Mosaic Mutations Research Articles

Somatic mosaicism in schizophrenia brains reveals prenatal mutational processes.

Germline mutations modulate the risk of developing schizophrenia (SCZ). Much less is known about the role of mosaic somatic mutations in the context of SCZ. Deep (239×) whole-genome sequencing (WGS) of brain neurons from 61 SCZ cases and 25 controls postmortem identified mutations occurring during prenatal neurogenesis. SCZ cases showed increased somatic variants in open chromatin, with increased mosaic CpG transversions (CpG>GpG) and T>G mutations at transcription factor binding sites (TFBSs) overlapping open chromatin, a result not seen in controls. Some of these variants alter gene expression, including SCZ risk genes and genes involved in neurodevelopment. Although these mutational processes can reflect a difference in factors indirectly involved in disease, increased somatic mutations at developmental TFBSs could also potentially contribute to SCZ.

Low-level mosaic GCK mutations in children with diazoxide-unresponsive congenital hyperinsulinism.

Some children with diazoxide-unresponsive congenital hyperinsulinism (HI) lack any detectable disease-causing mutation in peripheral blood DNA. To examine whether somatic post-zygotic mutations of known HI genes are responsible for disease in children with diazoxide-unresponsive HI requiring surgery with histology not classified as focal or Localized Islet Nuclear Enlargement (LINE), and without detectable mutations by standard genetic testing of peripheral blood DNA. Next-generation sequencing (NGS) was performed on specimens of pancreas from 10 children with diazoxide-unresponsive HI. Four unique GCK mutations were identified at low levels of mosaicism ranging from 4.4-10.1% in pancreatic DNA from five of these 10 children. The GCK mutations were not detectable in peripheral blood DNA by NGS in three cases from which peripheral blood DNA was available for testing. All four GCK mutations have been previously published as activating HI mutations. The histology was consistent with diffuse-HI in four of the five cases with mosaic GCK mutations. In one of these, hypomethylation of IC2 on chromosome 11p was identified in pancreatic and peripheral blood DNA. Histology of the fifth case revealed minor islet abnormalities suggestive of Beckwith Wiedemann Spectrum (BWSp) although molecular analysis for 11pUPD was negative in pancreas. These results indicate that post-zygotic somatic GCK mutations are responsible for some cases of non-focal diazoxide-unresponsive hyperinsulinism.

A Simple Model to Study Mosaic Gene Expression in 3D Endothelial Spheroids.

The goal of this study was to establish a simple model of 3D endothelial spheroids with mosaic gene expression using adeno-associated virus (AAV) transduction, with a future aim being to study the activity of post-zygotic mutations common to vascular malformations. In this study, 96-well U-bottom plates coated with a commercial repellent were seeded with two immortalized human endothelial cell lines and aggregation monitored using standard microscopy or live-cell analysis. The eGFP expression was used to monitor the AAV transduction. HUVEC-TERT2 could not form spheroids spontaneously. The inclusion of collagen I in the growth medium could stimulate cell aggregation; however, these spheroids were not stable. In contrast, the hCMEC/D3 cells aggregated spontaneously and formed reproducible, robust 3D spheroids within 3 days, growing steadily for at least 4 weeks without the need for media refreshment. The hCMEC/D3 spheroids spontaneously developed a basement membrane, including collagen I, and expressed endothelial-specific CD31 at the spheroid surface. Serotypes AAV1 and AAV2QUADYF transduced these spheroids without toxicity and established sustained, mosaic eGFP expression. In the future, this simple approach to endothelial spheroid formation combined with live-cell imaging could be used to rapidly assess the 3D phenotypes and drug and radiation sensitivities arising from mosaic mutations common to brain vascular malformations.

