Unraveling synonymous and deep intronic variants causing aberrant splicing in two genetically undiagnosed epilepsy families

Abstract Background Variants identified through parent–child trio-WES yield up to 28–55% positive diagnostic rate across a variety of Mendelian disorders, there remain numerous patients who do not receive a genetic diagnosis. Studies showed that some aberrant splicing variants, which are either not readily detectable by WES or could be miss-interpreted by regular detecting pipelines, are highly relevant to human diseases. Methods We retrospectively investigated the negative molecular diagnostics through trio-WES for 15 genetically undiagnosed patients whose clinical manifestations were highly suspected to be genetic disorders with well-established genotype–phenotype relationships. We scrutinized the synonymous variants from WES data and Sanger sequenced the suspected intronic region for deep intronic variants. The functional consequences of variants were analyzed by in vitro minigene experiments. Results Here, we report two abnormal splicing events, one of which caused exon truncating due to the activation of cryptic splicing site by a synonymous variant; the other caused partial intron retention due to the generation of splicing sites by a deep intronic variant. Conclusions We suggest that, despite initial negative genetic test results in clinically highly suspected genetic diseases, the combination of predictive bioinformatics and functional analysis should be considered to unveil the genetic etiology of undiagnosed rare diseases.

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Legal restrictions on pre-implantation genetic diagnostics in the framework of in vitro fertilization procedure: foreign experience and Russian perspective

10.21516/2413-1458-2019-18-2-162-168 ◽

2019 ◽

Author(s):

N.A. Altinnik , S.S. Zenin , V.V. Komarova et all

Keyword(s):

In Vitro Fertilization ◽

Assisted Reproductive Technologies ◽

Genetic Diseases ◽

Genetic Diagnosis ◽

Reproductive Technologies ◽

Legal Regulation ◽

Genetic Diagnostics ◽

Vitro Fertilization ◽

Medical Indications

The article discusses the factors that determine the content of the legal limitations of pre-implantation genetic diagnosis in the framework of the in vitro fertilization procedure, taking into account international experience and modern domestic regulatory legal regulation of the field of assisted reproductive technologies. The authors substantiates the conclusion that it is necessary to legislate a list of medical indications for preimplantation genetic diagnosis, as well as the categories of hereditary or other genetic diseases diagnosed in the framework of this procedure.

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Key diagnostic markers for autoimmune lymphoproliferative syndrome with molecular genetic diagnosis

Blood ◽

10.1182/blood.2020005486 ◽

2020 ◽

Vol 136 (17) ◽

pp. 1933-1945

Author(s):

Emese Molnár ◽

Nesrine Radwan ◽

Gábor Kovács ◽

Hajnalka Andrikovics ◽

Frances Henriquez ◽

...

Keyword(s):

