Frameshift Mutation In Exon Research Articles

A genotypic and phenotypic analysis of four unrelated Chinese patients with Pitt–Hopkins syndrome

BackgroundPitt–Hopkins syndrome (PTHS) is a rare genetic condition caused by a mutation in the transcription Factor 4 (TCF4) gene and characterized by its unique clinical presentations. At present, there is an incomplete understanding of the possible TCF4 mutations and their downstream consequences, and no reliable treatment exists for patients with PTHS. Elucidating the variations in TCF4 occurring in PTHS could lead to new treatment ideas.Case presentationWe described the clinical and genetic characteristics of four Chinese patients with PTHS. Genetic mutations related to central nervous system (CNS) disorders were identified via high-throughput sequencing. The patient’s mutations were subsequently confirmed with Sanger sequencing. Most patients had facial features typical of PTHS; however, Patient 2 demonstrated poor auricle shape. Each patient presented with differing levels of delayed development and intellectual impairment. The patients showed a splice site mutation in intron 15 of TCF4 (c.1452 + 3A > G), a frameshift mutation in exon 18 of TCF4 (c.1942delA), and two missense mutations in exon 19 of TCF4 (c. 2147C > T and c.2026A > G).ConclusionsWe discovered four new TCF4 mutations in Chinese children with PTHS. To our knowledge, the c.2026A > G and c.1942delA mutations have not yet been reported. The detection of these mutations can help accurately diagnose PTHS early, especially in patients whose clinical symptoms are not obvious. Exploring the genotype and phenotype of individuals with PTHS, will enrich our understanding and guide further research into the role of TCH4.

The first Japanese case of autosomal dominant cutis laxa with a frameshift mutation in exon 30 of the elastin gene complicated by small airway disease with 8 years of follow-up

BackgroundCutis laxa constitutes a diverse group of connective tissue diseases, both inherited and acquired, characterized by loose skin and varying systemic involvement, including pulmonary lesions. While cutis laxa has been linked to conditions like emphysema, asthma, and bronchiectasis, the specific pathological and radiological characteristics underlying pulmonary complications related to cutis laxa remain unclear.Case presentationA 36-year-old woman, diagnosed with cutis laxa at birth, presented to our outpatient clinic with severe obstructive ventilatory impairment, evident in pulmonary function tests (expiratory volume in one second (FEV1)/forced vital capacity (FVC): 34.85%; %residual volume [RV]: 186.5%; %total lung capacity [TLC]: 129.2%). Pulmonary function tests also indicated small airway disease (%FEF50%, 7.9%; %FEF75%, 5.7%; and %FEF25–75%, 6.8%). Computed tomography (CT) revealed the lack of normal increase in lung attenuation on expiratory CT scan, with no discernible emphysematous changes. Exome sequencing was performed to confirm the association between the pulmonary lesions and cutis laxa, revealing a frameshift variant in exon 30 of the elastin gene (ELN). Further analysis employing a parametric response map revealed a longitudinal increase in the percentage of functional small airway disease (fSAD) from 37.84% to 46.61% over the 8-year follow-up, despite the absence of overt changes in CT findings, specifically the lack of normal increase in lung attenuation on expiratory CT scan. Over the same follow-up interval, there was a modest reduction of 25.6 mL/year in FEV1 coupled with a significant increase in %RV. Pulmonary function test metrics, reflective of small airway disease, exhibited a continual decline; specifically, %FEF50%, %FEF75%, and %FEF25–75% diminished from 7.9% to 7.0%, 5.7% to 4.6%, and 6.8% to 5.4%, respectively.ConclusionsThis case highlighted an instance of autosomal dominant cutis laxa arising from a frameshift variant in exon 30 of ELN, accompanied by small airway disease. Comprehensive investigation, utilizing quantitative CT analysis, revealed a longitudinal increase in fSAD percentage with a mild reduction in FEV1. These findings indicate that elastin deficiency may not only diminish elastic fibers in the skin but also be implicated in small airway disease by impacting components of the extracellular matrix in the lungs.

