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Accumulation of mutant proteins in cells can induce proteinopathies and cause functional damage to organs. Recently, the Cingulin (CGN) protein has been shown to maintain the morphology of cuticular plates of inner ear hair cells and a frameshift mutation in CGN causes autosomal dominant non-syndromic hearing loss. Here, we find that the mutant CGN proteins form insoluble aggregates which accumulate intracellularly and lead to cell death. Expression of the mutant CGN in the inner ear results in severe hair cell death and hearing loss in mice, resembling the auditory phenotype in human patients. Interestingly, a human-specific residue (V1112) in the neopeptide generated by the frameshift mutation is critical for the aggregation and cytotoxicity of the mutant human CGN. Moreover, the expression of heat shock factor 1 (HSF1) decreases the accumulation of insoluble mutant CGN aggregates and rescues cell death. In summary, these findings identify mutant-specific toxic polypeptides as a disease-causing mechanism of the deafness mutation in CGN, which can be targeted by the expression of the cell chaperone response regulator HSF1., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest., (Copyright © 2024 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.)

Typical (or classic) Rett syndrome (RTT) is an X-linked neurodevelopmental disorder characterized by a period of regression, partial or complete loss of purposeful hand movements, and acquired speech, impaired gait, and stereotyped hand movements. In over 95% of typical RTT, a pathogenic variant is found in the methyl-CPG binding protein 2 gene (MECP2). Here, we describe a young woman with clinically diagnosed typical RTT syndrome who lacked a genetic diagnosis despite 20 years of investigation and multiple rounds of sequencing the MECP2 gene. Recently, additional genetic testing using next-generation sequencing was completed, which revealed a partial insertion of the BCL11A gene within exon 4 of MECP2, resulting in a small deletion in MECP2, causing likely disruption of MeCP2 function due to a frameshift. This case demonstrates the ever-changing limitations of genetic testing, as well as the importance of continual pursuit of a diagnosis as technologies improve and are more widely utilized., (© 2024 Wiley Periodicals LLC.)

We present the case of a 37-year-old woman who underwent bilateral penetrating keratoplasty for congenital hereditary endothelial dystrophy at the age of 10 years. Over the subsequent 27 years, the patient's vision slowly deteriorated. Our examination revealed decompensation of the right corneal graft. We addressed this with regraft surgery. We then learned that the patient had been suffering from progressive hearing loss since adolescence. Tonal audiometry revealed hearing per ceptive deafness of 25 dB, which was more prominent in the left ear. Because the patterns of progressive sensorineural hearing loss and congenital hereditary endothelial dystrophy have both been linked to the same gene, slc4a11, we tested our patient for mutations in this gene. The test was positive for a heterozygous slc4a11 gene fifth exon mutation on chromosome 20p13-p12, which causes a frameshift. A combined clinical and genetic evaluation confirmed a diagnosis of Harboyan syndrome. After the genetic diagnosis of the disease, she was evaluated for the need for a hearing aid due to her hearing loss. The patient was also informed about genetic counseling.

Brugada syndrome (BrS) is a rare autosomal dominant inherited channel disorder characterized by a specific electrocardiographic pattern of right precordial ST-segment elevation. Clinically, patients may experience polymorphic ventricular tachycardia and ventricular fibrillation, leading to recurrent syncope and sudden cardiac death (SCD) in the absence of structural cardiomyopathy. The A-kinase anchor protein 9 (AKAP9) gene, located on chromosome 7, encodes the AKAP9 protein, which plays a crucial role in regulating the phosphorylation of slowly activating delayed rectifier potassium channels (IKs). Here, we present a rare case of BrS associated with an insertion mutation in AKAP9, resulting in a frameshift mutation., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Filippi syndrome is a rare genetic disorder characterized by growth and neurodevelopmental delays, dysmorphism, and selective limb abnormalities. Although the syndrome was described approximately four decades ago, only a few families with molecularly confirmed diagnoses have been reported. In this article, we present three new patients of Filippi syndrome with unusual clinical and genetic aspects. These patients exhibited novel clinical features that have not previously been associated with Filippi syndrome, including renal hypoplasia/aplasia, renal cysts, renal cortical thinning, hypomelanotic, and hypermelanotic macules. All three patients had homozygous frameshift variants of the CKAP2L gene, specifically NM_152515.3: c.554_555del, c.981_982del, and c.1463_1467del, with the second being a novel variant. Given the limited number of reported Filippi syndrome patients to date and the ongoing discovery of new clinical aspects of the disease, exploring its potential connection with kidney and skin pigmentation abnormalities could be valuable for future research., (© 2024 Wiley Periodicals LLC.)

Background/objectives: Tooth agenesis (TA) is among the most common malformations in humans. Although several causative mutations have been described, the genetic cause often remains elusive. Here, we test whether whole genome sequencing (WGS) could bridge this diagnostic gap., Methods: In four families with TA, we assessed the dental phenotype using the Tooth Agenesis Code after intraoral examination and radiographic and photographic documentation. We performed WGS of index patients and subsequent segregation analysis., Results: We identified two variants of uncertain significance (a potential splice variant in PTH1R, and a 2.1 kb deletion abrogating a non-coding element in FGF7) and three pathogenic variants: a novel frameshift in the final exon of PITX2, a novel deletion in PAX9, and a known nonsense variant in WNT10A. Notably, the FGF7 variant was found in the patient, also featuring the WNT10A variant. While mutations in PITX2 are known to cause Axenfeld-Rieger syndrome 1 (ARS1) predominantly featuring ocular findings, accompanied by dental malformations, we found the PITX2 frameshift in a family with predominantly dental and varying ocular findings., Conclusion: Severe TA predicts a genetic cause identifiable by WGS. Final exon PITX2 frameshifts can cause a predominantly dental form of ARS1., (© 2023 The Authors. Oral Diseases published by Wiley Periodicals LLC.)

