Pathogenic Missense Variants Research Articles

Saturation genome editing-based functional evaluation and clinical classification of BRCA2 single nucleotide variants.

10511 Background: Germline BRCA2 loss-of function (LOF) variants identified by clinical genetic testing predispose to breast, ovarian, prostate and pancreatic cancer. However, variants of uncertain significance (VUS) (n > 4000) limit the clinical use of testing results. Thus, there is an urgent need for functional characterization and clinical classification of all BRCA2 variants. Here we report on comprehensive saturation genome editing (SGE)-based functional characterization of 97% of all possible single nucleotide variants (SNVs) in the BRCA2 DNA Binding Domain hotspot encoded by exons 15 to 26 for pathogenic missense variants. Methods: SGE of exons 15 to 26 of BRCA2(MANE transcript ENST00000380152.8) was performed in the haploid human HAP1 cell line. Each exon along with 10bp flanking intronic sequence was studied as an independent target region. The most efficient sgRNA for each target region was selected and cloned into a Cas9 expressing plasmid. Site-saturation mutagenesis (SSM) libraries for each of the 14 target regions were designed to target every nucleotide and were generated by site directed mutagenesis using NNN-tailed PCR primers. HAP1 cells were transfected and harvested at Day5 and 14. Cell survival in response to each variant was assessed based on deep sequence analysis of the surviving endogenously targeted HAP1 cells at Day 5 and Day 14. Results: A total of 7013 SNVs were characterized as functionally abnormal (n = 955), intermediate/uncertain, or functionally normal (n = 5224) based on 95% precision for ClinVar known pathogenic and benign standards. Results were validated relative to batches of nonsense and synonymous variants and variants evaluated using a homology directed repair (HDR) functional assay. Breast cancer case-control association studies showed that pooled SNVs encoding functionally abnormal missense variants were associated with increased risk of breast cancer (odds ratio (OR) 3.89, 95%CI: 2.77-5.51). The functional data were added to other sources of information in a ClinGen/ACMG/AMP-like model and 700 functionally abnormal SNVs, including 220 missense SNVs, were classified as pathogenic or likely pathogenic, while 4862 functionally normal SNVs, including 3084 missense SNVs, were classified as benign or likely benign. Conclusions: The production of functional maps for 97% of all SNVs in the BRCA2 DBD allowed for separation of nucleotide/protein-level functional aberrations. When coupled with genetic and clinical sources of evidence this study led to clinical classification of 5500 individual variants. These data will prove useful in the future, through integration with other datasets, for characterization and clinical classification of all variants in this region in individuals from all racial and ethnic backgrounds and for all BRCA2 associated forms of cancer.

Identification of a De novo pathogenic missense variant (c.559G>A) in CASZ1 associated with dilated cardiomyopathy.

Identification of a De novo pathogenic missense variant (c.559G>A) in CASZ1 associated with dilated cardiomyopathy.

#2305 Mice with a PAX2 missense variant are susceptible to experimental focal segmental glomerulosclerosis (FSGS) and display impaired glomerular repair