Identification of cellular retinoic acid binding protein 2 (CRABP2) as downstream target of nuclear factor I/X (NFIX): implications for skeletal dysplasia syndromes.

Nuclear factor I/X (NFIX) mutations are associated with 2 skeletal dysplasias, Marshall-Smith (MSS) and Malan (MAL) syndromes. NFIX encodes a transcription factor that regulates expression of genes, including Bobby sox (BBX) and glial fibrillary acidic protein (GFAP) in neural progenitor cells and astrocytes, respectively. To elucidate the role of NFIX mutations in MSS, we studied their effects in fibroblast cell lines obtained from 5 MSS unrelated patients and 3 unaffected individuals. The 5 MSS NFIX frameshift mutations in exons 6-8 comprised 3 deletions (c.819-732_1079-948del, c.819-471_1079-687del, c.819-592_1079-808del), an insertion (c.1037_1038insT), and a duplication (c.1090dupG). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot analyses using MSS and unrelated control fibroblasts and in vitro expression studies in monkey kidney fibroblast (COS-7) cells showed that frameshift mutations in NFIX exons 6-8 generated mutant transcripts that were not cleared by nonsense-mediated-decay mechanisms and encoded truncated NFIX proteins. Moreover, BBX or GFAP expression was unaffected in the majority of MSS fibroblasts. To identify novel NFIX downstream target genes, RNA sequencing and proteomics analyses were performed on mouse embryonic fibroblast (MEF) cells derived from control Nfix+/+, Nfix+/Del2, Nfix+/Del24, NfixDel24/Del24, Nfix+/Del140, and NfixDel140/Del140 mice, compared with NfixDel2/Del2 mice which had developmental, skeletal, and neural abnormalities. This identified 191 transcripts and 815 proteins misregulated in NfixDel2/Del2 MEFs with ≥2-fold-change (P <0 .05). Validation studies using qRT-PCR and western blot analyses confirmed that 2 genes, cellular retinoic acid binding protein 2 (Crabp2) and vascular cell adhesion molecule 1 (Vcam1), were misregulated at the RNA and protein levels in NfixDel2/Del2 MEFs, and that CRABP2 and VCAM1 expressions were altered in 60%-100% of MSS fibroblast cells. Furthermore, in vitro luciferase reporter assays confirmed that NFIX directly regulates CRABP2 promoter activity. Thus, these altered genes and pathways may represent possible targets for drugs as potential treatments and therapies for MSS.

Investigating the effects of a single ASPM variant (c.8508_8509) on brain architecture among siblings in a consanguineous Pakistani family.

Autosomal Recessive Primary Microcephaly (MCPH) is a rare, neurodevelopmental disorder associated with mild to severe mental retardation. It is characterized by reduced cerebral cortex that ultimately leads to reduction in skull size less than - 3S.D below the mean for normal individuals having same age and sex. Till date, 30 known loci have been reported for MCPH. In the present study, Sanger sequencing was performed followed by linkage analysis to validate the mutation in ASPM gene of the consanguineous Pakistani clans. Bioinformatics tools were also used to confirm the pathogenicity of the diseased variant in the gene. MRI scan was used to compare the brain structure of both the affected individuals (Aslam et al. in Kinnaird's 2nd International Conference on Science, Technology and Innovation, Lahore, 2023). Our study described a consanguineous family with two patients with a known ASPM (MCPH5) variant c.8508_8509delGA causing a frameshift mutation in exon 18 which located in calmodulin-binding IQ domain of the ASPM protein. The salient feature of this study is that a single variant led to significantly distinct changes in the architecture of brain of both siblings which is further confirmed by MRI results. The computation analysis showed that the change in the conservation of this residue cause this variant highly pathogenic. Carrier screening and genetic counselling were also remarkable features of this study (Aslam et al. in Kinnaird's 2nd International Conference on Science, Technology and Innovation, Lahore, 2023). This study explores the extraordinary influence of a single ASPM variant on divergent brain structure in consanguineous siblings and enable us to reduce the incidence of further microcephalic cases in this Pakistani family (Aslam et al. in Kinnaird's 2nd International Conference on Science, Technology and Innovation, Lahore, 2023).