Marfan syndrome (MFS) is a hereditary systemic connective tissue disorder with great clinical variability. It is caused by heterozygous pathogenic variants in the FBN1 gene. Cardinal manifestations involve the cardiovascular, ocular, and skeletal systems. Clinical diagnosis is based on the revised Ghent nosology. We present the case of a child with a Marfan systemic score of 9 whose genetic study revealed two pathogenic mosaic frameshift variants in the FBN1 gene. Mosaicism is very rare in patients diagnosed with MFS, and this is the first description of a patient with two pathogenic mosaic variants in the FBN1 gene. Both variants are present in cells derived from ectodermal (buccal swab) and mesodermal (leukocyte) tissues, suggesting a mutation prior to gastrulation. We propose a defective repair of the de novo variant in the complementary strand as the mechanism that led this individual to be a carrier of two different populations of mutant cells carrying adjacent variants., (© 2024 Wiley Periodicals LLC.)

L-2-Hydroxyglutaric aciduria (L2HGA) is a rare, autosomal recessive neurometabolic disease, which presents with elevated L-2-hydroxyglutarate acid. Generally, L2HGA appear as slowly progressing central nervous system function deterioration during infancy, and a rapid progression in adulthood is uncommon for the syndrome's classic phenotype., (© 2024. The Author(s) under exclusive licence to Belgian Neurological Society.)

The mediator complex subunit 13 (MED13) gene is implicated in neurodevelopmental disorders including autism spectrum disorder (ASD), intellectual disability, and speech delay with varying severity and course. Additional, extra central nervous system, features include eye or vision problems, hypotonia, congenital heart abnormalities, and dysmorphisms. We describe a 7-year- and 4-month-old girl evaluated for ASD whose brain magnetic resonance imaging was suggestive of multiple cortical tubers. The exome sequencing (ES - trio analysis) uncovered a unique, de novo, frameshift variant in the MED13 gene (c.4880del, D1627Vfs*17), with a truncating effect on the protein. This case report thus expands the phenotypic spectrum of MED13-related disorders to include brain abnormalities., (© 2024 Wiley Periodicals LLC.)

Purpose: Oocyte maturation defect (OOMD) is a rare cause of in vitro fertilization failure characterized by the production of immature oocytes. Compound heterozygous or homozygous PATL2 mutations have been associated with oocyte arrest at the germinal vesicle (GV), metaphase I (MI), and metaphase II (MII) stages, as well as morphological changes., Methods: In this study, we recruited three OOMD cases and conducted a comprehensive multiplatform laboratory investigation., Results: Whole exome sequence (WES) revealed four diagnostic variants in PATL2, nonsense mutation c.709C > T (p.R237*) and frameshift mutation c.1486_1487delinsT (p.A496Sfs*4) were novel mutations that have not been reported previously. Furthermore, the pathogenicity of these variants was predicted using in silico analysis, which indicated detrimental effects. Molecular dynamic analysis suggested that the A496S variant disrupted the hydrophobic segment, leading to structural changes that affected the overall protein folding and stability. Additionally, biochemical and molecular experiments were conducted on cells transfected with wild-type (WT) or mutant PATL2 (p.R237* and p.A496Sfs*4) plasmid vectors., Conclusions: The results demonstrated that PATL2 A496Sfs*4 and PATL2 R237* had impacts on protein size and expression level. Interestingly, expression levels of specific genes involved in oocyte maturation and early embryonic development were found to be simultaneously deregulated. The findings in our study expand the variation spectrum of the PATL2 gene, provide solid evidence for counseling on future pregnancies in affected families, strongly support the application of in the diagnosis of OOMD, and contribute to the understanding of PATL2 function., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Mismatch repair (MMR)-deficient cancer evolves through the stepwise erosion of coding homopolymers in target genes. Curiously, the MMR genes MutS homolog 6 (MSH6) and MutS homolog 3 (MSH3) also contain coding homopolymers, and these are frequent mutational targets in MMR-deficient cancers. The impact of incremental MMR mutations on MMR-deficient cancer evolution is unknown. Here we show that microsatellite instability modulates DNA repair by toggling hypermutable mononucleotide homopolymer runs in MSH6 and MSH3 through stochastic frameshift switching. Spontaneous mutation and reversion modulate subclonal mutation rate, mutation bias and HLA and neoantigen diversity. Patient-derived organoids corroborate these observations and show that MMR homopolymer sequences drift back into reading frame in the absence of immune selection, suggesting a fitness cost of elevated mutation rates. Combined experimental and simulation studies demonstrate that subclonal immune selection favors incremental MMR mutations. Overall, our data demonstrate that MMR-deficient colorectal cancers fuel intratumor heterogeneity by adapting subclonal mutation rate and diversity to immune selection., (© 2024. The Author(s).)

To identify genetic alterations associated with tongue cancer recurrence in young adults, whole exome sequencing of the primary tumor, recurrence, and whole blood samples from young patients with tongue cancer was performed. A frameshift mutation in the TP53 gene was detected in the primary tumor and recurrence tumor tissue. A mutation in the EPHB6 gene was detected in the recurrence and was absent in the primary tumor. In addition, the primary tumor and recurrence tongue cancer tissue harbored amplification of the 20p13 region containing C20orf96, DEFB125, DEFB126, DEFB127, DEFB128, DEFB129, DEFB132, and ZCCHC3 genes. Thus, genetic alterations have been identified that are associated with tongue cancer recurrence in young adults., (© 2024. Springer Science+Business Media, LLC, part of Springer Nature.)