Fas Ligand ◽

Molecular Genetic ◽

Group Identification ◽

Genetic Diagnosis ◽

Clinical Manifestations ◽

Functional Test ◽

Enzyme Linked Immunosorbent Assay ◽

Autoimmune Lymphoproliferative Syndrome ◽

Lymphoproliferative Syndrome

Abstract Autoimmune lymphoproliferative syndrome (ALPS) is a rare immunodeficiency caused by mutations in genes affecting the extrinsic apoptotic pathway (FAS, FASL, CASP10). This study evaluated the clinical manifestations, laboratory findings, and molecular genetic results of 215 patients referred as possibly having ALPS. Double-negative T-cell (DNT) percentage and in vitro apoptosis functional tests were evaluated by fluorescence-activated cell sorting; interleukin 10 (IL-10) and IL-18 and soluble FAS ligand (sFASL) were measured by enzyme-linked immunosorbent assay. Genetic analysis was performed by next-generation sequencing. Clinical background data were collected from patients’ records. Patients were categorized into definite, suspected, or unlikely ALPS groups, and laboratory parameters were compared among these groups. Of 215 patients, 38 met the criteria for definite ALPS and 17 for suspected ALPS. The definite and suspected ALPS patient populations showed higher DNT percentages than unlikely ALPS and had higher rates of lymphoproliferation. Definite ALPS patients had a significantly more abnormal in vitro apoptosis function, with lower annexin, than patients with suspected ALPS (P = .002) and patients not meeting ALPS criteria (P < .001). The combination of elevated DNTs and an abnormal in vitro apoptosis functional test was the most useful in identifying all types of ALPS patients; the combination of an abnormal in vitro apoptosis functional test and elevated sFASLs was a predictive marker for ALPS-FAS group identification. Lymphoproliferation, apoptosis functional test, and DNTs are the most sensitive markers; elevated IL-10 and IL-18 are additional indicators for ALPS. The combination of elevated sFASLs and abnormal apoptosis function was the most valuable prognosticator for patients with FAS mutations.

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Potential usefulness of preimplantation genetic diagnosis in the control and prevention of genetic diseases

Eastern Mediterranean Health Journal ◽

10.26719/1999.5.6.1134 ◽

1999 ◽

Vol 5 (6) ◽

pp. 1134-1139

Author(s):

M. A. El Hazmi

Keyword(s):

Prenatal Diagnosis ◽

Preimplantation Genetic Diagnosis ◽

Genetic Disorder ◽

Genetic Diseases ◽

Genetic Diagnosis ◽

Potential Usefulness ◽

Vitro Fertilization ◽

Polar Bodies ◽

Control And Prevention

Prenatal diagnosis of molecular mutations can be of immense value, since diagnosis followed by genetic counselling provides the most appropriate approach to genetic diseases control and prevention. However, ethical, psychosocial and religious considerations hamper adoption of prenatal diagnosis in communities where termination of a pregnancy may not be acceptable. Recently, preimplantation genetic diagnosis has attracted considerable interest. This involves in vitro fertilization, followed by genetic disorder diagnosis using polar bodies or cells extracted from a blastomere stage. The normal blastomere is implanted in the womb and pregnancy proceeds naturally. If an abnormality is diagnosed, the blastomere is not implanted, thus preventing pregnancy with the affected fetus. This paper outlines the potential usefulness of preimplantation genetic diagnosis in the control and prevention of genetic disease in our part of the world

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MITF p.Arg217Thr Variant Identified in a Han Chinese Family with Tietz/Waardenburg Syndrome

BioMed Research International ◽

10.1155/2021/4381272 ◽

2021 ◽

Vol 2021 ◽

pp. 1-6

Author(s):

Rong Yu ◽

Lv Liu ◽

Ya-Li Li ◽

Liang-Liang Fan

Keyword(s):

Hearing Loss ◽

Genetic Disorders ◽

Molecular Genetic ◽

Genetic Diagnosis ◽

Han Chinese ◽

White Hair ◽

Chinese Family ◽

Waardenburg Syndrome ◽

Genetic Lesion

Waardenburg syndrome (WS) is a group of rare genetic disorders characterized by hearing loss, changes in coloring of hair, skin, and eyes, and alterations in the shape of the face. Tietz syndrome is another rare disorder which presented similar phenotypes to WS. Patients with Tietz/Waardenburg syndrome often present with pale blue eyes, albino skin, and distinctive hair coloring, such as a patch of white hair or hair that prematurely turns gray. At present, more than six candidate genes are responsible for four types of Waardenburg syndrome and Tietz syndrome. This study is aimed at identifying the pathogenic gene variants in a three-generation Han Chinese family with hearing loss, blue-gray iris, albino skin, and white hair. In order to discover the molecular genetic lesion underlying the disease phenotype, whole exome sequencing in the proband, with Tietz/Waardenburg syndrome phenotypes, of a Han Chinese family from HeBei, China, was conducted. A novel heterozygous c.650G>C/p.Arg217Thr variant in melanocyte inducing transcription factor (MITF) was identified. Sanger sequencing further validated that this mutation existed in three affected individuals and absent in healthy family members. Bioinformatics analysis predicted that this mutation was deleterious. Our study further identified the genetic lesion of the family. Simultaneously, our study may also contribute to genetic counseling, embryonic screening of in vitro fertilized embryos, and prenatal genetic diagnosis of patients with Tietz/Waardenburg syndrome, especially for the proband, unmarried and unpregnant women, to reduce familial transmission in this Han Chinese family.