Abstract Background and Aims Focal segmental glomerulosclerosis (FSGS) is a histopathologic lesion observed in chronic kidney disease characterized by scarring of the glomerulus due to the injury and irreversible loss of highly specialized glomerular epithelial cells called podocytes. Recent studies suggest that podocytes may be regenerated by another population of glomerular epithelial cells called parietal epithelial cells (PECs), although this remains controversial. PAX2 is a transcription factor protein that regulates glomerular development and its expression persists in adult parietal epithelial cells (PECs), postulated to serve as a reservoir for podocytes in the event of injury and loss. Our team previously found that PAX2 pathogenic missense variants account for 4% of adult FSGS. In our present study, our aim is to determine whether PEC-mediated podocyte regeneration is impaired in mice with a pathogenic Pax2 missense variant (Pax2-MV). Method FSGS was induced in wildtype and Pax2-MV mice using Adriamycin. Urine samples were collected from mice every week for 28 days to measure proteinuria. At 28 days after Adriamycin injury, kidneys were collected for histopathology, transmission electron microscopy, and immunofluorescence staining for PAX2 and podocyte marker WT-1. Glomeruli from Adriamycin-injured mice were also isolated through intra-cardiac perfusion with Dynabeads and subjected to mass-spectrometry for proteomics analysis. Proteins with statistically-significant fold changes in expression levels were further analyzed by Gene Ontology to determine biological and molecular processes involved in pathogenesis. Kidney tissue from an FSGS patient with a pathogenic PAX2 missense variant was also examined by histopathology and PAX2 immunohistochemistry. Results Compared to wildtype, Pax2-MV mice were more susceptible to worsened FSGS after Adriamycin, as demonstrated by worsened glomerular scarring, increased podocyte injury and loss, and increased proteinuria, associated with increased morality in Pax2-MV mice (Fig. 1A-B). Wildtype mice showed fewer PAX2-expressing PECs after injury, accompanied by the occurrence of PAX2/WT-1 co-expressing glomerular tuft cells (Fig. 2A-B). These suggest that in Adriamycin-injured wildtype, PAX2-expressing PECs move to the glomerular tuft, where they trans-differentiate to a podocyte fate. In contrast, Pax2-MV mice showed no changes in numbers of PAX2-expressing PECs after injury, associated with fewer PAX2/WT-1 co-expressing glomerular tuft cells compared to injured wildtype (Fig. 2A-B). These suggest that in Adriamycin-injured Pax2-MV mice, PAX2-expressing PECs are unable to move to the glomerular tuft, resulting in decreased numbers of cells undergoing podocyte trans-differentiation. A subset of ectopic PAX2-expressing glomerular tuft cells after injury was increased in Pax2-MV mice (Fig. 2A), suggesting a pathologic process given the worse outcomes observed in these mice. Consistent with findings from injured Pax2-MV mice, we found that in an FSGS patient with a pathogenic PAX2 missense variant, PAX2-expressing glomerular tuft cells overlap with areas of glomerular scarring, while in a normal human kidney biopsy, PAX2 expression is only observed in PECs but not within glomerular tuft cells (Fig. 2A). Lastly, proteomics Gene Ontology analysis of isolated glomeruli demonstrated biological and molecular processes indicative of maladaptive glomerular repair in injured Pax2-MV mice, including decreased regulation of cell shape, decreased cell-cell junction assembly and decreased actin and cadherin binding compared to wildtype (Fig. 2C). Conclusion Altogether, our findings suggest that PAX2-expressing PECs migrate to the glomerular tuft to assist in podocyte regeneration in wildtype. However, this mechanism is impaired in Pax2-MV mice, leading to worsened podocyte loss and injury and further exacerbation of glomerular disease (Fig. 2C). Our findings provide a further understanding of normal and impaired regeneration mechanisms in FSGS, and our identified pathogenic mechanisms can be further harnessed for future therapeutic development.

Mutations in the tail and rod domains of the neurofilament heavy-chain gene increase the risk of ALS.

Neurofilament heavy-chain gene (NEFH) variants are associated with multiple neurodegenerative diseases, however, their relationship with ALS has not been robustly explored. Still, NEFH is commonly included in genetic screening panels worldwide. We therefore aimed to determine if NEFH variants modify ALS risk. Genetic data of 11,130 people with ALS and 7,416 controls from the literature and Project MinE were analysed. We performed meta-analyses of published case-control studies reporting NEFH variants, and variant analysis of NEFH in Project MinE whole-genome sequencing data. Fixed-effects meta-analysis found that rare (MAF <1%) missense variants in the tail domain of NEFH increase ALS risk (OR 4.55, 95% CI 2.13-9.71, p < 0.0001). In Project MinE, ultrarare NEFH variants increased ALS risk (OR 1.37 95% CI 1.14-1.63, p = 0.0007), with rod domain variants (mostly intronic) appearing to drive the association (OR 1.45 95% CI 1.18-1.77, pMadsen-Browning = 0.0007, pSKAT-O = 0.003). While in the tail domain, ultrarare (MAF <0.1%) pathogenic missense variants were also associated with higher risk of ALS (OR 1.94, 95% CI 0.86-4.37, pMadsen-Browning = 0.039), supporting the meta-analysis results. Finally, several tail in-frame deletions were also found to affect disease risk, however, both protective and pathogenic deletions were found in this domain, highlighting an intricate architecture that requires further investigation. We showed that NEFH tail missense and in-frame deletion variants, and intronic rod variants are risk factors for ALS. However, they are not variants of large effect, and their functional impact needs to be clarified in further studies. Therefore, their inclusion in routine genetic screening panels should be reconsidered.