Generation and characterization of a zebrafish knockout model of abcb4, a homolog of the human multidrug efflux transporter P-glycoprotein

The ATP-binding cassette subfamily B member 1 (ABCB1), encoding a multidrug transporter referred to as P-glycoprotein (Pgp), plays a critical role in the efflux of xenobiotics in humans and is implicated in cancer resistance to chemotherapy. Therefore, developing high-throughput animal models to screen for Pgp function and bioavailability of substrates and inhibitors is paramount. Here, we generated and validated a zebrafish knockout line of abcb4, a human Pgp transporter homolog. CRISPR/Cas9 genome editing technology was deployed to generate a frameshift mutation in exon 4 of zebrafish abcb4. The zebrafish abcb4 homozygous mutant exhibited elevated accumulation of fluorescent rhodamine 123, a substrate of human Pgp, in the intestine and brain area of embryos. Moreover, abcb4 knockout embryos were sensitized toward toxic compounds such as doxorubicin and vinblastine compared to the WT zebrafish. Immunostaining for zebrafish Abcb4 colocalized in the endothelial brain cells of adult zebrafish. Transcriptome profiling using Gene Set Enrichment Analysis uncovered that the 'cell cycle process,' 'mitotic cell cycles,' and 'microtubule-based process' were significantly downregulated in the abcb4 knockout brain with age. This study establishes and validates the abcb4 knockout zebrafish as an animal model to study Pgp function in vivo. Unexpectedly it reveals a potentially novel role for zebrafish abcb4 in age-related changes in the brain. The zebrafish lines generated here will provide a platform to aid in the discovery of modulators of Pgp function as well as the characterization of human mutants thereof.

Genetically engineered sheep: A new paradigm for future preclinical testing of biological heart valves

BackgroundHeart valve implantation in juvenile sheep to demonstrate biocompatibility and physiologic performance is the accepted model for regulatory approval of new biological heart valves (BHVs). However, this standard model does not detect the immunologic incompatibility between the major xenogeneic antigen, galactose-α-1,3-galactose (Gal), which is present in all current commercial BHVs, and patients who universally produce anti-Gal antibody. This clinical discordance leads to induced anti-Gal antibody in BHV recipients, promoting tissue calcification and premature structural valve degeneration, especially in young patients. The objective of the present study was to develop genetically engineered sheep that, like humans, produce anti-Gal antibody and mirror current clinical immune discordance. MethodsGuide RNA for CRISPR Cas9 nuclease was transfected into sheep fetal fibroblasts, creating a biallelic frame shift mutation in exon 4 of the ovine α-galactosyltransferase gene (GGTA1). Somatic cell nuclear transfer was performed, and cloned embryos were transferred to synchronized recipients. Cloned offspring were analyzed for expression of Gal antigen and spontaneous production of anti-Gal antibody. ResultsTwo of 4 surviving sheep survived long-term. One of the 2 was devoid of the Gal antigen (GalKO) and expressed cytotoxic anti-Gal antibody by age 2 to 3 months, which increased to clinically relevant levels by 6 months. ConclusionsGalKO sheep represent a new, clinically relevant advanced standard for preclinical testing of BHVs (surgical or transcatheter) by accounting for the first time for human immune responses to residual Gal antigen that persists after current BHV tissue processing. This will identify the consequences of immune disparity preclinically and avoid unexpected past clinical sequelae.

Determination of Factor II Codons Genotype in Southeastern Iranian Patients With Hereditary Deficiency of Factor II