Advances in the Treatment of Congenital Infiltrating Lipomatosis of the Face: The Role of PIK3CA Mutations and Microvascular Reconstruction

Congenital infiltrating lipomatosis of the face is a rare aggressive-benign disorder characterized by progressive hemifacial overgrowth and complex, often asymmetrical, facial differences. Recently linked with the PIK3CA-Related Overgrowth Spectrum, it arises from mosaic mutations in the PIK3CA gene. Treatment, largely supportive and tailored to individual clinical presentations, requires a multidisciplinary approach. This article reviews the longitudinal care of a young adult patient from 8 years of age, detailing his journey through combined orthognathic and microvascular reconstructive surgery and the outcomes thereof. In addition, it explores the treatment’s implications in the context of the PIK3CA mutation, offering insights into potential prognostic and therapeutic considerations.

Mutation in the mitochondrial chaperone TRAP1 leads to autism with more severe symptoms in males.

There is increasing evidence of mitochondrial dysfunction in autism spectrum disorders (ASD), but the causal relationships are unclear. In an ASD patient whose identical twin was unaffected, we identified a postzygotic mosaic mutation p.Q639* in the TRAP1 gene, which encodes a mitochondrial chaperone of the HSP90 family. Additional screening of 176 unrelated ASD probands revealed an identical TRAP1 variant in a male patient who had inherited it from a healthy mother. Notably, newly generated knock-in Trap1 p.Q641* mice display ASD-related behavioral abnormalities that are more pronounced in males than in females. Accordingly, Trap1 p.Q641* mutation also resulted in sex-specific changes in synaptic plasticity, the number of presynaptic mitochondria, and mitochondrial respiration. Thus, the TRAP1 p.Q639* mutation is the first example of a monogenic ASD caused by impaired mitochondrial protein homeostasis.

Constitutional mosaicism of pathogenic variants in SMARCB1 in a subset of patients with sporadic rhabdoid tumors.

Malignant rhabdoid tumors are aggressive malignancies predominantly affecting very young children. The characteristic genetic alteration is biallelic inactivation of SMARCB1. In approximately 30% of patients one SMARCB1 allele is constitutionally altered conferring a particularly unfavourable prognosis. Constitutional mosaicism for pathogenic SMARCB1 mutations has recently been reported in distinct cases of allegedly sporadic rhabdoid tumors. We aimed to systematically investigate the frequency and clinical impact of constitutional mosaicism in patients with sporadic rhabdoid tumors included in the EU-RHAB registry. We selected 29 patients with rhabdoid tumors displaying at least one pathogenic small variant in SMARCB1 in the tumor DNA and absence of a germline mutation. We re-screened blood-derived patient and control DNA for the respective small variant by PCR with unique molecular identifiers and ultra-deep next generation sequencing. Clinical data in patients with and without mosaicism and 174 EU-RHAB controls were compared. Employing an ultra-deep sequencing approach, we detected tumor-associated SMARCB1 variants in blood-derived DNA in 9/29 patients. In 6/29 patients (21%), whose variant allele frequency (VAF) exceeded 2%, constitutional mosaicism was assumed whereas tumor DNA contamination was documented in 1/3 patients with VAF below 1%. No significant differences were observed between 6 mosaic-positive and 20 -negative patients regarding age at diagnosis, presence of metastases, event-free or overall survival. Constitutional mosaicism for pathogenic small SMARCB1 variants is recurrent in patients with allegedly sporadic rhabdoid tumors. The clinical implications of such variants need to be determined in larger, prospective cohorts also including detection of structural variants of SMARCB1.

TP53 variants underlying pediatric low-hypodiploidy B-cell acute lymphoblastic leukemia demonstrate diverse origins and may persist as a hematopoietic clone in remission.

Pediatric low-hypodiploidy B-cell acute lymphoblastic leukemia (LH-ALL) with TP53 variants has been proposed to be considered a manifestation of Li-Fraumeni syndrome (LFS). However, our study demonstrates that of the majority the pathogenic variants in the TP53 gene are somatic (70.5%), and only 12.5% of patients with germline fulfilled the criteria of LFS. We also describe the first case of hypodiploid BCP-ALL with a mosaic pathogenic mutation in TP53 and the first case of the persistence of clonal hematopoiesis with the TР53 gene mutation in the child during 3-year minimal residual disease-negative remission, similar to what has been described in adults.