Expanding the Phenotype of the CACNA1C-Associated Neurological Disorders in Children: Systematic Literature Review and Description of a Novel Mutation.

Background: CACNA1C gene encodes the alpha 1 subunit of the CaV1.2 L-type Ca2+ channel. Pathogenic variants in this gene have been associated with cardiac rhythm disorders such as long QT syndrome, Brugada syndrome and Timothy syndrome. Recent evidence has suggested the possible association between CACNA1C mutations and neurologically-isolated (in absence of cardiac involvement) phenotypes in children, giving birth to a wider spectrum of CACNA1C-related clinical presentations. However, to date, little is known about the variety of both neurological and non-neurological signs/symptoms in the neurologically-predominant phenotypes. Methods and Results: We conducted a systematic review of neurologically-predominant presentations without cardiac conduction defects, associated with CACNA1C mutations. We also reported a novel de novo missense pathogenic variant in the CACNA1C gene of a children patient presenting with constructional, dressing and oro-buccal apraxia associated with behavioral abnormalities, mild intellectual disability, dental anomalies, gingival hyperplasia and mild musculoskeletal defects, without cardiac conduction defects. Conclusions: The present study highlights the importance of considering the investigation of the CACNA1C gene in children's neurological isolated syndromes, and expands the phenotype of the CACNA1C related conditions. In addition, the present study highlights that, even in absence of cardiac conduction defects, nuanced clinical manifestations of the Timothy syndrome (e.g., dental and gingival defects) could be found. These findings suggest the high variable expressivity of the CACNA1C gene and remark that the absence of cardiac involvement should not mislead the diagnosis of a CACNA1C related disorder.

Whole-exome sequencing revealed a likely pathogenic variant in NF1 causing neurofibromatosis type I and Arrhythmogenic Cardiomyopathy

BackgroundNeurofibromatosis type I (NF1) is a genetic disorder characterized by the tumor’s development in nerve tissue. Complications of NF1 can include pigmented lesions, skin neurofibromas, and heart problems such as cardiomyopathy. In this study, we performed whole-exome sequencing (WES) on an Iranian patient with NF1 to identify the genetic cause of the disease.MethodsFollowing clinical assessment, WES was used to identify genetic variants in a family with a son suffering from NF1. No symptomatic manifestations were observed in other family members. In the studied family, in silico and segregation analysis were applied to survey candidate variants.ResultsClinical manifestations were consistent with arrhythmogenic cardiomyopathy (ACM). WES detected a likely pathogenic heterozygous missense variant, c.3277G > A:p.Val1093Met, in the NF1 gene, confirmed by PCR and Sanger sequencing. The patient’s parents and brother had a normal sequence at this locus.ConclusionsAlthough there is no cure for NF1, genetic tests, such as WES, can detect at-risk asymptomatic family members. Furthermore, cardiac evaluation could also help these patients before heart disease development.

Recessive GNE Mutations in Korean Nonaka Distal Myopathy Patients with or without Peripheral Neuropathy.

Autosomal recessive Nonaka distal myopathy is a rare autosomal recessive genetic disease characterized by progressive degeneration of the distal muscles, causing muscle weakness and decreased grip strength. It is primarily associated with mutations in the GNE gene, which encodes a key enzyme of sialic acid biosynthesis (UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase). This study was performed to find GNE mutations in six independent distal myopathy patients with or without peripheral neuropathy using whole-exome sequencing (WES). In silico pathogenic prediction and simulation of 3D structural changes were performed for the mutant GNE proteins. As a result, we identified five pathogenic or likely pathogenic missense variants: c.86T>C (p.Met29Thr), c.527A>T (p.Asp176Val), c.782T>C (p.Met261Thr), c.1714G>C (p.Val572Leu), and c.1771G>A (p.Ala591Thr). Five affected individuals showed compound heterozygous mutations, while only one patient revealed a homozygous mutation. Two patients revealed unreported combinations of combined heterozygous mutations. We observed some specific clinical features, such as complex phenotypes of distal myopathy with distal hereditary peripheral neuropathy, an earlier onset of weakness in legs than that of hands, and clinical heterogeneity between two patients with the same set of compound heterozygous mutations. Our findings on these genetic causes expand the clinical spectrum associated with the GNE mutations and can help prepare therapeutic strategies.