Hypertrophic cardiomyopathy (HCM) is frequently caused by mutations in the MYPBC3 gene, which encodes the cardiac myosin-binding protein C (cMyBP-C). Most pathogenic variants in MYPBC3 are either nonsense mutations or result in frameshifts, suggesting that the primary disease mechanism involves reduced functional cMyBP-C protein levels within sarcomeres. However, a subset of MYPBC3 variants are missense mutations, and the molecular mechanisms underlying their pathogenicity remain elusive. Upon in vitro differentiation into cardiomyocytes, induced pluripotent stem cells (iPSCs) derived from HCM patients represent a valuable resource for disease modeling. In this study, we generated two iPSC lines from peripheral blood mononuclear cells (PBMCs) of a female with early onset and severe HCM linked to the MYBPC3: c.772G > A variant. Although this variant was initially classified as a missense mutation, recent studies indicate that it interferes with splicing and results in a frameshift. The generated iPSC lines exhibit a normal karyotype and display hallmark characteristics of pluripotency, including the ability to undergo trilineage differentiation. These novel iPSCs expand the existing repertoire of MYPBC3-mutated cell lines, broadening the spectrum of resources for exploring how diverse mutations induce HCM. They additionally offer a platform to study potential secondary genetic elements contributing to the pronounced disease severity observed in this individual., (© 2024. The Author(s).)

PURA syndrome is a congenital developmental disorder caused by de novo mutations in the PURA gene, which encodes a DNA/RNA-binding protein essential for transcriptional and translational regulation. We present the case of an 11-year-old patient with a de novo frameshift variant in the PURA gene, identified through whole exome sequencing (WES). In addition to the classical PURA deficiency phenotype, our patient exhibited pronounced sialorrhea and seizures, which were effectively treated with the ketogenic diet (KD). Our integrative approach, combining a literature review and bioinformatics data, has led to the first documented clinical case showing improvement in both sialorrhea and seizures with KD treatment, a phenomenon not previously reported. Although a direct relationship between the de novo PURA mutation and the KD was not established, we identified a novel frameshift deletion associated with a new clinical phenotype.

Background: Familial hypercholesterolemia (FH) is a common inherited metabolic disease that causes premature atherosclerosis, cardiovascular disease, and even death at a young age. Approximately 95% of FH-causing genetic variants that have been identified are in the LDLR gene. However, only 10% of the FH population worldwide has been diagnosed and adequately treated, due to the existence of numerous unidentified variants, uncertainties in the pathogenicity scoring of many variants, and a substantial number of individuals lacking access to genetic testing., Objective: The aim of this study was to identify a novel variant in the LDLR gene that causes FH in a Chinese family, thereby expanding the spectrum of FH-causing variants., Methods: Patients were recruited from Beijing Anzhen Hospital, Capital Medical University. FH diagnosis was made according to the Dutch Lipid Clinical Network (DLCN) criteria. Whole-exome sequencing (WES) was conducted to identify the FH-causing variant in the proband, and amplicon sequencing was used to verify the variant in his family members., Results: A three-generation Chinese family was recruited, and two FH patients were clinically diagnosed, both without known FH-causing variants. These two FH patients and another possible patient carried a novel variant, NC_000019.9(NM_000527.5):c.89_92dup (NP_000518.1:p.Phe32Argfs*21), in the ligand-binding domain of the low-density lipoprotein (LDL) receptor that led to a frameshift. The FH adults in the family showed severe clinical symptoms and statin therapy resistance., Conclusion: This study identified a novel pathogenic LDLR variant, c.89_92dup, associated with severe FH clinical manifestations and statin therapy resistance., (© 2024. The Author(s).)

Silver-Russell syndrome (SRS) is a representative imprinting disorder characterized by pre- and postnatal growth failure. We encountered two Japanese SRS cases with a de novo pathogenic frameshift variant of HMGA2 (NM_003483.6:c.138_141delinsCT, p.(Lys46Asnfs*16)) and a de novo ~ 3.4 Mb microdeletion at 12q14.2-q15 involving HMGA2, respectively. Furthermore, we compared clinical features in previously reported patients with various genetic conditions leading to compromised IGF2 expression, i.e., HMGA2 aberrations, PLAG1 aberrations, IGF2 aberrations, and H19/IGF2:IG-DMR epimutations (hypomethylations). The results provide further support for HMGA2 being involved in the development of SRS and imply some characteristic features in patients with HMGA2 aberrations., (© 2024. The Author(s).)

Somatic variants in the NOTCH pathway regulator FBXW7 are frequently seen in a variety of malignancies. Heterozygous loss-of-function germline variants in FBXW7 have recently been described as causative for a neurodevelopmental syndrome. Independently, FBXW7 was also considered as a susceptibility gene for Wilms tumor due to a few observations of heterozygous germline variants in patients with Wilms tumor. Whether the same FBXW7 variants are implicated in both, neurodevelopmental delay and Wilms tumor formation, remained unclear. By clinical testing, we now observed a patient with neurodevelopmental delay due to a de novo constitutional mosaic FBXW7 splice site pathogenic variant who developed Wilms tumor. In the tumor, we identified a second hit frameshift variant in FBXW7. Immunohistochemical staining was consistent with mosaic loss of FBXW7 protein expression in the tumor. Our data support the role of constitutional FBXW7 pathogenic variants in both, neurodevelopmental disorder and the etiology of Wilms tumor. Therefore, Wilms tumor screening should be considered in individuals with constitutional or germline pathogenic variants in FBXW7 and associated neurodevelopmental syndrome., (© 2024 The Authors. American Journal of Medical Genetics Part A published by Wiley Periodicals LLC.)

Myogenic transcription factors with a basic helix-loop-helix (bHLH) such as MYOD, myogenin, MRF4, and MYF5 contribute to muscle differentiation and regulation. The MYF5 gene located on chromosome 12 encodes for myogenic factor 5 (MYF5), which has a role in skeletal and extraocular muscle development and rib formation. Variants in MYF5 were found to cause external ophthalmoplegia with rib and vertebral anomalies (EORVA), a rare recessive condition. To date, three homozygous variants in MYF5 have been reported to cause EORVA in six members of four unrelated families. Here, we present a novel homozygous MYF5 frameshift variant, c.596dupA p. (Asn199Lysfs*49), causing premature protein termination and presenting with external ophthalmoplegia, ptosis, and scoliosis in three siblings from a consanguineous family of Pakistani origin. With four MYF5 variants now discovered, genetic testing and paediatric assessment for extra-ocular features should be considered in all cases of congenital ophthalmoplegia.