Mosaic SUFU mutation associated with a mild phenotype of multiple hereditary infundibulocystic basal cell carcinoma syndrome.

Multiple hereditary infundibulocystic basal cell carcinoma syndrome (MHIBCC), an autosomal dominant disorder caused by variants in SUFU, is characterized by numerous infundibulocystic basal cell carcinomas (IBCCs). In this report, we present a possible case of mosaic MHIBCC. A 57-year-old woman underwent the removal of four papules on her face, which were diagnosed as IBCCs. Exome sequencing revealed a SUFU c.1022+1G>A mutation within the skin tumor. The same mutation was detected in her blood but at a lower allele frequency. TA cloning revealed that the allele frequency of the mutation in the blood was 0.07. Additionally, tumor assessment revealed loss of heterozygosity (LOH) in chromosome 10, including the SUFU locus. These results indicate the patient had mosaicism for the SUFU mutation in normal tissues, aligning with the mosaic MHIBCC diagnosis. This, combined with LOH, likely contributed to IBCC development. Mosaic MHIBCC may present with milder symptoms. However, it may still increase the risk of developing brain tumors and more aggressive basal cell carcinoma. The possibility of mosaicism should be investigated in mild MHIBCC cases, where standard genetic tests fail to detect SUFU germline variants.

Phenotypic and genotypic characteristics of children with PCDH19 clustering epilepsy in China

PurposePCDH19 gene variants, termed PCDH19 clustering epilepsy, represent a distinct etiology of epilepsy. This study aimed to elucidate the clinical manifestations and explore the genotypes and phenotypes of children affected by PCDH19 clustering epilepsy. MethodsThis retrospective study included medical history, magnetic resonance imaging, video-electroencephalography, and genetic analysis of patients diagnosed with PCDH19 Clustering Epilepsy at the Neurology Department of Beijing Children's Hospital from 2015 to 2023. Chi-square tests and logistic regression analyses were conducted to study the factors associated with developmental delay in patients. ResultsThe age at seizure onset ranged from 5 to 61 months among all 30 patients (median 14 months; IQR 9.25–22.5 months). Among the 30 patients, 29 were female and 1 was male. Clusters of seizures and fever-triggered seizures were observed, with the most prevalent seizure types being focal to bilateral tonic-clonic seizures (FBTCS). Seizures were successfully controlled in 15 patients. Unfortunately, one patient experienced a sudden unexpected death in epilepsy (SUDEP). Additionally, 14 patients had hereditary mutations, 14 had de novo mutations, 1 had both hereditary and de novo mutations, and 1 male patient had a mosaic component mutation of 0.64 due to a somatic mutation. Developmental delays were identified in 17 patients (56.7 %), and 6 patients (20 %) were diagnosed with autism spectrum disorder (ASD). Among the 17 patients, 9 experienced developmental delays before the onset of epilepsy, while 8 were initially normal but later developed developmental delays during disease progression. Statistical analysis revealed that the presence of drug-resistant epilepsy was an independent risk factor for the occurrence of developmental delays (P = 0.020, OR = 9.758, 95 % CI (1.440–66.111)). ConclusionIn this study, 13 new potential rare pathogenic variations in PCDH19 clustering epilepsy were identified. The clinical features observed in patients are consistent with known phenotypic features, and we found that patients with drug-resistant epilepsy are more likely to have developmental delays. The severity of the phenotype in patients with PCDH19 variants ranged from drug-responsive seizures to refractory epilepsy.

Sotorasib for Vascular Malformations Associated with KRAS G12C Mutation.