A novel pathogenic SLC12A5 missense variant in epilepsy of infancy with migrating focal seizures causes impaired KCC2 chloride extrusion.

The potassium-chloride co-transporter 2, KCC2, is a neuron-specific ion transporter that plays a multifunctional role in neuronal development. In mature neurons, KCC2 maintains a low enough intracellular chloride concentration essential for inhibitory neurotransmission. During recent years, pathogenic variants in the KCC2 encoding gene SLC12A5 affecting the functionality or expression of the transporter protein have been described in several patients with epilepsy of infancy with migrating focal seizures (EIMFS), a devastating early-onset developmental and epileptic encephalopathy. In this study, we identified a novel recessively inherited SLC12A5 c.692G>A, p. (R231H) variant in a patient diagnosed with severe and drug-resistant EIMFS and profound intellectual disability. The functionality of the variant was assessed in vitro by means of gramicidin-perforated patch-clamp experiments and ammonium flux assay, both of which indicated a significant reduction in chloride extrusion. Based on surface immunolabeling, the variant showed a reduction in membrane expression. These findings implicate pathogenicity of the SLC12A5 variant that leads to impaired inhibitory neurotransmission, increasing probability for hyperexcitability and epileptogenesis.

In-Silico Method for Predicting Pathogenic Missense Variants Using Online Tools: AURKA Gene as a Model.

In-silico analysis provides a fast, simple, and cost-free method for identifying potentially pathogenic single nucleotide variants. To propose a simple and relatively fast method for the prediction of variant pathogenicity using free online in-silico (IS) tools with AURKA gene as a model. We aim to propose a methodology to predict variants with high pathogenic potential using computational analysis, using AURKA gene as model. We predicted a protein model and analyzed 209 out of 64,369 AURKA variants obtained from Ensembl database. We used bioinformatic tools to predict pathogenicity. The results were compared through the VarSome website, which includes its own pathogenicity score and the American College of Medical Genetics (ACMG) classification. Out of the 209 analyzed variants, 16 were considered pathogenic, and 13 were located in the catalytic domain. The most frequent protein changes were size and hydrophobicity modifications of amino acids. Proline and Glycine amino acid substitutions were the most frequent changes predicted as pathogenic. These bioinformatic tools predicted functional changes, such as protein up or down-regulation, gain or loss of molecule interactions, and structural protein modifications. When compared to the ACMG classification, 10 out of 16 variants were considered likely pathogenic, with 7 out of 10 changes at Proline/Glycine substitutions. This method allows quick and cost-free bulk variant screening to identify variants with pathogenic potential for further association and/or functional studies.

Menke-Hennekam syndrome; delineation of domain-specific subtypes with distinct clinical and DNA methylation profiles

CREB-binding protein (CBP, encoded by CREBBP) and its paralog E1A-associated protein (p300, encoded by EP300) are involved in histone acetylation and transcriptional regulation. Variants that produce a null allele or disrupt the catalytic domain of either protein cause Rubinstein-Taybi syndrome (RSTS), while pathogenic missense and in-frame indel variants in parts of exons 30 and 31 cause phenotypes recently described as Menke-Hennekam syndrome (MKHK). To distinguish MKHK subtypes and define their characteristics, molecular and extended clinical data on 82 individuals (54 unpublished) with variants affecting CBP (n=71) or p300 (n=11) (NP_004371.2 residues 1705-1875 and NP_001420.2 residues 1668-1833, respectively) were summarized. Additionally, genome-wide DNA methylation profiles were assessed in DNA extracted from whole peripheral blood from 54 individuals. Most variants clustered closely around the zinc binding residues of two zinc finger domains (ZZ and TAZ2) and within the first α-helix of the fourth intrinsically disordered linker (ID4) of CBP/p300. Domain-specific methylation profiles were discerned for the ZZ domain in CBP/p300 (found in 9/10 tested individuals) and TAZ2 domain in CBP (in 14/20), while a domain-specific diagnostic episignature was refined for the ID4 domain in CBP/p300 (in 21/21). Phenotypes including intellectual disability of varying degree, and distinct physical features were defined for each of the regions. These findings demonstrate existence of at least three MKHK subtypes, which are domain-specific (MKHK-ZZ, MKHK-TAZ2, and MKHK-ID4) rather than gene-specific (CREBBP/EP300). DNA methylation episignatures enable stratification of molecular pathophysiologic entities within a gene or across a family of paralogous genes.