Introduction: Approximately 80% of primary hyperoxaluria cases are caused by primary hyperoxaluria type 1 (PH1, OMIM# 259900), which is characterized by pathogenic variants in the AGXT gene, resulting in deficiency of the liver-specific enzyme alanine-glyoxylate aminotransferase (AGT). This leads to increased production of oxalate, which cannot be effectively eliminated from the body, resulting in its accumulation primarily in the kidneys and other organs. Subjects and Methods: This study included 17 PH1 Egyptian patients from 12 unrelated families, recruited from the Inherited Kidney Disease Outpatient Clinic and the Dialysis Units, Cairo University Hospitals, during the period from January 2018 to December 2019, aiming to identify the pathogenic variants in the AGXT gene. Results: Six different variants were detected. These included three frameshift and three missense variants, all found in homozygosity within the respective families. The most common variant was c.121G>A;p.(Gly41Arg) detected in four families, followed by c.725dup;p.(Asp243GlyfsTer12) in three families, c.33dup;p.(Lys12Glnfs156) in two families, and c.731T >C;p.(Ile244Thr), c.33delC;p.(Lys12Argfs34), and c.568G>A;p.(Gly190Arg) detected in one family each. Conclusion: Consanguineous Egyptian families with history of renal stones or renal disease suspicious of primary hyperoxaluria should undergo AGXT genetic sequencing, specifically targeting exons 1 and 7, as variants in these two exons account for >75% of disease-causing variants in Egyptian patients with confirmed PH1.

Amelogenesis imperfecta (AI) represents a group of clinically and genetically heterogeneous disorders that affect enamel formation and mineralization. Although AI is commonly considered a monogenic disorder, digenic inheritance is rarely reported. In this study, we recruited two nonconsanguineous Chinese families exhibiting diverse phenotypes of enamel defects among affected family members. Digenic variants were discovered in both probands. In family 1, the proband inherited a paternal frameshift variant in LAMA3 (NM_198129.4:c.3712dup) and a maternal deletion encompassing the entire AMELX gene. This resulted in a combined hypoplastic and hypomineralized AI phenotype, which was distinct from the parents' manifestations. In family 2, whole-exome sequencing analysis revealed the proband carried a maternal heterozygous splicing variant in COL17A1 (NC_000010.11 (NM_000494.3): c.4156 + 2dup) and compound heterozygous variants in RELT (paternal: NM_032871.4:c.260A > T; maternal: NM_032871.4:c.521 T > G). These genetic changes caused the abundant irregular enamel defects observed in the proband, whereas other affected family members carrying heterozygous variants in both COL17A1 and RELT displayed only horizontal grooves as their phenotype. The pathogenicity of the novel COL17A1 splice site variant was confirmed through RT-PCR and minigene assay. This study enhances our understanding by highlighting the potential association between the co-occurrence of variants in two genes and variable phenotypes observed in AI patients., (© 2023 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Objectives: Oculo-facio-cardio-dental (OFCD) syndrome is a rare X-linked genetic disorder caused by mutations in the BCL6 co-repressor (BCOR) and is mainly characterized by radiculomegaly (elongated dental roots). All BCOR mutations reported to date have been associated with premature termination codons, indicating that nonsense-mediated mRNA decay (NMD) might play a vital role in the pathogenesis of OFCD syndrome. However, the molecular mechanisms underlying NMD remain unclear. In this study, we investigated the involvement of up-frameshift protein 1 (UPF1), which plays a central role in NMD, in the hyperactive root formation caused by BCOR mutations., Methods: Periodontal ligament cells, isolated from a Japanese woman with a c.3668delC frameshift mutation in BCOR, and primary human periodontal ligament fibroblasts (HPdLFs) were used for an RNA immunoprecipitation assay to confirm the binding of UPF1 to mutated BCOR. Additionally, the effects of UPF1 on the BCOR transcription levels and corresponding gene expression were determined by performing relative quantitative real-time polymerase chain reactions., Results: RNA immunoprecipitation revealed that UPF1 binds to exon 9 of mutated BCOR. Additionally, UPF1 knockdown via siRNA upregulated the transcription of BCOR, whereas overexpression of wild-type and mutated BCOR with the same frameshift mutation in HPdLFs altered bone morphogenetic protein 2 (BMP2) expression., Conclusions: Our findings indicate that BCOR mutations regulate the transcription of BCOR via UPF1, which may in turn regulate the expression of BMP2. NMD, caused by a c.3668delC mutation, potentially leads to an OFCD syndrome phenotype, including elongated dental roots., Competing Interests: Declaration of competing interest The authors declare that no conflicts of interest exist., (Copyright © 2024 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.)

The synthesis of proteins as encoded in the genome depends critically on translational fidelity. Nevertheless, errors inevitably occur, and those that result in reading frame shifts are particularly consequential because the resulting polypeptides are typically nonfunctional. Despite the generally maladaptive impact of such errors, the proper decoding of certain mRNAs, including many viral mRNAs, depends on a process known as programmed ribosomal frameshifting. The fact that these programmed events, commonly involving a shift to the -1 frame, occur at specific evolutionarily optimized "slippery" sites has facilitated mechanistic investigation. By contrast, less is known about the scope and nature of error (i.e., nonprogrammed) frameshifting. Here, we examine error frameshifting by monitoring spontaneous frameshift events that suppress the effects of single base pair deletions affecting two unrelated test proteins. To map the precise sites of frameshifting, we developed a targeted mass spectrometry-based method called "translational tiling proteomics" for interrogating the full set of possible -1 slippage events that could produce the observed frameshift suppression. Surprisingly, such events occur at many sites along the transcripts, involving up to one half of the available codons. Only a subset of these resembled canonical "slippery" sites, implicating alternative mechanisms potentially involving noncognate mispairing events. Additionally, the aggregate frequency of these events (ranging from 1 to 10% in our test cases) was higher than we might have anticipated. Our findings point to an unexpected degree of mechanistic diversity among ribosomal frameshifting events and suggest that frameshifted products may contribute more significantly to the proteome than generally assumed., Competing Interests: Competing interests statement:J.W.H. is a consultant and founder of Caraway Therapeutics and is a founding board member of Interline Therapeutics. Other authors declare no competing interests.