KRAS gain-of-function mutations are frequently observed in sporadic arteriovenous malformations. The mechanisms underlying the progression of such KRAS-driven malformations are still incompletely understood, and no treatments for the condition are approved. Here, we show the effectiveness of sotorasib, a specific KRAS G12C inhibitor, in reducing the volume of vascular malformations and improving survival in two mouse models carrying a mosaic Kras G12C mutation. We then administered sotorasib to two adult patients with severe KRAS G12C-related arteriovenous malformations. Both patients had rapid reductions in symptoms and arteriovenous malformation size. Targeting KRAS G12C appears to be a promising therapeutic approach for patients with KRAS G12C-related vascular malformations. (Funded by the European Research Council and others.).

Effects of centrifugation treatment before electroporation on gene editing in pig embryos.

Genetic mosaicism, characterized by multiple genotypes within an individual, is considered an obstacle to CRISPR/Cas9 genome editing in animal models. Despite the various strategies for minimizing mosaic mutations, no definitive methods exist to eliminate them. This study aimed to enhance gene editing efficiency in porcine zygotes using CRISPR/Cas9, which targets specific genes through centrifugation and zona pellucida removal before electroporation. Centrifugation at 2000 × g did not adversely affect blastocyst formation rates in zygotes electroporated with gRNA targeting the GGTA1 gene; instead, it led to increased total and monoallelic mutation rates compared with control zygotes without centrifugation. However, the groups had no significant differences in biallelic mutation rates. In zygotes electroporated with gRNA targeting the CMAH gene, centrifugation treatments exceeding 1000 × g significantly increased both biallelic mutation rates and mutation efficiency. The combination of centrifugation and zona pellucida removal did not have a detrimental effect on blastocyst formation rates. It led to a higher rate of double biallelic mutations in embryos targeting both GGTA1 and CMAH compared to embryos without centrifugation treatment. In summary, our results demonstrate that pre-electroporation treatments, including centrifugation and zona pellucida removal, positively influenced the reduction of mosaic mutations, with the effectiveness of centrifugation depending on the specific gRNA used.

Sperm Mosaic Variants and Their Influence on the Offspring

Genomic mosaicism arising from mosaic variants is a phenomenon that describes the presence of a cell or cell populations with different genome compositions from the germline cells of an individual. It comprises all types of genetic variants. A large proportion of childhood genetic disorders are defined as being de novo, meaning that the disease-causing mutations are only detected in the proband, not in any of the parents. Population studies show that 80% of the de novo mutations arise from the paternal haplotype, that is, from paternal sperm mosaicism. This review provides a summary of the types and detection strategies of sperm mosaicism. In addition, it provides discussions on how recent studies demonstrated that genomic mosaic mutations in parents, especially those in the paternal sperms, could be inherited by the offspring and cause childhood disorders. According to the previous findings of the author's research team, sperm mosaicism derived from early embryogenesis and primordial germ cell stages can explain 5% to 20% of the de novo mutations related to clinical phenotypes and can serve as an important predictor of both rare and complex disorders. Sperm mosaicism shows great potential for clinical genetic diagnosis and consultations. Based on the published literature, the author suggests that, large-scale screening for de novo sperm mosaic mutations and population-based genetic screening should be conducted in future studies, which will greatly enhance the risk assessment in the offspring and effectively improve the genetic health at the population level. Implementation of direct sperm detection for de novo mutations will significantly increase the efficiency of the stratification of patient cohorts and improve recurrence risk assessment for future births. Future research in the field should be focused on the impact of environmental and lifestyle factors on the health of the offspring through sperms and their modeling of mutation signatures. In addition, targeted in vitro modeling of sperm mutations will also be a promising direction.