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High allele frequency of the p.Q258X mutation and identification of a novel mis-splicing mutation in the STAR gene in Korean patients with congenital lipoid adrenal hyperplasia

Acta Endocrinologica ◽

10.1530/eje-11-0597 ◽

2011 ◽

Vol 165 (5) ◽

pp. 771-778 ◽

Cited By ~ 22

Author(s):

Jae-Min Kim ◽

Jin-Ho Choi ◽

Jung Hyun Lee ◽

Gu-Hwan Kim ◽

Beom Hee Lee ◽

...

Keyword(s):

Intron Retention ◽

Mrna Level ◽

Site Directed Mutagenesis ◽

Adrenal Hyperplasia ◽

Aberrant Splicing ◽

Korean Patients ◽

Compound C ◽

Congenital Lipoid Adrenal Hyperplasia ◽

Steroidogenic Acute Regulatory

ObjectiveSteroidogenic acute regulatory (STAR) protein plays a crucial role in steroidogenesis, and mutations in the STAR gene cause congenital lipoid adrenal hyperplasia (CLAH). This study investigated the STAR mutation spectrum and functionally analyzed a novel STAR mutation in Korean patients with CLAH.MethodsMutation analysis of STAR was carried out in 25 unrelated Korean CLAH patients. A region of STAR comprising exons 4–7 was cloned from human genomic DNA into an expression vector, followed by site-directed mutagenesis and transient expression in COS7 cells. The splicing pattern was analyzed by in vitro transcription, and each transcript was functionally characterized by measuring pregnenolone production in COS7 cells cotransfected with the cholesterol side chain cleavage system.ResultsMutation p.Q258X was identified in 46 of 50 alleles (92%); mutation c.653C>T was detected in two alleles (4%); and mutations p.R182H and c.745–6_810del were found in one allele (2%). Reverse transcriptase-PCR products amplified from a patient heterozygous for compound c.653C>T and c.745–6_810del mutation revealed multiple alternatively spliced mRNAs. In vitro expression analysis of a minigene consisting of exons 4–7 containing the c.653C>T yielded two transcripts in which exon 6 or exons 5 and 6 were skipped. The encoded proteins exhibited defective pregnenolone-producing ability. The c.745–6_810del mutation led to full and partial intron retention.Conclusionsp.Q258X is the most common STAR mutation in Korea. A previously reported c.653C>T variant was found to cause aberrant splicing at the mRNA level, resulting in perturbation of STAR function. The c.745–6_810del mutation also resulted in aberrant splicing.

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Antisense Oligonucleotide-Based Rescue of Aberrant Splicing Defects Caused by 15 Pathogenic Variants in ABCA4

International Journal of Molecular Sciences ◽

10.3390/ijms22094621 ◽

2021 ◽

Vol 22 (9) ◽

pp. 4621

Author(s):

Tomasz Z. Tomkiewicz ◽

Nuria Suárez-Herrera ◽

Frans P. M. Cremers ◽

Rob W. J. Collin ◽

Alejandro Garanto

Keyword(s):

Antisense Oligonucleotide ◽

Mrna Splicing ◽

Stargardt Disease ◽

Aberrant Splicing ◽

Suitable Candidate ◽

Pathogenic Variants ◽

Splice System ◽

Splicing Defects ◽

Intronic Variants

The discovery of novel intronic variants in the ABCA4 locus has contributed significantly to solving the missing heritability in Stargardt disease (STGD1). The increasing number of variants affecting pre-mRNA splicing makes ABCA4 a suitable candidate for antisense oligonucleotide (AON)-based splicing modulation therapies. In this study, AON-based splicing modulation was assessed for 15 recently described intronic variants (three near-exon and 12 deep-intronic variants). In total, 26 AONs were designed and tested in vitro using a midigene-based splice system. Overall, partial or complete splicing correction was observed for two variants causing exon elongation and all variants causing pseudoexon inclusion. Together, our results confirm the high potential of AONs for the development of future RNA therapies to correct splicing defects causing STGD1.