KDM4B (MIM*609765, NM_015015.3, formerly JMJD2B) encodes a histone demethylase and regulates gene expression via demethylation, mainly of H3K9 tri-methylation. Heterozygous KDM4B loss-of-function variants cause autosomal dominant intellectual developmental disorder 65 (MIM#619320), which is characterized by global developmental delay, intellectual disability, language and gross motor delays, structural brain anomalies, characteristic facial features, and clinodactyly. Although the majority of reported patients have de novo pathogenic variants, some patients inherit pathogenic variants from affected parents. To our knowledge, only 23 patients with heterozygous KDM4B variants have been reported to date, and there are no reports of patients with biallelic KDM4B pathogenic variants. Herein, we report a female patient with a biallelic KDM4B frameshift variant (NM_015015.3: c.1384_1394delinsGGG, p.(Leu462Glyfs*43)) located at exon 12 of 23 protein-coding exons, which is thought to be subject to nonsense-mediated mRNA decay and no protein production. She presented developmental and language delays and a hypotonic and characteristic face. The patient's phenotype was more obvious than that of her mother, who is heterozygous for the same variant. Although declining birth rate (embryonic lethality in male mice) in homozygous knockout mice has been demonstrated, our report suggests that homozygous KDM4B frameshift variants can be viable in humans at least female., (© 2023 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Coffin-Siris syndrome (CSS) is a rare congenital disorder characterized by coarse facial features, intellectual disability or developmental delay, and aplasia or hypoplasia of the tips of the fifth finger and/or toes. Mutations in genes affecting the switch/sucrose non-fermenting ATP-dependent chromatin remodeling complex are reported to cause CSS. Here, we describe three CSS patients. Two girls aged 3 and 2 years old presented with global developmental delay, poor growth, and a dysmorphic face. Whole-exome sequencing (WES) was performed and they were diagnosed with CSS due to heterozygous frameshift variants (c.3443_3444del, p.Lys1148ArgfsTer9 and c.2869_2890del, p.Pro957CysfsTer20) in ARID1B A 2-year-old girl presented with gross motor delay and dysmorphic face. She was diagnosed with CSS due to a novel heterozygous frameshift variant (c.4942_4943del: p.Gln1648GlyfsTer8) in ARID2 ., (© 2023 by the Association of Clinical Scientists, Inc.)

Human monkeypox virus (hMPXV) has caused sporadic outbreaks intermittently across countries in recent years, with the largest outbreak in 2022. However, the underlying mechanisms remain unclear. This study searched for recently developed structural variants of the viral genome. A total of 22 hMPXV whole genome sequences were randomly selected from the National Center for Biotechnology Information GenBank sequence database for initial screening. As a result, a recent frameshift mutation based on a 2-base insertion in a coding region was identified at the 3' terminal of the OPG191 gene, which encodes MPXVgp168 (B7R) protein. With this insertion, the protein was prematurely truncated, and the last 11 amino acids were missing, with 3 alternative amino acids added. Among the hMPXV genome sequences registered in the GenBank database as of January 2023, 61 sequences lacked the 2-base insertion and 3,362 sequences were inserted. All 61 sequences without mutations were collected before 2020, whereas 3,358 (99.9%) of the 3,362 sequences with the insertion were collected during or after 2022. These findings imply that a 2-base insertion has recently emerged and has been fixed among the virus population that prevailed in 2022. In summary, a recently emerged frameshift mutation with a 2-base insertion was identified in hMPXV OPG191 gene. Although the structural and functional consequences of this mutation on virulence and infectivity are unknown, research on the possible associations between this mutation and recent hMPXV outbreaks is warranted.

Restrictive dermopathy (RD) is a lethal condition caused by biallelic loss-of-function mutations in ZMPSTE24, whereas mutations preserving residual enzymatic activity of the ZMPSTE24 protein lead to the milder mandibuloacral dysplasia with type B lipodystrophy (MADB) phenotype. Remarkably, we identified a homozygous, presumably loss-of-function mutation in ZMPSTE24 [c.28_29insA, p.(Leu10Tyrfs*37)] in two consanguineous Pakistani families segregating MADB. To clarify how lethal consequences are prevented in affected individuals, functional analysis was performed. Expression experiments supported utilization of two alternative translation initiation sites, preventing complete loss of protein function consistent with the relatively mild phenotypic outcome in affected patients. One of these alternative start codons is newly formed at the insertion site. Our findings indicate that the creation of new potential start codons through N-terminal mutations in other disease-associated genes should generally be taken into consideration in the variant interpretation process., (© 2023 The Authors. Clinical Genetics published by John Wiley & Sons Ltd.)