Objectives: Congenital prothrombin (factor II) deficiency is an inherited rare bleeding disorder with an autosomal recessive manner. The prevalence of this disorder is about one in 2000000 people in general population, but it is more common in areas with a high rate of consanguinity. To date, there is no report on the absence of prothrombin, which is a life-threating disorder. Considering the importance of factor II in body homeostasis, this study aimed to find any possible mutation of coagulation factor II codons in patients with inherited factor II deficiency in southeastern Iran. Materials andMethods: This study was conducted on 12 patients with inherited deficiency of prothrombin. Early diagnosis was based on clinical symptoms, laboratory evaluation, and family history. Then, the function level of prothrombin was measured, the initial diagnosis of disease was confirmed, and polymerase chain reaction (PCR) analysis was performed. Finally, gene sequencing and genotyping of factor II was done. Results: Molecular analysis indicated a point mutation in exon 7 in three patients and a frameshift mutation in exon 14 due to addition of a thymine base at position 1760-1761 in one patient, both of which have been reported for the first time. Conclusions: Molecular methods performed on patients from Southeastern Iranian population in terms of coagulation factor II deficiency revealed a substitution mutation in exon 7 in three patients and a frameshift mutation in exon 14 in one patient, both of which were reported for the first time. Considering the significant difference between the clinical symptoms of the present study and previous studies, probably the type of mutations reported in this study (for the first time) caused these clinical symptoms, but statistical studies did not show any relationship between the type of mutation and the occurrence of clinical symptoms. And it needs more investigations on more patients, with a larger population.

A novel frameshift mutation in TRPV6 is associated with hereditary pancreatitis.

Introduction: Hereditary pancreatitis (HP) is a rare debilitating disease with incompletely understood etio-pathophysiology. The reduced penetrance of genes such as PRSS1 associated with hereditary pancreatitis indicates a role for novel inherited factors. Methods: We performed whole-exome sequencing of three affected members of an Indian family (Father, Son, and Daughter) with chronic pancreatitis and compared variants with those seen in the unaffected mother. Results: We identified a novel frameshift mutation in exon 11 of TRPV6 (c.1474_1475delGT; p.V492Tfs*136), a calcium channel, in the patients. Functional characterization of this mutant TRPV6 following heterologous expression revealed that it was defective in calcium uptake. Induction of pancreatitis in mice induced Trpv6 expression, indicating that higher expression levels of the mutant protein and consequent dysregulation of calcium levels in patients with chronic pancreatitis could aggravate the disease. Discussion: We report a novel frameshift mutation in TRPV6 in an Indian family with HP that renders the mutant protein inactive. Our results emphasize the need to expand the list of genes used currently for evaluating patients with hereditary pancreatitis.

Somatic NGS Analysis of DNA Damage Response (DDR) Genes ATM, MRE11A, RAD50, NBN, and ATR in Locally Advanced Rectal Cancer Treated with Neoadjuvant Chemo-Radiotherapy.

Neoadjuvant chemo-radiotherapy (nCRT) represents the standard of care for locally advanced rectal cancer (LARC); however, there exists no biomarker that can predict the cancer's response to treatment as less than 20% of patients experience pathological complete response (pCR). Ionizing radiations induce double strand breaks (DSBs) and trigger a DNA damage response (DDR) involving ATM, ATR, and the MRN complex (MRE11, Rad50, and NBS1). In this study, we performed an extensive mutational analysis of the genes involved in the DDR pathway in LARC patients who have undergone nCRT. 13 LARC patients with pCR and 11 LARC patients with partial response (pPR) were investigated using a NGS dedicated panel, designed for formalin-fixed paraffin-embedded (FFPE) samples, containing ATR, ATM, and MRE11-RAD50-NBN genes. The identified variants were classified according to guidelines' recommendations. Eight non-benign variants, six of which were observed in 3 (23%) out of 13 pCR patients, were identified. In particular, a pCR patient carried out a pathogenetic frameshift mutation in exon 21 of the RAD50 gene. The two remaining non-benign missense variants were found in 2 (18%) out of 11 patients in the pPR group. Our data show that the genes involved in the Homologous Recombination (HR) pathway are rarely mutated in LARC; however, given the identification of a missense mutation in RAD 50 in one case of pCR, it could be worth exploring its potential role as a biomarker in larger series.

Evaluation of CRISPR/Cas9 exon-skipping vector for choroideremia using human induced pluripotent stem cell-derived RPE.

