Homozygous Intronic Variant Research Articles

Targeting ATP2B1 impairs PI3K/Akt/FOXO signaling and reduces SARS-COV-2 infection and replication

ATP2B1 is a known regulator of calcium (Ca2+) cellular export and homeostasis. Diminished levels of intracellular Ca2+ content have been suggested to impair SARS-CoV-2 replication. Here, we demonstrate that a nontoxic caloxin-derivative compound (PI-7) reduces intracellular Ca2+ levels and impairs SARS-CoV-2 infection. Furthermore, a rare homozygous intronic variant of ATP2B1 is shown to be associated with the severity of COVID-19. The mechanism of action during SARS-CoV-2 infection involves the PI3K/Akt signaling pathway activation, inactivation of FOXO3 transcription factor function, and subsequent transcriptional inhibition of the membrane and reticulum Ca2+ pumps ATP2B1 and ATP2A1, respectively. The pharmacological action of compound PI-7 on sustaining both ATP2B1 and ATP2A1 expression reduces the intracellular cytoplasmic Ca2+ pool and thus negatively influences SARS-CoV-2 replication and propagation. As compound PI-7 lacks toxicity in vitro, its prophylactic use as a therapeutic agent against COVID-19 is envisioned here.

Erythrocyte Disorders Mimicking Congenital Dyserythropoietic Anemia Based on Bone Marrow Pathology Exposed By Genetic Evaluation

Congenital dyserythropoietic anemias (CDAs) are a rare group of heterogeneous genetic diseases characterized by hemolysis, ineffective erythropoiesis predisposing to iron overload, and the pathologic finding of bi- and multinucleated erythroblasts in the bone marrow (BM). With the identification of causative genetic mutations over the last two decades, the current classification includes the following types with the causative gene in parenthesis: Ia ( CDAN1), Ib ( CDIN1); II ( SEC23B); IIIa ( KIF23), IIIb ( RACGAP1); IV ( KLF1) ; and XLTD ( GATA1). Clinical presentation can vary widely even within the same type, ranging from mild cases of anemia to severe cases in which patients are subject to lifelong transfusion dependency. The CDA registry (CDAR) in North America (NCT02964494) was established to allow natural history studies on patients with CDA. Participants without a genetic diagnosis are offered whole genome sequencing (WGS) for patients and parents and may elect to provide samples to the CDAR biorepository to support collaborative mechanistic studies. Currently 169 subjects from 73 different families are enrolled in CDAR (85 patients, 84 unaffected family members). After genetic evaluation, 13 patients were found to be affected not by CDA, but rather by other causes of hereditary hemolytic anemia with secondary dyserythropoiesis. Three patients were diagnosed with pyruvate kinase deficiency (PKD). Patient (pt) 1 had a history of chronic anemia requiring sporadic transfusions since birth and was diagnosed with CDA-II based on BM studies. WGS revealed that the patient was heterozygous for a known PKLR pathogenic variant c.1022G>C p.(Gly341Ala)and for a novel variant of uncertain significance (c.695-3C>G).PK activity assay confirmed the diagnosis of PKD and the patient is now treated with the PK activator mitapivat. Pt 2 had severe transfusion dependent anemia since birth and iron overload. The patient underwent splenectomy at the age of 4 with mild improvement of symptoms. He was diagnosed with CDA II based on BM pathology. Genetic testing was performed and he was found to be compound heterozygous for 2 pathogenic variants in PKLR: c.644dup p.(Arg216fs) and c.994_1003dup p.(Val335fs). Pt 3 had a history of anemia and jaundice since birth; she has not had frequent transfusions but was splenectomized at age 17. The patient was found to be homozygous for c.1436G>A p.(Arg510Gln), a known pathogenic variant in PKLR. Four patients were diagnosed with HBB pathogenic variants causing frameshift and transcription of an elongated β-globin chain, i.e. unstable hemoglobinopathy. Pt 4 was first diagnosed with CDA II at 12 years of age based on BM studies performed due to chronic hemolytic anemia. After enrolling in CDAR, WES revealed heterozygous HBB p.(Lys96Asnfs*63). Pts 5, 6, and 7 are all symptomatic participants from the same family (mother and children). The mother (Pt 5) had frequent transfusion requirement, misdiagnosed in childhood as CDA IV due to BM dyserythropoiesis and the finding of high fetal hemoglobin. Her children had a similar phenotype requiring episodic transfusions initially, transitioning to scheduled transfusions by 8 y.o. WES in pt 5 revealed heterozygous HBB c.348_349delinsG (Hb Grand Junction) with follow-up HBB sequencing confirming that the children also carry the same variant . Three more patients were diagnosed with SPTA1 variants causing RBC membrane disorders. Pt 8 was found to have homozygous c.4339-99C>T (alpha-LEPRA) and heterozygous c.4347G>T p.(Lys1449Asn) with a phenotype of hereditary spherocytosis. Pts 9 and 10 are sisters, who presented with chronic, transfusion-dependent anemia since birth. They were diagnosed with CDA-II based on binucleated erythroblasts in BM studies performed in early childhood. They had splenectomy at 5 years of age and became transfusion-independent but continued to have mild anemia and reticulocytosis. WGS performed for them and their parents revealed a novel homozygous intronic variant in SPTA1 c.5834-18A>G. Phenotypic analysis was compatible with hereditary spherocytic pyropoikilocytosis. Hemolytic anemias with stress erythropoiesis may mimic the clinical presentation and pathologic findings of CDAs, making the phenotypic diagnosis challenging. Including genetic evaluation in the diagnostic workup of these patients can improve accuracy, timing of diagnosis, and management by specific therapy, if available.