Background: Osteogenesis imperfecta (OI) is a heterogeneous group of inherited disorders characterised by susceptibility to fractures, primarily due to defects in type 1 collagen. The aim of this study is to present a novel OI phenotype and its causative candidate gene., Methods: Whole-exome sequencing and clinical evaluation were performed in five patients from two unrelated families. PHLDB1 mRNA expression in blood and fibroblasts was investigated by real-time PCR, and western blot analysis was further performed on skin fibroblasts., Results: The common findings among the five affected children were recurrent fractures and/or osteopaenia, platyspondyly, short and bowed long bones, and widened metaphyses. Metaphyseal and vertebral changes regressed after early childhood, and no fractures occurred under bisphosphonate treatment. We identified biallelic NM_001144758.3:c.2392dup and NM_001144758.3:c.2690_2693del pathogenic variants in PHLDB1 in the affected patients, respectively, in the families; parents were heterozygous for these variants. PHLDB1 encodes pleckstrin homology-like domain family B member-1 (PHLDB1) protein, which has a role in insulin-dependent Akt phosphorylation. Compared with controls, a decrease in the expression levels of PHLDB1 in the blood and skin fibroblast samples was detected. Western blot analysis of cultured fibroblasts further confirmed the loss of PHLDB1., Conclusion: Two biallelic frameshift variants in the candidate gene PHLDB1 were identified in independent families with a novel, mild-type, autosomal recessive OI. The demonstration of decreased PHLDB1 mRNA expression levels in blood and fibroblast samples supports the hypothesis that PHLDB1 pathogenic variants are causative for the observed phenotype., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Mitochondrial membrane protein‒associated neurodegeneration (MPAN) is a rare genetic disease characterized by progressive neurodegeneration with brain iron accumulations combined with neuronal α-synuclein and tau aggregations. Mutations in C19orf12 have been associated with both autosomal recessive and autosomal dominant inheritance patterns of MPAN., Methods: We present clinical features and functional evidence from a Taiwanese family with autosomal dominant MPAN caused by a novel heterozygous frameshift and nonsense mutation in C19orf12, c273_274 insA (p.P92Tfs*9). To verify the pathogenicity of the identified variant, we examined the mitochondrial function, morphology, protein aggregation, neuronal apoptosis, and RNA interactome in p.P92Tfs*9 mutant knock-in SH-SY5Y cells created with CRISPR-Cas9 technology., Results: Clinically, the patients with the C19orf12 p.P92Tfs*9 mutation presented with generalized dystonia, retrocollis, cerebellar ataxia, and cognitive decline, starting in their mid-20s. The identified novel frameshift mutation is located in the evolutionarily conserved region of the last exon of C19orf12. In vitro studies revealed that the p.P92Tfs*9 variant is associated with impaired mitochondrial function, reduced ATP production, aberrant mitochondria interconnectivity and ultrastructure. Increased neuronal α-synuclein and tau aggregations, and apoptosis were observed under conditions of mitochondrial stress. Transcriptomic analysis revealed that the expression of genes in clusters related to mitochondrial fission, lipid metabolism, and iron homeostasis pathways was altered in the C19orf12 p.P92Tfs*9 mutant cells compared to control cells., Conclusion: Our findings provide clinical, genetic, and mechanistic insight revealing a novel heterozygous C19orf12 frameshift mutation to be a cause of autosomal dominant MPAN, further strengthening the importance of mitochondrial dysfunction in the pathogenesis of MPAN., Competing Interests: Declaration of competing interest All authors report no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Protein phosphatase 1 regulatory subunit 35 (PPP1R35) encodes a centrosomal protein required for recruiting microtubule-binding elongation machinery. Several proteins in this centriole biogenesis pathway correspond to established primary microcephaly (MCPH) genes, and multiple model organism studies hypothesize PPP1R35 as a candidate MCPH gene. Here, using exome sequencing (ES) and family-based rare variant analyses, we report a homozygous, frameshifting indel deleting the canonical stop codon in the last exon of PPP1R35 [Chr7: c.753_*3delGGAAGCGTAGACCinsCG (p.Trp251Cysfs*22)]; the variant allele maps in a 3.7 Mb block of absence of heterozygosity (AOH) in a proband with severe MCPH (-4.3 SD at birth, -6.1 SD by 42 months), pachygyria, and global developmental delay from a consanguineous Turkish kindred. Droplet digital PCR (ddPCR) confirmed mutant mRNA expression in fibroblasts. In silico prediction of the translation of mutant PPP1R35 is expected to be elongated by 18 amino acids before encountering a downstream stop codon. This complex indel allele is absent in public databases (ClinVar, gnomAD, ARIC, 1000 genomes) and our in-house database of 14,000+ exomes including 1800+ Turkish exomes supporting predicted pathogenicity. Comprehensive literature searches for PPP1R35 variants yielded two probands affected with severe microcephaly (-15 SD and -12 SD) with the same homozygous indel from a single, consanguineous, Iranian family from a cohort of 404 predominantly Iranian families. The lack of heterozygous cases in two large cohorts representative of the genetic background of these two families decreased our suspicion of a founder allele and supports the contention of a recurrent mutation. We propose two potential secondary structure mutagenesis models for the origin of this variant allele mediated by hairpin formation between complementary GC rich segments flanking the stop codon via secondary structure mutagenesis., (© 2023 Wiley Periodicals LLC.)

Competing Interests: Conflict of interest The authors declare that they have no conflict of interest.

A family, with two affected identical twins with early-onset recessive inherited retinal degeneration, was analyzed to determine the underlying genetic cause of pathology. Exome sequencing revealed a rare and previously reported causative variant (c.1923_1969delinsTCTGGG; p.Asn643Glyfs*29) in the PDE6B gene in the affected twins and their unaffected father. Further investigation, using genome sequencing, identified a novel ∼7.5-kb deletion (Chr 4:670,405-677,862del) encompassing the ATP5ME gene, part of the 5' UTR of MYL5 , and a 378-bp (Chr 4:670,405-670,782) region from the 3' UTR of PDE6B in the affected twins and their unaffected mother. Both variants segregated with disease in the family. Analysis of the relative expression of PDE6B , in peripheral blood cells, also revealed a significantly lower level of PDE6B transcript in affected siblings compared to a normal control. PDE6B is associated with recessive rod-cone degeneration and autosomal dominant congenital stationary night blindness. Ophthalmic evaluation of these patients showed night blindness, fundus abnormalities, and peripheral vision loss, which are consistent with PDE6B -associated recessive retinal degeneration. These findings suggest that the loss of PDE6B transcript resulting from the compound heterozygous pathogenic variants is the underlying cause of recessive rod-cone degeneration in the study family., (© 2022 Sangermano et al.; Published by Cold Spring Harbor Laboratory Press.)

