially targetable alterations in a subset of cases with recurrence.

The Pathogenic Mechanism of the ATP2C1 p.Ala109_Gln120del Mutation in Hailey-Hailey Disease.

BackgroundHailey–Hailey disease (HHD) is an autosomal dominant cutaneous disorder that manifests as repeated blisters and erosions on flexural or intertriginous skin areas. The calcium-transporting ATPase type 2C member 1 gene (ATP2C1) encodes the secretory pathway Ca2+/Mn2+-ATPase 1 (SPCA1), whose deficiency is responsible for HHD. An ATP2C1 splice-site mutation (c.325-2A>G, p.Ala109_Gln120del) was previously identified in a Han Chinese family with HHD.MethodsIn this study, the identified ATP2C1 splice-site mutation (c.325-2A>G, p.Ala109_Gln120del) was investigated in transfected human embryonic kidney 293 cells to analyze its pathogenic mechanism in HHD patients by using cycloheximide chase assay, CCK8 assay and in silico modeling of SPCA1 mutant.ResultsCycloheximide chase assay showed that the degradation rate of the SPCA1 mutant was not obviously faster than that of the normal SPCA1. CCK8 assay showed that cell proliferation rates in the wild-type, A109_Q120del, and empty vector control groups all decreased in the gradient Mn2+ solutions in a dose-dependent manner. The cell proliferation rate in the wild-type was lower than that in the A109_Q120del and empty vector control (both P < 0.01), indicating overexpression of normal SPCA1 may rather induce Golgi stress, and even cell death. The cell proliferation rate in the A109_Q120del was lower than that in the empty vector control (P < 0.01), indicating that overexpression of the mutated SPCA1 may decrease its detoxification capability. Three-dimensional (3D) structure model of SPCA1 built by SWISS-MODEL and PyMOL showed that absence of the 12 amino acids from p.Ala109 to p.Gln120 in the SPCA1 mutant can cause obviously shortened transmembrane 2, which may affect correct localization of SPCA1 on the Golgi.ConclusionThese results demonstrate that the ATP2C1 mutation (c.325-2A>G, p.Ala109_Gln120del) may cause impaired SPCA1 capability to detoxify Mn2+ and abnormal SPCA1 structure, which reveals a new side for the pathogenesis of HHD.

Identification of a novel CACNA1F mutation in a Chinese family with CORDX3.

X-linked cone-rod dystrophy (CORDX) is one form of inherited retinal disorders (IRDs) characterized by progressive dysfunction of photoreceptor. Three types of CORDX were reported and CACNA1F gene defect can cause CORDX3. The aim of this study was to investigate the pathogenic variant in a Chinese family with IRD. The two affected subjects including the proband and his elder sister underwent ophthalmic examinations. Whole exome sequencing (WES) was performed in the proband at first, then co-segregation analysis was performed in the family by Sanger sequencing. Minigene approach was used to verify the effect of the mutation on the splicing of CACNA1F. X-chromosomal inactivation assay was performed to evaluate the inactivation patterns of the female carriers. The ophthalmic examination results of the proband fit the clinical description of CORDX3, and the female patient presented with only mild symptoms due to mildly skewed X-chromosomal inactivation (ratio 67: 33). Molecular genetic testing identified a novel splice-site mutation c.3847-2A > G in CACNA1F (NM_005183.4) gene in the patients, which inherited from their asymptomatic mother. Minigene approach confirmed that c.3847-2A > G could affect the splicing of CACNA1F. Our study identified a novel splice-site mutation in the CACNA1F gene, which expanded the mutational spectrum of CACNA1F-releated diseases and demonstrated the importance of combining clinical and genetic testing in the diagnosis of IRDs.