A Novel Leptin Receptor LEPR Variant in a Toddler With Early-Onset Fatal Obesity.

Monogenic obesity generally results in severe early-onset obesity associated with abnormal feeding behavior and endocrine disorders. We report here an extremely severe case of early-onset obesity associated with hyperphagia in an 11-month-old boy without other signs of a syndromic obesity. He developed severe obstructive sleep apnea, dyslipidemia, hepatic steatosis with cytolysis, and acanthosis nigricans with insulin resistance in the first months of life. Laboratory investigations showed an elevated serum leptin level (80.03 ng/mL, normal range 2.45-6.55 ng/mL). Next-generation sequencing of obesity genes panel identified a novel homozygous intronic variant in leptin receptor gene (LEPR), c.703 + 5G>A, predicting affected splicing that resulted in a frameshift, premature stop, and truncation of the protein beyond the cytokine receptor homology domain 1. The child died at 27 months of age in the absence of available specific drug therapy.

Identification and functional characterization of a novel homozygous intronic variant in the fumarylacetoacetate hydrolase gene in a Chinese patient with tyrosinemia type 1

BackgroundHereditary tyrosinemia type 1 (HT1; OMIM# 276700) is a genetic metabolism disorder caused by disease-causing variants in the fumarylacetoacetate hydrolase (FAH) gene encoding the last enzyme of the tyrosine catabolic pathway. Herein, we describe the clinical features and genetic characteristics of HT1 in a five years and seven months old Chinese patient.MethodsAfter clinical diagnosis of the proband with HT1, genetic testing was performed by Sanger sequencing of the FAH gene in all family members. Functional analysis of the disease-causing variant was performed by cDNA sequencing to understand the effect of the variant on FAH transcript. To further predict the variant effect, we used Human Splicing Finder (HSF) and PyMol in silico analysis.ResultsWe identified a novel previously undescribed intronic variant in the FAH gene (c.914-1G>A). It was detected in a child who was homozygous for the variant and had the clinical presentation of HT1. cDNA sequencing showed that this splice-junction variant affected the transcription of FAH by formation of two different transcripts. Our observations and laboratory experiments were in line with in silico methods.ConclusionsOur study provides new insight into the HT1 variant spectrum and a better understanding of this disease in the Chinese population. This will be useful for molecular diagnosis in our country in cases where premarital screening, prenatal diagnosis and preimplantation genetic diagnosis are planned.

Expanding the genetic spectrum of the pyruvate carboxylase deficiency with novel missense, deep intronic and structural variants

IntroductionPathogenic variants in the pyruvate carboxylase (PC) gene cause a wide spectrum of recessive phenotypes, ranging from the early-onset fatal encephalopathy to the adult-onset benign form. ResultsPatient 1 is a 6 y.o. boy with ataxia, hypoglycemia and episodes of lactic acidosis. WGS revealed the novel heterozygous missense variant c.1372A > G (p.Asn458Asp) in the PC gene. Additional analysis revealed discordant reads mapped to chromosomes 11 and 1, so a reciprocal translocation disrupted the PC gene was suspected. The translocation was validated via FISH-analysis and Sanger sequencing of its boundaries.Patient 2 is a 13 y.o. girl with psychomotor delay, episodes of lactic acidosis and ketonuria. WES revealed the novel homozygous intronic variant c.1983-116C > T. The PC's mRNA analysis demonstrated the exonization of several intron 16 sequences and some residual amount of WT mRNA isoform.Two other patients had more severe course of the disease. Their genotype represents missense variants in compound heterozygous and homozygous state (c.1876C > T (p.Arg626Trp), c.2606G > C (p.Gly869Ala), c.2435C > A (p.Ala812Asp). ConclusionIn patients with metabolic crises, lactic acidosis and hypoglycemia analysis of PC gene is recommended. WGS with deep bioinformatic analysis should be taken into consideration when none or the only one pathogenic variant in the PC gene is found.