Abstract 994: Single analyte profiling of the mutational, immune and microbiome landscape in african american colon cancer patients

Abstract Colorectal cancer (CRC) disproportionately affects African American (AA) patients who experience a higher incidence and mortality than any other demographic groups in the United States. Yet molecular profiling in this population is scarce. Here, we performed whole-exome sequencing (WES) of 342 immediately snap-frozen tissues comprising AA CRC and matched, distant normal colon and identified distinct genomic drivers, DNA signatures, and, surprisingly, mosaic pathogenic mutations that are not present in the patient-matched CRC. We inferred T cell infiltration and tissue-resident microbiome (TR-M) directly from WES, enabling integrative analyses. Compared to normal colon, T cell infiltration is strongly decreased in tumours with microsatellite stability (MSS), while microsatellite unstable (MSI) and Polymerase Epsilon (POLE) mutant tumours had similar levels, suggesting immune evasion in MSS rather than increased T cell infiltration in MSI/POLE-mutants. Analyses of matched normal and tumour tissues revealed the evolving TR-M at unprecedented granularity and identified distinct niches of interactions with genomic and tumour-microenvironmental features. Together, this study represents the largest comprehensive genomic analysis of CRC in AA and provides a framework for performing a multi-layered analysis from a single analyte, thus, affording a cost-effective approach to expedite medical discoveries in underrepresented ancestries. Citation Format: Carino Gurjao, Edmond Chan, Michael J. Lee, Sho Hangai, Raul Rabadan, Tal Korem, Hanina Hibshoosh, Benjamin Izar. Single analyte profiling of the mutational, immune and microbiome landscape in african american colon cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 994.

Clinical Characteristics of Cryopyrin-Associated Periodic Syndrome and Long-Term Real-World Efficacy and Tolerability of Canakinumab in Japan: Results of a Nationwide Survey.

We assess the clinical characteristics of patients with cryopyrin-associated periodic syndrome (CAPS) in Japan and evaluate the real-world efficacy and safety of interleukin-1 (IL-1) inhibitors, primarily canakinumab. Clinical information was collected retrospectively, and serum concentrations of canakinumab and cytokines were analyzed. A total of 101 patients were included, with 86 and 15 carrying heterozygous germline and somatic mosaic mutations, respectively. We identified 39 mutation types, and the common CAPS-associated symptoms corresponded with those in previous reports. Six patients (5.9% of all patients) died, with four of the deaths caused by CAPS-associated symptoms. Notably, 73.7% of patients (100%, 79.6%, and 44.4% of familial cold autoinflammatory syndrome, Muckle-Wells syndrome, and chronic infantile neurological cutaneous articular syndrome/neonatal onset multisystem inflammatory disease, respectively) achieved complete remission with canakinumab, and early therapeutic intervention was associated with better auditory outcomes. In some patients, canakinumab treatment stabilized the progression of epiphysial overgrowth and improved height gain, visual acuity, and renal function. However, 23.7% of patients did not achieve inflammatory remission with crucial deterioration of organ damage, with two dying while receiving high-dose canakinumab treatment. Serological analysis of canakinumab and cytokine concentrations revealed that the poor response was not related to canakinumab shortage. Four inflammatory nonremitters developed inflammatory bowel disease (IBD)-unclassified during canakinumab treatment. Dual biologic therapy with canakinumab and anti-tumor necrosis factor-α agents was effective for IBD- and CAPS-associated symptoms not resolved by canakinumab monotherapy. This study provides one of the largest epidemiologic data sets for CAPS. Although early initiation of anti-IL-1 treatment with canakinumab is beneficial for improving disease prognosis, some patients do not achieve remission despite a high serum concentration of canakinumab. Moreover, IBD may develop in CAPS after canakinumab treatment.