Exon-skipping is a powerful genetic tool, especially when delivering genes using an AAV-mediated full-length gene supplementation strategy is difficult owing to large length of genes. Here, we used engineered human induced pluripotent stem cells and artificial intelligence to evaluate clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9-based exon-skipping vectors targeting genes of the retinal pigment epithelium (RPE). The model system was choroideremia; this is an X-linked inherited retinal disease caused by mutation of the CHM gene. We explored whether artificial intelligence detected differentiation of human OTX2, PAX6 and MITF (hOPM) cells, in which OTX2, PAX6 and MITF expression was induced by doxycycline treatment, into RPE. Plasmid encoding CHM exon-skipping modules targeting the splice donor sites of exons 6 were constructed. A clonal hOPM cell line with a frameshift mutation in exon 6 was generated and differentiated into RPE. CHM exon 6-skipping was induced, and the effects of skipping on phagocytic activity, cell death and prenylation of Rab small GTPase (RAB) were evaluated using flow cytometry, an in vitro prenylation assay and western blotting. Artificial intelligence-based evaluation of RPE differentiation was successful. Retinal pigment epithelium cells with a frameshift mutation in exon 6 showed increased cell death, reduced phagocytic activity and increased cytosolic unprenylated RABs only when oxidative stress was in play. The latter two phenotypes were partially rescued by exon 6-skipping of CHM. CHM exon 6-skipping contributed to RPE phagocytosis probably by increasing RAB38 prenylation under oxidative stress.

A CASE OF DOCK8 DEFICIENCY TREATED WITH DUPILUMAB

<h3>Introduction</h3> Dedicator of cytokinesis 8 gene (DOCK8) deficiency is an immune disorder associated with autosomal recessive hyper-IgE syndrome, characterized by elevated IgE levels, atopic dermatitis, and predisposition to recurrent skin and lung infections. <h3>Case Description</h3> A 12-year-old male presented to Immunology clinic for DOCK8 deficiency. He was diagnosed in infancy due to diffuse papulopustular eruption positive for candidiasis and known family history. Past medical history included severe eczematous dermatitis, asthma, interstitial lung disease, food allergies, and poor growth, with cutaneous HSV and fungal infections, HSV keratitis, and MRSA bacteremia. Family history was notable for two siblings with DOCK8 deficiency, both deceased. Physical exam demonstrated severe generalized eczematous papules and plaques and intermittent coarse breath sounds. IgE ranged from 22.9–14,730 kU/L. Absolute eosinophil count fluctuated between 30–10,350 cells/mcL. Sequencing of the <i>DOCK8</i> gene revealed a novel 2 base pair deletion resulting in a frame shift mutation in exon 14 at codon 510. He was managed on weekly subcutaneous immunoglobulin replacement, prophylactic Bactrim and valacyclovir, and followed by Immunology, Dermatology, Pulmonology, and Ophthalmology. Family declined bone marrow transplantation. Due to the severity of his eczematous dermatitis, monthly dupilumab injections were initiated. Within a year, he reported significant improvement in cutaneous and respiratory symptoms, with increase in lung function from FEV1 of 32% to 63%. <h3>Discussion</h3> In patients with DOCK8 deficiency, hematopoietic stem cell transplantation is curative. For patients who are unable or unwilling to undergo or awaiting transplant, dupilumab is a treatment option for improvement of both cutaneous and respiratory manifestations.

A case of Dravet syndrome with focal cortical myoclonus

BackgroundDravet syndrome (DS) is a severe epileptic encephalopathy in children dominated by polymorphic seizures. Focal cortical myoclonus indicated on conventional electroencephalogram (EEG) was rarely observed in DS.Case presentationThe child, boy, thirteen months old, suffered from clonic seizures during bathing at two months old. Later he suffered from recurrent afebrile or febrile generalized tonic–clonic seizures often developing into status epilepticus. A genetic analysis of the SCN1A gene revealed a de novo heterozygous frame shift mutation in exon 21(c.3836_c.3837del AT).His myoclonic jerks of unilateral arm occurred spontaneously in response to movement. A spike wave from right central-parietal cortex immediately preceded a left myoclonic muscle activity, while a spike wave from left immediately preceded a right myoclonic muscle activity. The onset of the detected spike preceded the onset of myoclonic muscle activity by 42 ms using jerk-locked back-averaging of electroencephalogram data. The focal cortical myoclonus was not noted when one year old.ConclusionsFocal cortical myoclonus could be a form of seizures during the first year of life in DS, which may broaden the types of seizures of DS and may provide some diagnostic clues for DS.