Genome Sequencing of Idiopathic Speech Delay

Genetic investigations of people with speech and language disorders can provide windows into key aspects of human biology. Most genomic research into impaired speech development has so far focused on childhood apraxia of speech (CAS), a rare neurodevelopmental disorder characterized by difficulties with coordinating rapid fine motor sequences that underlie proficient speech. In 2001, pathogenic variants of FOXP2 provided the first molecular genetic accounts of CAS aetiology. Since then, disruptions in several other genes have been implicated in CAS, with a substantial proportion of cases being explained by high-penetrance variants. However, the genetic architecture underlying other speech-related disorders remains less well understood. Thus, in the present study, we used systematic DNA sequencing methods to investigate idiopathic speech delay, as characterized by delayed speech development in the absence of a motor speech diagnosis (such as CAS), a language/reading disorder, or intellectual disability. We performed genome sequencing in a cohort of 23 children with a rigorous diagnosis of idiopathic speech delay. For roughly half of the sample (ten probands), sufficient DNA was also available for genome sequencing in both parents, allowing discovery of de novo variants. In the thirteen singleton probands, we focused on identifying loss-of-function and likely damaging missense variants in genes intolerant to such mutations. We found that one speech delay proband carried a pathogenic frameshift deletion in SETD1A, a gene previously implicated in a broader variable monogenic syndrome characterized by global developmental problems including delayed speech and/or language development, mild intellectual disability, facial dysmorphisms, and behavioural and psychiatric symptoms. Of note, pathogenic SETD1A variants have been independently reported in children with CAS in two separate studies. In other probands in our speech delay cohort, likely pathogenic missense variants were identified affecting highly conserved amino acids in key functional domains of SPTBN1 and ARF3. Overall, this study expands the phenotype spectrum associated with pathogenic SETD1A variants, to also include idiopathic speech delay without CAS or intellectual disability, and suggests additional novel potential candidate genes that may harbour high-penetrance variants that can disrupt speech development.

Expanding the clinical spectrum of biglycan-related Meester-Loeys syndrome

Pathogenic loss-of-function variants in BGN, an X-linked gene encoding biglycan, are associated with Meester-Loeys syndrome (MRLS), a thoracic aortic aneurysm/dissection syndrome. Since the initial publication of five probands in 2017, we have considerably expanded our MRLS cohort to a total of 18 probands (16 males and 2 females). Segregation analyses identified 36 additional BGN variant-harboring family members (9 males and 27 females). The identified BGN variants were shown to lead to loss-of-function by cDNA and Western Blot analyses of skin fibroblasts or were strongly predicted to lead to loss-of-function based on the nature of the variant. No (likely) pathogenic missense variants without additional (predicted) splice effects were identified. Interestingly, a male proband with a deletion spanning the coding sequence of BGN and the 5’ untranslated region of the downstream gene (ATP2B3) presented with a more severe skeletal phenotype. This may possibly be explained by expressional activation of the downstream ATPase ATP2B3 (normally repressed in skin fibroblasts) driven by the remnant BGN promotor. This study highlights that aneurysms and dissections in MRLS extend beyond the thoracic aorta, affecting the entire arterial tree, and cardiovascular symptoms may coincide with non-specific connective tissue features. Furthermore, the clinical presentation is more severe and penetrant in males compared to females. Extensive analysis at RNA, cDNA, and/or protein level is recommended to prove a loss-of-function effect before determining the pathogenicity of identified BGN missense and non-canonical splice variants. In conclusion, distinct mechanisms may underlie the wide phenotypic spectrum of MRLS patients carrying loss-of-function variants in BGN.