A glycogen storage disease type 1a patient with type 2 diabetes

BackgroundGlycogen storage disease type 1a (GSD1a) is an inborn genetic disease caused by glucose-6-phosphatase-α (G6Pase-α) deficiency and is often observed to lead to endogenous glucose production disorders manifesting as hypoglycemia, hyperuricemia, hyperlipidemia, lactic acidemia, hepatomegaly, and nephromegaly. The development of GSD1a with diabetes is relatively rare, and the underlying pathogenesis remains unclear.Case presentationHere we describe a case of a 25-year-old Chinese female patient with GSD1a, who developed uncontrolled type 2 diabetes mellitus (T2DM) as a young adult. The patient was diagnosed with GSD1a disease at the age of 10 and was subsequently treated with an uncooked cornstarch diet. Recently, the patient was treated in our hospital for vomiting and electrolyte imbalance and was subsequently diagnosed with T2DM. Owing to the impaired secretory function of the patient’s pancreatic islets, liver dysfunction, hypothyroidism, severe hyperlipidemia, and huge hepatic adenoma, we adopted diet control, insulin therapy, and hepatic adenoma resection to alleviate this situation. The WES discovered compound heterozygous mutations at the exon 5 of G6PC gene at 17th chromosome in the patient, c.648G>T (p.L216 L, NM_000151.4, rs80356484) in her father and c.674T>C (p.L225 P, NM_000151.4, rs1555560128) in her mother. c.648G>T is a well-known splice-site mutation, which causes CTG changing to CTT at protein 216 and creates a new splicing site 91 bp downstream of the authentic splice site, though both codons encode leucine. c.674T>C is a known missense mutation that causes TGC to become CGC at protein 225, thereby changing from coding for leucine to coding for proline.ConclusionWe report a rare case of GSD1a with T2DM. On the basis of the pathogenesis of GSD1a, we recommend attentiveness to possible development of fasting hypoglycemia caused by GSD and postprandial hyperglycemia from diabetes. As the disease is better identified and treated, and as patients with GSD live longer, this challenge may appear more frequently. Therefore, it is necessary to have a deeper and more comprehensive understanding of the pathophysiology of the disease and explore suitable treatment options.

A novel mutation in RS1 and clinical manifestations in a Chinese twin family with congenital retinoschisis.

Purpose: We aim to analyze the clinical and genetic features in a Chinese family with congenital retinoschisis by whole-exome sequencing and comprehensive clinical examination. Methods: Six members were recruited from a Chinese family. Three of them were diagnosed as congenital retinoschisis, including two twin siblings. All subjects received a full eye examination. Whole-exome sequencing (WES) and Sanger sequencing were performed on two twin probands and all participants, respectively. Results: A novel splice site mutation RS1.c.53-1G>A was identified in a Chinese congenital retinoschisis family. The mean onset age was 16.7 ± 2.4 years old. The average BCVA in patients was 0.37 ± 0.05. A typical spoke-wheel pattern was observed in all affected eyes. OCT examination results showed fovea schisis and schisis cavities were located in the inner nuclear layer in 100% eyes (6/6). ERG b/a ratio was decreased markedly, but was still more than 1 in the four eyes that were available. Conclusion: The present study discovered a new pathogenic splice cite variant of RS1 in congenital retinoschisis, which expands the mutational spectrum. In contrast to previous research, the phenotype of patients with the same mutation within one family was highly similar. Early molecular testing is crucial for early diagnosis, clinical management, and genetic counseling of patients with congenital retinoschisis.

A novel homozygous mutation in ERLIN1 gene causing spastic paraplegia 62 and literature review

Hereditary spastic paraplegia (HSP) is a group of genetic neurodegenerative disorders, which is characterized by the presence of progressive spasticity and weakness in bilateral lower limbs. Spastic paraplegia 62 (SPG62) caused by the endoplasmic reticulum lipid raft associated 1 (ERLIN1) gene mutation is a rare subtype of HSP. Herein, we report the case of the first Chinese SPG62 patient, explore the potential pathogenic mechanism and review ERLIN1-related HSP patients. A 23-year-old man had progressive difficulty in walking and gait abnormalities for more than 11 years. Physical examination showed slightly reduced muscle strength (5-/5) and elevated muscle tone in the lower limbs and hyperreflexia in four limbs. Genetic analysis identified a novel splicing site mutation in ERLIN1 gene (c.504+1G > A), which was predicted to disturb the normal splicing process of mRNA by bioinformatic tools. Minigene experiment further confirmed the mutation c.504+1G > A could cause erroneous deletion of Exon 7 in the mRNA, which may change the conserved prohibitin (PHB) domain of erlin-1 and affect the function of erlin1/2 complex. Thus, we identified a pathogenic mutation of ERLIN1 splicing site causing delayed-onset pure HSP. This study widened the genetic and phenotypic spectrum of SPG62.

A novel germline mutation of the &lt;i&gt;PALB&lt;/i&gt; gene in a young Yakut breast cancer woman