A Novel Splice Site Mutation in Anthrax Toxin Receptor 2 (Capillary Morphogenesis Protein 2) Gene Results in Systemic Hyalinosis

Infantile Systemic Hyalinosis, now included under the unifying term, “hyaline fibromatosis syndrome” (HFS) is a rare, progressive and fatal autosomal recessive disorder characterized by various dermatological manifestations such as thickened skin, papules and nodules, hyperpigmentation over bony prominences, gingival hypertrophy and systemic manifestations such as joint contractures, osteopenia, recurrent infections, diarrhea, and short stature. Histopathological examination of the affected tissue shows deposition of amorphous hyaline material. Mutations in the gene encoding capillary morphogenesis protein-2/anthrax toxin receptor 2 (ANTXR2) are implicated in the pathogenesis. Here, we report a 16-month-old toddler with HFS whose genetic analysis revealed a novel homozygous frameshift mutation in exon 13 of ANTXR2 gene.

A novel missense mutation in the folliculin gene associated with the renal tumor-only phenotype of Birt-Hogg-Dubé syndrome

Birt-Hogg-Dubé syndrome is an autosomal dominant disease caused by germline mutations in the folliculin gene (FLCN), characterized by skin fibrofolliculomas, pulmonary cysts, and multiple renal tumors. We report the case of a 51-year-old woman with multiple bilateral renal tumors resected by bilateral open partial nephrectomy. Following pathological diagnosis of hybrid oncocytic/chromophobe tumors, targeted next-generation sequencing of FLCN of the patient's blood revealed a novel missense mutation (c.602A>C, p.Gln201Pro) in exon 6. Sanger sequencing revealed that this mutation was heterozygous. In silico prediction programs consistently indicated the mutation as pathogenic. Western blot analysis and immunohistochemistry revealed suppressed FLCN expression and the upregulation of glycoprotein nonmetastatic B, a downstream target negatively regulated by FLCN, in the tumor tissue, suggesting that the mutation resulted in reduction of functional FLCN expression. Whole-genome sequencing of one of the tumors identified another frameshift mutation in exon 4, suggesting a “second hit” leading to tumorigenesis. We recommend that gene sequencing should be considered in patients with multiple renal tumors to identify their genetic predisposition to renal tumors.

239 New de novo mutation in CACNA1a gene leading to episodic ataxia type 2

Introduction andAimEpisodic ataxia (EA) is a group of inherited ion channel disorders affecting the cer- ebellum characterised by sporadic episodes of ataxia (Riant et al, 2011; Tomlinson et al, 2013). At least 7 different types have been identified, type 2 being the most common. We describe a patient present- ing with recurrent episodes of dizziness and gait disturbance with a novel mutation in CACNA1a gene (c.586del) leading to episodic ataxia type 2.Clinical presentationA 65-year old male presented with recurrent episodes of dizziness and gait distur- bance, which had been present since childhood but increased in severity over last 2 years and leading to hospitalisation. He also reported mild cognitive impairment. Examination showed bilateral horizontal jerky nyastagmus with an otherwise normal neurological examination.InvestigationsNeuroimaging studies showed mild small vessel disease. Genetic sequencing revealed a heterogenicity for the c.586del likely pathogenic frameshift mutation in exon 4 of the CACNA1A gene, resulting in targeting of the mRNA for nonsense- mediated decay or the production of a truncated protein leading to the diagnosis of autosomal dominant episodic ataxic type 2 in this patient.SummaryWe report a new de novo mutation in CACNA1a gene leading to episodic ataxia type 2. The patient was given acetazolamide (250mg/day) which improved his symptoms. Episodic ataxia should be considered in patients presenting with a history of intermittent dizziness.kitwu@doctors.org.uk