A TBK1 variant causes autophagolysosomal and motoneuron pathology without neuroinflammation in mice.

Heterozygous mutations in the TBK1 gene can cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The majority of TBK1-ALS/FTD patients carry deleterious loss-of-expression mutations, and it is still unclear which TBK1 function leads to neurodegeneration. We investigated the impact of the pathogenic TBK1 missense variant p.E696K, which does not abolish protein expression, but leads to a selective loss of TBK1 binding to the autophagy adaptor protein and TBK1 substrate optineurin. Using organelle-specific proteomics, we found that in a knock-in mouse model and human iPSC-derived motor neurons, the p.E696K mutation causes presymptomatic onset of autophagolysosomal dysfunction in neurons precipitating the accumulation of damaged lysosomes. This is followed by a progressive, age-dependent motor neuron disease. Contrary to the phenotype of mice with full Tbk1 knock-out, RIPK/TNF-α-dependent hepatic, neuronal necroptosis, and overt autoinflammation were not detected. Our in vivo results indicate autophagolysosomal dysfunction as a trigger for neurodegeneration and a promising therapeutic target in TBK1-ALS/FTD.

Clinical feature, GALC variant spectrum, and genotype-phenotype correlation in Korean Krabbe disease patients: Multicenter experience over 13 years.

Krabbe disease (KD) is an autosomal recessive neurodegenerative disorder caused by deficiency of the galactocerebrosidase (GALC) due to variants in the GALC gene. Here, we provide the first and the largest comprehensive analysis of clinical and genetic characteristics, and genotype-phenotype correlations of KD in Korean in comparison with other ethnic groups. From June 2010 to June 2023, 10 patients were diagnosed with KD through sequencing of GALC. Clinical features, and results of GALC sequencing, biochemical test, neuroimaging, and neurophysiologic test were obtained from medical records. An additional nine previously reported Korean KD patients were included for review. In Korean KD patients, the median age of onset was 2 years (3 months-34 years) and the most common phenotype was adult-onset (33%, 6/18) KD, followed by infantile KD (28%, 5/18). The most frequent variants were c.683_694delinsCTC (23%) and c.1901T>C (23%), while the 30-kb deletion was absent. Having two heterozygous pathogenic missense variants was associated with later-onset phenotype. Clinical features were similar to those of other ethnic groups. In Korean KD patients, the most common phenotype was the adult-onset type and the GALC variant spectrum was different from that of the Caucasian population. This study would further our understanding of KD.

Mono-allelic KCNB2 variants lead to a neurodevelopmental syndrome caused by altered channel inactivation

Ion channels mediate voltage fluxes or action potentials that are central to the functioning of excitable cells such as neurons. The KCNB family of voltage-gated potassium channels (Kv) consists of two members (KCNB1 and KCNB2) encoded by KCNB1 and KCNB2, respectively. These channels are major contributors to delayed rectifier potassium currents arising from the neuronal soma which modulate overall excitability of neurons. In this study, we identified several mono-allelic pathogenic missense variants in KCNB2, in individuals with a neurodevelopmental syndrome with epilepsy and autism in some individuals. Recurrent dysmorphisms included a broad forehead, synophrys, and digital anomalies. Additionally, we selected three variants where genetic transmission has not been assessed, from two epilepsy studies, for inclusion in our experiments. We characterized channel properties of these variants by expressing them in oocytes of Xenopus laevis and conducting cut-open oocyte voltage clamp electrophysiology. Our datasets indicate no significant change in absolute conductance and conductance-voltage relationships of most disease variants as compared to wild type (WT), when expressed either alone or co-expressed with WT-KCNB2. However, variants c.1141A>G (p.Thr381Ala) and c.641C>T (p.Thr214Met) show complete abrogation of currents when expressed alone with the former exhibiting a left shift in activation midpoint when expressed alone or with WT-KCNB2. The variants we studied, nevertheless, show collective features of increased inactivation shifted to hyperpolarized potentials. We suggest that the effects of the variants on channel inactivation result in hyper-excitability of neurons, which contributes to disease manifestations.