A novel leaky splice variant in centromere protein J (CENPJ)-associated Seckel syndrome.

Primary microcephaly and Seckel syndrome are rare genetically and clinically heterogenous brain development disorders. Several exonic/splicing mutations are reported for these disorders to date, but ∼40% of all cases remain unexplained. We aimed to uncover the genetic correlate(s) in a family of multiple siblings with microcephaly. A novel homozygous intronic variant (NC_000013.10:g.25459823T>C) in CENPJ (13q12) segregating with all four affected male siblings was identified by exome sequencing and validated by targeted linkage approach (logarithm of the odds score 1.8 at θ 0.0). RT-PCR of CENPJ in affected siblings using their EBV derived cell lines showed aberrant transcripts suggestive of exon skipping confirmed by Sanger sequencing. Significantly reduced wild type transcript/protein in the affected siblings having the splice variant indicates a leaky gene expression of pathological relevance. Based on known CENPJ function, assessing for mitotic alterations revealed defect in centrosome duplication causing mono/multicentrosome(s) at prophase, delayed metaphase, and unequal chromosomal segregation in patient cells. Clinical features witnessed in this study expand the spectrum of CENPJ-associated primary microcephaly and Seckel syndrome. Furthermore, besides the importance of regulatory variants in classical monogenic disorders these findings provide new insights into splice site biology with possible implications for ASO-based therapies.

A splice site and copy number variant responsible for TTC25-related primary ciliary dyskinesia

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder of motile cilia. With few exceptions, PCD is an autosomal recessive condition, and there are over 40 genes associated with the condition. We present a case of a newborn female with clinical features of PCD, specifically the Kartagener syndrome phenotype, due to variants in TTC25. This gene has been previously associated with PCD in three families. Two multi-gene panels performed as a neonate and at two years of age were uninformative. Exome sequencing was performed by the Care4Rare Canada Consortium on a research basis, and an apparent homozygous intronic variant (TTC25:c.1145+1G > A) was identified that was predicted to abolish the canonical splice donor activity of exon 8. The child's mother was a heterozygous carrier of the variant. The paternal sample did not show the splice variant, and homozygosity was observed across the paternal locus. Microarray analysis showed a 50 kb heterozygous deletion spanning the genes TTC25 and CNP. This is the first example of a pathogenic gross deletion in trans with a splice variant, resulting in TTC25-related PCD.

Intronic variant in POU1F1 associated with canine pituitary dwarfism

The anterior pituitary gland secretes several endocrine hormones, essential for growth, reproduction and other basic physiological functions. Abnormal development or function of the pituitary gland leads to isolated or combined pituitary hormone deficiency (CPHD). At least 30 genes have been associated with human CPHD, including many transcription factors, such as POU1F1. CPHD occurs spontaneously also in mice and dogs. Two affected breeds have been reported in dogs: German Shepherds with a splice defect in the LHX3 gene and Karelian Bear Dogs (KBD) with an unknown genetic cause. We obtained samples from five KBDs presenting dwarfism and abnormal coats. A combined analysis of genome-wide association and next-generation sequencing mapped the disease to a region in chromosome 31 and identified a homozygous intronic variant in the fourth exon of the POU1F1 gene in the affected dogs. The identified variant, c.605-3C>A, resided in the splice region and was predicted to affect splicing. The variant's screening in three new prospective cases, related breeds, and ~ 8000 dogs from 207 breeds indicated complete segregation in KBDs with a carrier frequency of 8%, and high breed-specificity as carriers were found at a low frequency only in Lapponian Herders, a related breed. Our study establishes a novel canine model for CPHD with a candidate POU1F1 defect.

A Novel Homozygous Intronic Variant in TNNT2 Associates With Feline Cardiomyopathy.

BackgroundHypertrophic cardiomyopathy (HCM) is a genetic disease of the heart and the most common cause of sudden cardiac death in the young. HCM is considered a disease of the sarcomere owing to the large number of mutations in genes encoding sarcomeric proteins. The riddle lies in discovering how these mutations lead to disease. As a result, treatments to prevent and/or treat HCM are limited to invasive surgical myectomies or ablations. The A31P variant of cardiac myosin binding protein-C, encoded by MYBPC3, was found to be more prevalent in a cohort of Maine Coon cats with HCM. However, other mutations in MYBPC3 and MYH7 have also been associated with HCM in cats of other breeds. In this study, we expand the spectrum of genes associated with HCM in cats.ResultsNext Generation Whole Genome sequencing was performed using DNA isolated from peripheral blood of a Maine Coon with cardiomyopathy that tested negative for the MYBPC3 A31P variant. Through risk stratification of variants, we identified a novel, homozygous intronic variant in cardiac troponin T (TNNT2). In silico analysis of the variant suggested that it may affect normal splicing of exon 3 of TNNT2. Both parents tested heterozygous for the mutation, but were unaffected by the disease. Echocardiography analyses revealed that the proband had shown early onset congestive heart failure, which is managed with a treatment regime including ACE and aldosterone inhibitors.ConclusionIn summary, we are the first to demonstrate the association between TNNT2 mutations and HCM in felines, suggesting that this gene should be included in the testing panel of genes when performing genetic testing for HCM in cats.