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The Power of Options: The Solution for Healthier Society and Better Economy

Research in Health Science ◽

10.22158/rhs.v2n4p350 ◽

2017 ◽

Vol 2 (4) ◽

pp. 350

Author(s):

Dr. Saleh A. S. AL-Abdulhadi

Keyword(s):

Reproductive Medicine ◽

Genetic Disorders ◽

Genetic Diagnosis ◽

Community Services ◽

Human Oocyte ◽

Genetic Composition ◽

Vitro Fertilization ◽

Genetic Abnormalities ◽

New Frontier

<p><em>Preimplantation Genetic Diagnosis (PGD) testing is the practice of obtaining a cellular biopsy sample from a developing human oocyte or embryo, acquired via a cycle of In Vitro Fertilization (IVF); evaluating the genetic composition of this sample; and using this information to determine which embryos will be optimal for subsequent uterine transfer. PGD has become an increasingly useful adjunct to IVF procedures. The ability to provide couples who are known carriers of genetic abnormalities the opportunity to deliver healthy babies has opened a new frontier in reproductive medicine. The purpose of the PGD is enables us to choose which embryos will be implanted into the mother. In the present study we investigate the frequency of application of this technique in the kingdom and GCC to find out, the population awareness of these technique and the advantages which could apply to the community. We also interested to know frequencies of centers and studies in this field. This epidemiological study helps to improve social awareness and community services toward reducing frequencies of genetic disorders. </em></p>

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Medically Assisted Procreation and International Human Rights Law

The Italian Yearbook of International Law Online ◽

10.1163/22116133-02201008 ◽

2013 ◽

Vol 22 (1) ◽

pp. 155-183

Author(s):

Ilja Richard Pavone

Keyword(s):

Human Rights ◽

Genetic Diseases ◽

Genetic Diagnosis ◽

Private Life ◽

In Vitro Fertilisation ◽

Special Focus ◽

International Human Rights Law ◽

Medically Assisted Procreation ◽

Assisted Procreation

Since the birth of Louise Brown in 1978, the first human baby resulting from in vitro fertilisation (IVF), developments in reproductive medicine have opened up new opportunities to solve problems related to sterility/infertility and to avoid the transmission of serious genetic diseases to offspring. This article evaluates some challenges to human rights protection arising from medically assisted procreation (MAP), with particular reference to artificial insemination from a donor (AID) and preimplantation genetic diagnosis (PGD). It analyses the regulation of MAP at the international, regional and domestic level. Specific attention is paid to two landmark judgments of the European Court of Human Rights (ECtHR) on MAP (S.H. v. Austria and Costa and Pavan v. Italy), with a special focus on the interpretation of the concept of family and private life contained therein and on the effects of the ECtHR rulings on the Italian legal order. It concludes that national legislation concerning MAP should be minimal, i.e. should afford substantial freedom and autonomy to the couples in their procreative choices, in accordance with their right to respect for private and family life.

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Aberrant Splicing Events Associated to CDH23 Noncanonical Splice Site Mutations in a Proband with Atypical Usher Syndrome 1

Genes ◽

10.3390/genes10100732 ◽

2019 ◽

Vol 10 (10) ◽

pp. 732 ◽

Cited By ~ 3

Author(s):

Rebeca Valero ◽

Marta de Castro-Miró ◽

Sofía Jiménez-Ochoa ◽

Juan José Rodríguez-Ezcurra ◽

Gemma Marfany ◽

...