Background The ever-increasing capacity of short-read sequencing instruments is driving the adoption of whole genome sequencing (WGS) as a universal approach to the diagnosis of rare genetic disorders. However, many challenging genomic regions remain, for which alternative technologies must be deployed in order to address the clinical question satisfactorily. Methods Here we report the use of long-read sequencing to resolve ambiguity over a suspected diagnosis of Angelman syndrome. Results Despite a normal chromosomal microarray result and methylation studies at the imprinted 15q11q13 locus, the continued clinical suspicion of Angelman Syndrome prompted trio WGS of the proband and his parents. A de novo heterozygous frameshift variant, c.2370_2373del (NM_130838.2) p.(Asp790Glufs*7), in UBE3A was identified. To determine the parental allele on which this variant arose, long-read sequencing of the flanking genomic region was performed. Comparison of the resulting haplotypes allowed us to determine that the pathogenic frameshift variant arose on the maternal allele, confirming a diagnosis of Angelman syndrome in this case. Conclusion Long-read nanopore sequencing provides significant clinical utility when assessing the parental origin of de novo variants., Competing Interests: Competing interests: Dr CMW has received travel expenses to speak at an Oxford Nanopore Technologies organised conference., (© Author(s) (or their employer(s)) 2022. No commercial re-use. See rights and permissions. Published by BMJ.)

Hereditary myopathies are well documented in dogs, whereas hereditary dyserythropoietic anemias are rarely seen. The aim of this study was to further characterize the clinical and clinicopathological features of and to identify the causative genetic variant for a dyserythropoietic anemia and myopathy syndrome (DAMS) in English springer spaniel dogs (ESSPs). Twenty-six ESSPs, including five dogs with DAMS and two puppies that died perinatally, were studied. Progressive weakness, muscle atrophy-particularly of the temporal and pelvic muscles-trismus, dysphagia, and regurgitation due to megaesophagus were observed at all ages. Affected dogs had a non-regenerative, microcytic hypochromic anemia with metarubricytosis, target cells, and acanthocytes. Marked erythroid hyperplasia and dyserythropoiesis with non-orderly maturation of erythrocytes and inappropriate microcytic metarubricytosis were present. Muscle biopsies showed centralized nuclei, central pallor, lipocyte infiltrates, and fibrosis, which was consistent with centronuclear myopathy. The genome sequencing of two affected dogs was compared to 782 genomes of different canine breeds. A homozygous frameshift single-base deletion in EHBP1L1 was identified; this gene was not previously associated with DAMS. Pedigree analysis confirmed that the affected ESSPs were related. Variant genotyping showed appropriate complete segregation in the family, which was consistent with an autosomal recessive mode of inheritance. This study expands the known genotype-phenotype correlation of EHBP1L1 and the list of potential causative genes in dyserythropoietic anemias and myopathies in humans. EHBP1L1 deficiency was previously reported as perinatally lethal in humans and knockout mice. Our findings enable the genetic testing of ESSP dogs for early diagnosis and disease prevention through targeted breeding strategies.

Rahman syndrome (RMNS) is a rare genetic disorder characterized by mild to severe intellectual disability, hypotonia, anxiety, autism spectrum disorder, vision problems, bone abnormalities and dysmorphic facies. RMNS is caused by de novo heterozygous mutations in the histone linker gene H1-4; however, mechanisms underlying impaired neurodevelopment in RMNS are not understood. All reported mutations associated with RMNS in H1-4 are small insertions or deletions that create a shared frameshift, resulting in a H1.4 protein that is both truncated and possessing an abnormal C-terminus frameshifted tail (H1.4 CFT). To expand understanding of mutations and phenotypes associated with mutant H1-4, we identified new variants at both the C- and N-terminus of H1.4. The clinical features of mutations identified at the C-terminus are consistent with other reports and strengthen the support of pathogenicity of H1.4 CFT. To understand how H1.4 CFT may disrupt brain function, we exogenously expressed wild-type or H1.4 CFT protein in rat hippocampal neurons and assessed neuronal structure and function. Genome-wide transcriptome analysis revealed ~ 400 genes altered in the presence of H1.4 CFT. Neuronal genes downregulated by H1.4 CFT were enriched for functional categories involved in synaptic communication and neuropeptide signaling. Neurons expressing H1.4 CFT also showed reduced neuronal activity on multielectrode arrays. These data are the first to characterize the transcriptional and functional consequence of H1.4 CFT in neurons. Our data provide insight into causes of neurodevelopmental impairments associated with frameshift mutations in the C-terminus of H1.4 and highlight the need for future studies on the function of histone H1.4 in neurons., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Fused in sarcoma (FUS) is the most common causative gene in juvenile-onset amyotrophic lateral sclerosis (jALS). We presented a case of a 15-year-old Chinese girl with atypical and extremely rare bilateral abducens palsy was caused by a heterozygous c.1520del (p.Gly507Alafs*22) pathogenic frameshift mutation in the FUS gene revealed by whole-exome sequencing. This is the first jALS case presenting with bilateral abducens palsy and carrying de novo FUS genetic variant.

PTEN is a tumour suppressor gene known for regulating apoptosis, cell growth, and many other pathways. It is one of the most frequently mutated genes comprising the phosphatase domain (PD) and C terminal domain (C2). Direct therapeutic methods are not applicable for targeting PTEN because once gets mutated, it needs restoration. For mutant detection and restoration using PTEN mRNA there is a need to explore various mutations taking place in PTEN, identify their particular domains, and study their interactions within the cellular system. Here, we have tried to highlight a few such regions in the mutated PTEN of breast cancer patients. In this study, we have selected the top-most-occurring PTEN mutation in breast cancer and compared them to determine the specific properties of each mutation and its effect on functionality. Molecular dynamic simulation for 50 ns was performed on five structures to compare the structural behaviour of mutated PTEN in the system. Our finding suggests that frameshift mutations are more damaging and affect the c2 domain. Frameshift mutant fs_ACTT is the highest occurring as well as the most damaging mutation in all the compared structures. Docking study shows that substitution mutations D92H and R130Q causes loss of binding ability towards PIP2 in normal PTEN, interfering the dephosphorylation process. Overall, the C2 domain is more frequently mutated, and the amino acid residues in the C2 domain show more fluctuations compared to the other regions. Our study can provide the basis for selecting frequently mutated C2 domain as a potential therapeutic marker.Communicated by Ramaswamy H. Sarma.