Red cell adenylate kinase deficiency in China: molecular study of 2 new mutations (413G > A, 223dupA)

BackgroundAdenylate kinase (AK) is a monomolecular enzyme widely found in a variety of organisms. It mainly catalyses the reversible transfer of adenosine nucleotide phosphate groups and plays an important role in maintaining energy metabolism. AK deficiency is a rare genetic disorder that is related to haemolytic anaemia. Chronic haemolytic anaemia associated with AK deficiency is a rare condition, and only 14 unrelated families have been reported thus far. Moreover, only 11 mutations have been identified in the AK1 gene, with only 3 cases of psychomotor impairment.Case presentationThe patient was a 3-year-old boy with severe haemolytic anaemia and psychomotor retardation. A molecular study of the patient’s AK gene revealed 2 different mutations: a heterozygous missense mutation in exon 6 (c.413G > A) and a heterozygous frameshift mutation in exon 5 (c.223dupA). Molecular modelling analyses indicated that AK gene inactivation resulted in a lack of AK activity. The patient recovered after regular blood transfusion therapy.ConclusionsAK1 deficiency was diagnosed on the basis of low enzymatic activity and the identification of a mutation in the AK1 gene located on chromosome 9q. Here, we report the first case of moderate red cell AK1 deficiency associated with chronic nonspherocytic haemolytic anaemia (CNSHA) in China. The genetic mutations were confirmed by Sanger sequencing. The variants were classified as pathogenic by bioinformatics tools, such as ACMG/AMP guidelines, Mutation Taster, SIFT, MACP, REVEL and PolyPhen2.2. Based on our evidence and previous literature reports, we speculate that the site of the AK1 gene c.413G > A (p.Arg138His) mutation may be a high-frequency mutation site and the other mutation (c.223dupA) might be related to the neuropathogenicity caused by AK1 deficiency. NGS should be a part of newborn to early childhood screening to diagnose rare and poorly diagnosed genetic diseases as early as possible.

Editing TINF2 as a potential therapeutic approach to restore telomere length in dyskeratosis congenita

AbstractMutations in the TINF2 gene, encoding the shelterin protein TIN2, cause telomere shortening and the inherited bone marrow (BM) failure syndrome dyskeratosis congenita (DC). A lack of suitable model systems limits the mechanistic understanding of telomere shortening in the stem cells and thus hinders the development of treatment options for BM failure. Here, we endogenously introduced TIN2-DC mutations in human embryonic stem cells (hESCs) and human hematopoietic stem and progenitor cells (HSPCs) to dissect the disease mechanism and identify a gene-editing strategy that rescued the disease phenotypes. The hESCs with the T284R disease mutation exhibited the short telomere phenotype observed in DC patients. Yet, telomeres in mutant hESCs did not trigger DNA damage responses at telomeres or show exacerbated telomere shortening when differentiated into telomerase-negative cells. Disruption of the mutant TINF2 allele by introducing a frameshift mutation in exon 2 restored telomere length in stem cells and the replicative potential of differentiated cells. Similarly, we introduced TIN2-DC disease variants in human HSPCs to assess the changes in telomere length and proliferative capacity. Lastly, we showed that editing at exon 2 of TINF2 that restored telomere length in hESCs could be generated in TINF2-DC patient HSPCs. Our study demonstrates a simple genetic intervention that rescues the TIN2-DC disease phenotype in stem cells and provides a versatile platform to assess the efficacy of potential therapeutic approaches in vivo.

Generation of heterozygous (MRli003-A-3) and homozygous (MRli003-A-4) TRPM4 knockout human iPSC lines

TRPM4 is a Ca2+-activated channel mediating the transport of monovalent cations across the cell membrane. Mutations in the TRPM4 gene have been associated with cardiac arrhythmias in humans. Using CRISPR/Cas9 gene editing technology, we established two TRPM4 knockout human iPSC lines – one heterozygous (MRli003-A-3) and one homozygous (MRli003-A-4) – by inserting a frameshift mutation in exon 2 of the TRPM4 gene. Both lines maintained pluripotency, a normal karyotype, parental cell morphology, and the ability to differentiate into the three germ layers.