Induced pluripotent stem cell (iPSC) modeling validates reduced GBE1 enzyme activity due to a novel variant, p.Ile694Asn, found in a patient with suspected glycogen storage disease IV

BackgroundGlycogen Storage disease type 4 (GSD4), a rare disease caused by glycogen branching enzyme 1 (GBE1) deficiency, affects multiple organ systems including the muscles, liver, heart, and central nervous system. Here we report a GSD4 patient, who presented with severe hepatosplenomegaly and cardiac ventricular hypertrophy. GBE1 sequencing identified two variants: a known pathogenic missense variant, c.1544G>A (p.Arg515His), and a missense variant of unknown significance (VUS), c.2081T>A (p. Ile694Asn). As a liver transplant alone can exacerbate heart dysfunction in GSD4 patients, a precise diagnosis is essential for liver transplant indication. To characterize the disease-causing variant, we modeled patient-specific GBE1 deficiency using CRISPR/Cas9 genome-edited induced pluripotent stem cells (iPSCs). MethodsiPSCs from a healthy donor (iPSC-WT) were genome-edited by CRISPR/Cas9 to induce homozygous p.Ile694Asn in GBE1 (iPSC-GBE1-I694N) and differentiated into hepatocytes (iHep) or cardiomyocytes (iCM). GBE1 enzyme activity was measured, and PAS-D staining was performed to analyze polyglucosan deposition in these cells. ResultsiPSCGBE1-I694N differentiated into iHep and iCM exhibited reduced GBE1 protein level and enzyme activity in both cell types compared to iPSCwt. Both iHepGBE1-I694N and iCMGBE1-I694N showed polyglucosan deposits correlating to the histologic patterns of the patient's biopsies. ConclusionsiPSC-based disease modeling supported a loss of function effect of p.Ile694Asn in GBE1. The modeling of GBE1 enzyme deficiency in iHep and iCM cell lines had multi-organ findings, demonstrating iPSC-based modeling usefulness in elucidating the effects of novel VUS in ultra-rare diseases.

Mice with a Pax2 missense variant display impaired glomerular repair.

PAX2 regulates kidney development, and its expression persists in parietal epithelial cells (PECs), potentially serving as a podocyte reserve. We hypothesized that mice with a Pax2 pathogenic missense variant (Pax2A220G/+) have impaired PEC-mediated podocyte regeneration. Embryonic wild-type mouse kidneys showed overlapping expression of PAX2/Wilms' tumor-1 (WT-1) until PEC and podocyte differentiation, reflecting a close lineage relationship. Embryonic and adult Pax2A220G/+ mice have reduced nephron number but demonstrated no glomerular disease under baseline conditions. Pax2A220G/+ mice compared with wild-type mice were more susceptible to glomerular disease after adriamycin (ADR)-induced podocyte injury, as demonstrated by worsened glomerular scarring, increased podocyte foot process effacement, and podocyte loss. There was a decrease in PAX2-expressing PECs in wild-type mice after adriamycin injury accompanied by the occurrence of PAX2/WT-1-coexpressing glomerular tuft cells. In contrast, Pax2A220G/+ mice showed no changes in the numbers of PAX2-expressing PECs after adriamycin injury, associated with fewer PAX2/WT-1-coexpressing glomerular tuft cells compared with injured wild-type mice. A subset of PAX2-expressing glomerular tuft cells after adriamycin injury was increased in Pax2A220G/+ mice, suggesting a pathological process given the worse outcomes observed in this group. Finally, Pax2A220G/+ mice have increased numbers of glomerular tuft cells expressing Ki-67 and cleaved caspase-3 compared with wild-type mice after adriamycin injury, consistent with maladaptive responses to podocyte loss. Collectively, our results suggest that decreased glomerular numbers in Pax2A220G/+ mice are likely compounded with the inability of their mutated PECs to regenerate podocyte loss, and together these two mechanisms drive the worsened focal segmental glomerular sclerosis phenotype in these mice.NEW & NOTEWORTHY Congenital anomalies of the kidney and urinary tract comprise some of the leading causes of kidney failure in children, but our previous study showed that one of its genetic causes, PAX2, is also associated with adult-onset focal segmental glomerular sclerosis. Using a clinically relevant model, our present study demonstrated that after podocyte injury, parietal epithelial cells expressing PAX2 are deployed into the glomerular tuft to assist in repair in wild-type mice, but this mechanism is impaired in Pax2A220G/+ mice.