Background. Breast cancer (BC) is the most common female malignancy worldwide. partner and localizer of BRCA2 gene (PALB2) is directly involved in DNA damage response. germline mutation in PALB2 has been identified in breast cancer and familial pancreatic cancer cases, accounting for approximately 1–2% and 3–4%, respectively. the goal of this report was to describe new PALB2 mutation in a young Yakut breast cancer patient with family history of cancer. Material and methods. Genomic DNA were isolated from blood samples and used to prepare libraries using a capture-based target enrichment kit, Hereditary Cancer Solution™ (SOPHIA GENETICS, Switzerland), covering 27 genes (ATM, APC, BARD1, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, EPCAM, FAM175A, MLH1, MRE11A, MSH2, MSH6, MUTYH, NBN, PALB2, PIK3CA, PMS2, PMS2CL, PTEN, RAD50, RAD51C, RAD51D, STK11, TP53 and XRCC2). paired-end sequencing (2 × 150 bp) was conducted using NextSeq 500 system (Illumina, USA). Results. Here we describe a case of a never-before-reported mutation in the PALB gene that led to the early onset breast cancer. We report the case of a 39-year-old breast cancer Yakut woman with a family history of pancreatic cancer. Bioinformatics analysis of the NGS data revealed the presence of the new PALB2 gene germinal frameshift deletion (NM_024675:exon1:c.47dela:p.K16fs). in accordance with dbPubMed ClinVar, new mutation is located in codon of the PALB2 gene, where the likely pathogenic donor splice site mutation (NM_024675.3:c.48+1delG) associated with hereditary cancer-predisposing syndrome has been earlier described. Conclusion. We found a new never-before-reported mutation in PALB2 gene, which probably associated with early onset breast cancer in Yakut indigenous women with a family history of pancreatic cancer.

Lab resource: Single cell line generation and characterization of a human-derived induced pluripotent stem cell line (IGIBi005-A) from a patient with spastic paraplegia/ataxia/ALS phenotype due to the mutation of the gene Kinesin Family Member 5A (KIF5A)

Human Kinesin Family Member 5A (KIF5A) gene mutations have been identified as a putative genetic cause of amyotrophic lateral sclerosis (ALS). Disease modelling using human-induced pluripotent stem cells (HiPSCs) is the next-generation approach to studying numerous human diseases. For the current investigation, we report the generation of patient-specific KIF5A iPSC lines with a mutation at the splice site mutation (c.3020 + 3 A > T) in the intronic region. The resulting line displayed markers for pluripotency, a healthy karyotype, the ability to differentiate into three germ layers in vitro, vector clearance, the KIF5A mutation, STR-based genomic identity, and contamination-free culture.

Palmoplantar Keratoderma: A Molecular Genetic Analysis of Family Cases.

Palmoplantar keratoderma is a clinically polymorphic disorder with a heterogeneous etiology characterized by marked hyperkeratotic lesions on the surface of palms and soles. Hereditary forms of palmoplantar keratoderma usually have autosomal dominant inheritance and are caused by mutations in dozens of genes, most of which belong to the keratin family. We carried out clinical and molecular genetic analysis of the affected and healthy members of four families with autosomal dominant palmoplantar keratoderma. In three out of four family cases of autosomal dominant palmoplantar keratoderma, the following molecular genetic causes were established: in two families—previously non-described missense mutations in the AQP5 gene (NM_001651.4): c.369C>G (p.(Asn123Lys)) and c.103T>G (p.(Trp35Gly)); in one family—a described splice site mutation in the KRT9 gene (NM_000226.4): c.31T>G. In one family, the possible cause of palmoplantar keratoderma was detected—a variant in the KRT1 gene (NM_006121.4): c.931G>A (p.(Glu311Lys)).

Prenatal phenotype analysis and mutation identification of a fetus with meckel gruber syndrome.

Ciliopathies are a class of inherited severe human disorders that occur due to defective formation or function of cilia. The RPGRIP1L (retinitis pigmentosa GTPase regulator-interacting protein1-like) gene encodes for a ciliary protein involved in regulating cilia formation and function. Mutations in RPGRIP1L cause ciliopathies associated with severe embryonic defects, such as Meckel-Gruber Syndrome (MKS). Here we report RPGRIP1L mutation analysis in a family diagnosed with MKS. The clinical manifestations of the fetus included thoraco-lumbar open neural tube defect with associated Chiari type II malformation and hydrocephalus, bilateral club feet, and single right kidney/ureter. Analysis of the parental DNA samples revealed that the father carried a previously reported mutation R1236C/+ whereas the mother had a novel splice site mutation IVS6+1 G > A/+ in RPGRIP1L. The splice site mutation resulted in the exclusion of in-frame exon 6 of RPGRIP1L (RPGRIP1L-∆Ex6) but expressed a stable protein in fibroblasts derived from the parents’ skin biopsies. The GFP-RPGRIP1L-∆Ex6 mutant protein exhibited relatively reduced ciliary localization in transiently-transfected cultured RPE-1 cells. Taken together, this study identifies a novel RPGRIP1L variant RPGRIP1L-∆Ex6, which in combination with RPGRIP1L-R1236C is associated with MKS. We also suggest that the deletion of exon 6 of RPGRIP1L leads to reduced ciliary localization of RPGRIP1L, indicating a plausible mechanism of associated disease.