Keyword(s):

Splice Site ◽

Usher Syndrome ◽

Functional Characterization ◽

Genetic Diagnosis ◽

Functional Evaluation ◽

Syndrome Type ◽

Intronic Variants ◽

Usher Syndrome Type

Aims: The aim of this study was the genetic diagnosis by next generation sequencing (NGS) of a patient diagnosed with Usher syndrome type 2 and the functional evaluation of the identified genetic variants to establish a phenotype–genotype correlation. Methods: Whole exome sequencing (WES) analysis identified two heterozygous intronic variants in CDH23, a gene responsible of Usher syndrome type 1. Evaluation of the putative splicing effects was performed in vivo, in whole blood samples, and in vitro, by transfection of midigene constructs in HEK293T cells. Results: Two intronic variants were identified in intron 45 of CDH23—one novel, c.6050-15G>A, and the other, c.6050-9G>A, already reported as a noncanonical splice site (NCSS) mutation—with partial functional characterization. In vivo and in vitro analyses showed aberrant transcripts by the addition of 13 and 7 nucleotides to exon 46, respectively. Transcript degradation by nonsense mediated decay (NMD) in blood cells could only be prevented by cycloheximide treatment. Midigene constructs showed that the two variants contributed to exon skipping and generated aberrantly spliced transcripts. Conclusions: A combination of in vivo and in vitro assays provided a comprehensive view of the physiological effects of NCSS variants, which in this case led to a clinical reassignment of the proband as affected with atypical USH1 syndrome.

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Loss of UGP2 in brain leads to a severe epileptic encephalopathy, emphasizing that bi-allelic isoform-specific start-loss mutations of essential genes can cause genetic diseases

Acta Neuropathologica ◽

10.1007/s00401-019-02109-6 ◽

2019 ◽

Vol 139 (3) ◽

pp. 415-442 ◽

Cited By ~ 6

Author(s):

Elena Perenthaler ◽

Anita Nikoncuk ◽

Soheil Yousefi ◽

Woutje M. Berdowski ◽

Maysoon Alsagob ◽

...

Keyword(s):

Developmental Delay ◽

Genetic Disorders ◽

Genetic Diseases ◽

Intractable Epilepsy ◽

Stem Cell Differentiation ◽

Visual Disturbance ◽

Epileptic Encephalopathy ◽

Start Codon

AbstractDevelopmental and/or epileptic encephalopathies (DEEs) are a group of devastating genetic disorders, resulting in early-onset, therapy-resistant seizures and developmental delay. Here we report on 22 individuals from 15 families presenting with a severe form of intractable epilepsy, severe developmental delay, progressive microcephaly, visual disturbance and similar minor dysmorphisms. Whole exome sequencing identified a recurrent, homozygous variant (chr2:64083454A > G) in the essential UDP-glucose pyrophosphorylase (UGP2) gene in all probands. This rare variant results in a tolerable Met12Val missense change of the longer UGP2 protein isoform but causes a disruption of the start codon of the shorter isoform, which is predominant in brain. We show that the absence of the shorter isoform leads to a reduction of functional UGP2 enzyme in neural stem cells, leading to altered glycogen metabolism, upregulated unfolded protein response and premature neuronal differentiation, as modeled during pluripotent stem cell differentiation in vitro. In contrast, the complete lack of all UGP2 isoforms leads to differentiation defects in multiple lineages in human cells. Reduced expression of Ugp2a/Ugp2b in vivo in zebrafish mimics visual disturbance and mutant animals show a behavioral phenotype. Our study identifies a recurrent start codon mutation in UGP2 as a cause of a novel autosomal recessive DEE syndrome. Importantly, it also shows that isoform-specific start-loss mutations causing expression loss of a tissue-relevant isoform of an essential protein can cause a genetic disease, even when an organism-wide protein absence is incompatible with life. We provide additional examples where a similar disease mechanism applies.

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