FOXG1 variants can be associated with milder phenotypes than congenital Rett syndrome with unassisted walking and language development.

Since 2008, FOXG1 haploinsufficiency has been linked to a severe neurodevelopmental phenotype resembling Rett syndrome but with earlier onset. Most patients are unable to sit, walk, or speak. For years, FOXG1 sequencing was only prescribed in such severe cases, limiting insight into the full clinical spectrum associated with this gene. Next-generation sequencing (NGS) now enables unbiased diagnostics. Through the European Reference Network for Rare Malformation Syndromes, Intellectual and Other Neurodevelopmental Disorders, we gathered data from patients with heterozygous FOXG1 variants presenting a mild phenotype, defined as able to speak and walk independently. We also reviewed data from three previously reported patients meeting our criteria. We identified five new patients with pathogenic FOXG1 missense variants, primarily in the forkhead domain, showing varying nonspecific intellectual disability and developmental delay. These features are not typical of congenital Rett syndrome and were rarely associated with microcephaly and epilepsy. Our findings are consistent with a previous genotype-phenotype analysis by Mitter et al. suggesting the delineation of five different FOXG1 genotype groups. Milder phenotypes were associated with missense variants in the forkhead domain. This information may facilitate prognostic assessments in children carrying a FOXG1 variant and improve the interpretation of new variants identified with genomic sequencing.

The first Iranian patient with You-Hoover-Fong syndrome and a review of the literature on 27 cases: expanding the genotypic and phenotypic spectrum.

The ultra-rare autosomal recessive genetic disorder, You-Hoover-Fong Syndrome (YHFS), is caused by defects in the TELO2 gene and is characterized by intellectual disability, developmental delay, and ocular impairments. This study aims to contribute to a better understanding of YHFS by reviewing previous cases and introducing a novel variant in a new case. Whole exome sequencing (WES) was conducted on the proband to identify genetic variants, and Sanger sequencing was used to confirm variants within the family. This article presents a comprehensive collection of reported cases of YHFS, incorporating both molecular and clinical data, through an extensive literature search and analysis of English-language studies published until June 2023. Using WES, a novel homozygous missense variant, c.1799A > G (p. Tyr600Cys), was identified in the TELO2 gene in a 4-year-old Iranian male patient. Novel clinical features, including choanal atresia and clubfoot, were also identified. A comprehensive literature review identified 27 patients with YHFS, with 20 variants in the TELO2 gene. Missense pathogenic variants were the most common type of pathogenic variant, and the most common features were microcephaly and intellectual impairment. This study presents the first case of pathogenic variants in TELO2 genein Iran, expands the genotypic and phenotypic spectrum of YHFS and contributes to the growing body of literature pertaining to YHFS. Furthermore, our findings highlight the importance of genetic testing for non-consanguineous carrier screening, as compound heterozygosity may be a significant factor in the development of YHFS. Further research is needed to clarify the molecular mechanisms underlying YHFS pathogenesis.

The analyses of human MCPH1 DNA repair machinery and genetic variations.

Causal mutations in the MCPH1 gene have been associated with disorders like microcephaly, and recently congenital hearing impairment. This study examined the MCPH1 DNA repair machinery and identified genetic variations of interest in gnomAD database to discuss the biological roles and effects of rare variants in MCPH1-related diseases. Notably, MCPH1 coordinates two of the seven known mechanisms of DNA repair which confirmed its roles in neurogenesis and chromatin condensation. A pathogenic missense variant in MCPH1 p.Gly753Arg, and two pathogenic frameshifts MCPH1 p.Asn189LysfsTer15 and p.Cys624Ter identified in this study, already had entries in ClinVar and were associated with microcephaly. A pathogenic frameshift in MCPH1 p.Val10SerfsTer5 with a loss-of-function flag and a pathogenic stop gained p.Ser571Ter variants with ultra-rare allele frequency (MAF ≤ 0.001) were identified but have not been linked to any phenotype. The predicted pathogenic ultra-rare variants identified in this study, warranty phenotypic discovery, and also positioned these variants or nearby deleterious variants candidate for screening in MCPH1-associated rare diseases.