Variants in MFSD8 can cause neuronal ceroid lipofuscinoses (NCLs) as well as nonsyndromic retinopathy. The mutation spectrum includes mainly missense and stop variants, but splice sites and frameshift variants have also been reported. To date, apparently synonymous substitutions have not been shown to cause MFSD8 -associated diseases. We report two closely related subjects from a consanguineous Turkish family who presented classical features of NCLs but demonstrated marked intrafamilial variability in age at the onset and severity of symptoms. In fact, the difference in the onset of first neurologic symptoms was 15 years and that of ophthalmologic symptoms was 12 years. One subject presented an intellectual disability and a considerable cerebellar ataxia syndrome, while the other subject showed no intellectual disability and only a mild atactic syndrome. The diagnostic genetic testing of both subjects based on genome sequencing prioritized a novel, apparently synonymous variant in MFSD8 , which was found in homozygosity in both subjects. The variant was not located within an integral part of the splice site consensus sequences. However, the bioinformatic analyses suggested that the mutant allele is more likely to cause exon skipping due to an altered ratio of exonic splice enhancer and silencer motifs. Exon skipping was confirmed in vitro by minigene assays and in vivo by RNA analysis from patient lymphocytes. The mutant transcript is predicted to result in a frameshift and, if translated, in a truncated protein. Synonymous variants are often given a low priority in genetic diagnostics because of their expected lack of functional impact. This study highlights the importance of investigating the impact of synonymous variants on splicing.

GP1bβ is a component of the von Willebrand factor (vWF) receptor complex that is necessary for platelet formation and activation. A novel frameshift variant in GP1BB has been identified in a family with macrothrombocytopenia. The variant leads to a protein that is 101 amino acids longer than wild type with loss of the transmembrane domain. As there is no defect in platelet aggregation, the family are classified as heterozygous carriers of a Bernard-Soulier syndrome-related mutation. The levels of the vWF receptor on platelets are reduced to 50% of the controls, with the presence of large platelets but normal platelet aggregation demonstrating that decreased vWF receptor expression impacts proplatelet formation but not platelet function.

Variants in the pleckstrin homology domain-interacting protein (PHIP) gene are implicated in the clinical phenotype of Chung-Jansen syndrome, which includes dysmorphic features, cognitive dysfunction, aberrant behavior, and childhood onset obesity. Following a systematic literature review, 35 patients are reported to have unique PHIP variants impacting the encoded protein product. We summarize the status and frequency of these variants and relationship to clinical presentation. We also describe an additional patient with a rare, pathogenic variant due to a five base pair deletion leading to an altered codon at I307 but with a stop codon at 22 codons downstream; notably, a variant was identified at the same location as seen previously at protein position I307 in one other subject and a frameshift change at that protein position. We compare the clinical characteristics between the two patients and analyze whether certain types of gene defects impact clinical presentation in previously reported individuals. In addition, we predict structural protein models, which yielded unique differences between the wild-type and I307P-related mutant truncated proteins. Protein-protein interactions indicate involvement of POMC and related proteins with potential contribution to obesity, congenital, neuromuscular, and lipid disorders with heart, gastrointestinal, and rheumatoid diseases. With its surrounding proline-rich region, the I307P point mutation increases susceptibility to conformational rigidity and thermodynamic stability, ultimately impacting function as well as a stop codon downstream. Furthermore, the frameshift mutation seen in our patient may result in a truncated protein with a short abnormal region prior to the stop codon due to a five base pair deletion at I307 or target the protein for nonsense-mediated mRNA decay., (© 2021 Wiley Periodicals LLC.)

Background: Familial hypercholesterolemia (FH) is characterized by high low-density lipoprotein-cholesterol levels and it is primarily caused by pathogenic/likely pathogenic variants (P/LPVs) in LDLR, APOB or PCSK9 genes. Next generation sequencing (NGS) technology allows the evaluation of more genes simultaneously, rising the diagnostic throughput of genomics laboratories., Materials and Methods: We report a Ukrainian 37-year-old woman hypercholesterolemic since 2010. Despite a suggestive family history, FH was suspected only when the patient referred to the Endocrine and Metabolic Diseases Center of the Fondazione Policlinico Universitario A. Gemelli IRCCS in Rome. After specialist advice, genetic testing was offered to the patient at our Molecular and Genomic Diagnostics Unit., Results: A targeted NGS-based pipeline highlighted a novel out-of-frame deletion in the LDLR gene. This variant has a clear deleterious effect on the LDLR protein and it can be classified as PV., Conclusions: The ideal model of care for FH is an evidence-based system aimed to provide the highest-quality health services to all FH patients. In fact, this study reports that the integrated care pathway adopted in our hospital for FH patients led successfully to the discovery of a novel LDLR PV in an Ukrainian patient. The finding of this LDLR variant allowed the clinical FH diagnosis in this patient and in her family, expanding the knowledge of FH-related genetic variants in the Ukrainian population., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

In his influential book " Evolution by Gene Duplication ", Ohno postulated that frameshift mutation could lead to a new function after duplication, but frameshift mutation is generally thought to be deleterious, and thus drew little attention in functional innovation in duplicate evolution. To this end, we here report an exhaustive survey of the genomes of human, mouse, zebrafish, and fruit fly. We identified 80 duplicate genes that involved frameshift mutations after duplication. The frameshift mutation preferentially located close to the C-terminus in most cases (55/88), which indicated that a frameshift mutation that changed the reading frame in a small part at the end of a duplicate may likely have contributed to adaptive evolution (e.g., human genes NOTCH2NL and ARHGAP11B ) otherwise too deleterious to survive. A few cases (11/80) involved multiple frameshift mutations, exhibiting various patterns of modifications of the reading frame. Functionality of duplicate genes involving frameshift mutations was confirmed by sequence characteristics and expression profile, suggesting a potential role of frameshift mutation in creating functional novelty. We thus showed that genomes have non-negligible numbers of genes that have experienced frameshift mutations following gene duplication. Our results demonstrated the potential importance of frameshift mutations in molecular evolution, as Ohno verbally argued 50 years ago.