Frequency of GRN ‐associated cases in a Russian cohort of patients with frontotemporal dementia

Frontotemporal dementia (FTD) is the second most common cause of early-onset neurodegenerative dementia. Up to 40% of FTD cases are inherited. Mutations in GRN gene are frequent genetic cause of FTD in different population (1-5% of sporadic cases and 5-20% of familial cases). The frequency of this mutation in a Russian cohort of FTD patients has not been studied before. We analyzed DNA samples of patients with FTD for the period from 2014 to 2019 (N=57); 42% of patients had positive family history of dementia. All patients were examined at the Research Center of Neurology and I.M. Sechenov First Moscow State Medical University (Moscow). The distribution by phenotypes is shown in Table 1. GRN mutation screening was performed by Sanger sequencing. In total, 4 pathogenic heterozygous mutations in GRN gene were identified in 5 patients. Two frameshift mutations in exon 9 have not been previously described (Gly384fs*, Cys315fs*). One of these mutations (Gly384fs*) was detected in two unrelated patients with non-fluent variant of primary progressive aphasia (nfvPPA) with the same age at onset and positive family history of dementia, but different rates of the disease progression. Mutation Cys315fs* was detected in one patient with behavioral variant of FTD (bvFTD) without family history. Two other mutations (Gly35fs* and IVS9+1G>A) were previously described, and our patients with these mutations had bvFTD and nfvPPA, respectively. Thus, the frequency of mutation in GRN gene was 8.8%: 12.5% in familial FTD cases and 6% in sporadic cases. GRN mutations were more common in nfvPPA group (17%) compared to bvFTD (7%). Presented are the first data of GRN mutation frequency in Russian patients with FTD. The frequency found is comparable to the data described in other populations. The most frequent disease phenotype in GRN mutation carriers is nfvPPA. Two new frameshift mutations in GRN gene were identified.

Primary breast CD20-positive extranodal NK/T cell lymphoma with stomach involvement: a case report and literature review

BackgroundWe present a unique case of primary breast CD20-positive extranodal NK/T cell lymphoma with stomach involvement in a young Chinese female patient.Case presentationThe patient presented with a mass in her right breast that rapidly increased in size over approximately 2 months. Upper gastrointestinal endoscopy showed a giant serpentine ulcer in the stomach. Biopsy was performed, and microscopic inspection revealed that the fibrous tissue was diffusely involved by medium to large abnormal lymphocytes. The cytoplasm was low to moderate. The tumor cells had irregular nuclei and inconspicuous nucleoli. The lymphoid cells were strongly immunoreactive to CD20, CD3, CD4, CD56, TIA-1, EBER, and Ki-67 (90%). Epstein-Barr virus genomes were also found in tumor cells by in situ hybridization. A whole-body positron emission tomography (PET)-CT scan revealed intense FDG uptake in the right breast and greater curvature of the stomach. Monoclonal rearrangements of the T cell receptor (TCR-γ) and immunoglobulin heavy chain (IgH) were identified by genetic analysis. Whole-genome next-generation sequencing was performed, and up to 12 gene mutations, including a frameshift mutation in exon 4 of the BCOR (G97Rfs*87; 44.3%) gene and a base substitution mutation (Q61H 17.6%) in exon 3 of the KRAS gene, were detected. Kyoto Encyclopedia of Genes and Genomes pathway analyses were performed using the database for annotation, visualization, and integrated discovery, which showed that rare primary breast CD20-positive extranodal NK/T cell lymphoma had a unique genetic background compared with diffuse large B cell lymphoma and extranodal NK/T cell lymphoma without CD20 expression. The patient received four cycles of the modified SMILE regimen. The second whole-body PET-CT scan revealed that the right breast mass was significantly smaller than before; additionally, FDG uptake in the stomach wall disappeared.ConclusionsSystemic examination, extensive immunohistochemistry, and molecular profiling are essential for an accurate diagnosis. More similar cases are required to clarify the biological pathways and even the potential molecular mechanisms of rare lymphomas, which may help direct further treatment.