Homozygous intronic variants in TPM2 cause recessively inherited Escobar variant of multiple pterygium syndrome and congenital myopathy

Pathogenic variants in TPM2 have been associated with a variable clinical spectrum, including congenital myopathies and distal arthrogryposis, all but one with dominant inheritance. We report the second case of recessively inherited TPM2-related Escobar variant of multiple pterygium syndrome and congenital myopathy in a patient from a consanguineous family. Ultra-structural examination of the biopsy revealed few cores/mini-cores and sparse nemaline rods. We found a novel homozygous intronic sequence variant, c.564–2A>C in TPM2. This variant is predicted to abolish the consensus acceptor splice site for exon 6b of TPM2 gene. Parents of the proband, both healthy adults with no clinical features, were heterozygous for the variant. Here we establish a homozygous intronic variant in TPM2 as the likely cause of Escobar variant of multiple pterygium syndrome and congenital myopathy, with sparse nemaline rods.

Whole-Exome Sequencing Identifies an Intronic Cryptic Splice Site in SERPINF1 Causing Osteogenesis Imperfecta Type VI.

ABSTRACTThe heritable disorder osteogenesis imperfecta (OI) is characterized by bone fragility and low bone mass. OI type VI is an autosomal recessive form of the disorder with moderate to severe bone fragility. OI type VI is caused by mutations in the serpin peptidase inhibitor, clade F, member 1 (SERPINF1), the gene coding for pigment epithelium‐derived factor (PEDF). Here, we report a patient with OI type VI caused by a novel homozygous intronic variant in SERPINF1 identified by whole‐exome sequencing (WES). The mutation was not identified using a low bone mass gene panel based on next‐generation sequencing. This variant creates a novel consensus splice donor site (AGGC to AGGT) in intron 4. Analysis of cDNA generated from fibroblasts revealed retention of a 32‐bp intronic fragment between exons 4 and 5 in the cDNA, a result of alternative splicing from the novel splice‐donor site. As a result, the aberrant insertion of this intronic fragment generated a frameshift pathogenic variant and induced nonsense‐mediated decay. Furthermore, gene expression by quantitative PCR showed SERPINF1 expression was dramatically reduced in patient fibroblasts, and PEDF level was also significantly reduced in the patient's plasma. In conclusion, we report a novel homozygous variant that generates an alternative splice‐donor in intron 4 of SERPINF1 which gives rise to severe bone fragility. The work also demonstrates clinical utility of WES analysis, and consideration of noncoding variants, in the diagnostic setting of rare bone diseases. © 2018 The Authors. JBMR Plus is published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Idiopathic Superficial Thrombophlebitis and the Incidence of Cancer in Primary Care Patients

The association of spontaneous venous thromboembolism with occult malignancy is well established. Less clear is the incidence of subsequent cancer in patients with superficial thrombophlebitis. We wanted to determine the incidence of cancer after an episode of spontaneous superficial thrombophlebitis in a large general practice population. The objective of this study was to assess the incidence of newly diagnosed malignancies in patients within 2 years after the diagnosis of a spontaneous episode of superficial thrombophlebitis and to compare this incidence with nonexposed matched control patients and the Dutch population. The patients and their controls were identified by a search in the electronic patient records of 5 primary health care centers in Amsterdam, the Netherlands. A standardized morbidity ratio was calculated using data of the Dutch cancer registry. A total number of 277 patients with superficial thrombophlebitis were identified, of which 250 patients had no cancer at study entry. In 5 of these 250 patients (2%; 95% confidence interval [CI], 1%-5%), a new malignancy was diagnosed within 2 years after their superficial thrombophlebitis compared with 2% (95% CI, 1%-4%) in the control group. The standardized morbidity ratio was 1.1 (95% CI, 0.5-2.7). A recurrent episode of superficial thrombophlebitis was observed in 18 of the 250 patients, and in 1 patient cancer was diagnosed within 24 months after the first episode of superficial thrombophlebitis. We conclude that a single episode of unprovoked superficial thrombophlebitis diagnosed by a family physician is not associated with an increased risk of subsequent cancer.