O06 Clinical phenotype of the Klippel-Trenaunay syndrome with mosaic PIK3R1 mutations

Abstract Segmental overgrowth syndromes with cutaneous vascular malformations (Klippel-Trenaunay syndrome (KTS), CLOVES syndrome) are mainly due to mosaic PIK3CA mutations, but the GNAQ, GNA11, HRAS, KRAS, MAP2K1, AKT1, AKT3, and PIK3R1 genes may also be involved. We sought to determine the frequency and phenotype of patients with PIK3R1-related segmental overgrowth. Patients were ascertained nationwide in the French MUSTARD cohort of skin mosaic phenotypes. We searched for PIK3R1 mutations by next-generation sequencing on affected tissue in 297 patients with a clinical presentation suggestive of PIK3CA related overgrowth (PROS), but without PIK3CA variants. We retrospectively studied the clinical phenotype of PIK3R1-positive patients. We identified three missense variants and six in-phase indels of PIK3R1 in 15 patients (5.0%), exclusively in affected tissue. Variant allele fraction was 2.0% to 12.0%. We found extensive pale or dark capillary malformations in 14 patients, segmental overgrowth in 11, venous truncal anomalies (varicose veins or persistent lateral marginal vein) in 10, lymphatic malformation in six and a café-­au-lait macule or naevus spilus in three, thus defining phacomatosis pigmento-vascularis (PPV). One patient had limb hypoplasia. None had MCAP (Megalencephaly-Capillary malformation). PIKR1 mutations account for a minority of patients with KTS/CLOVES (5%). They are about 20 times less common than PIK3CA mutations. Truncal venous malformations, limb hypoplasia and PPV (spilorosea or melanorosea type) may be more common in PIK3R1-related KTS. No patient had severe or life-threatening involvement. Whether Sirolimus or Alpelisib are treatment options remains to be determined.

VEGF Secretion Drives Bone Formation in Classical MAP2K1+ Melorheostosis.

Patients with classical melorheostosis exhibit exuberant bone overgrowth in the appendicular skeleton, resulting in pain and deformity with no known treatment. Most patients have somatic, mosaic mutations in MAP2K1 (encoding the MEK1 protein) in osteoblasts and overlying skin. As with most rare bone diseases, lack of affected tissue has limited the opportunity to understand how the mutation results in excess bone formation. The aim of this study was to create a cellular model to study melorheostosis. We obtained patient skin cells bearing the MAP2K1 mutation (affected cells), and along with isogenic control normal fibroblasts reprogrammed them using the Sendai virus method into induced pluripotent stem cells (iPSCs). Pluripotency was validated by marker staining and embryoid body formation. iPSCs were then differentiated to mesenchymal stem cells (iMSCs) and validated by flow cytometry. We confirmed retention of the MAP2K1 mutation in iMSCs with polymerase chain reaction (PCR) and confirmed elevated MEK1 activity by immunofluorescence staining. Mutation-bearing iMSCs showed significantly elevated vascular endothelial growth factor (VEGF) secretion, proliferation and collagen I and IV secretion. iMSCs were then differentiated into osteoblasts, which showed increased mineralization at 21 days and increased VEGF secretion at 14 and 21 days of differentiation. Administration of VEGF to unaffected iMSCs during osteogenic differentiation was sufficient to increase mineralization. Blockade of VEGF by bevacizumab reduced mineralization in iMSC-derived affected osteoblasts and in affected primary patient-derived osteoblasts. These data indicate that patient-derived induced pluripotent stem cells recreate the elevated MEK1 activity, increased mineralization, and increased proliferation seen in melorheostosis patients. The increased bone formation is driven, in part, by abundant VEGF secretion. Modifying the activity of VEGF (a known stimulator of osteoblastogenesis) represents a promising treatment pathway to explore. iPSCs may have wide applications to other rare bone diseases. © 2023 American Society for Bone and Mineral Research (ASBMR).

A case ofcryopyrin-associated periodic syndrome due to somatic mosaic mutation complicated with recurrent circinate erythematous psoriasis.

Cryopyrin-associated periotic syndrome (CAPS) is a rare autoinflammatory disease caused by genetic variants in innate immunity genes. Autoinflammatory diseases, including CAPS, mediate proinflammatory cytokines such as interleukin (IL)-1 and IL-18 and result in severe systemic inflammation. A gain-of-function mutation in the NLR family pyrin domain-containing 3 (NLRP3) gene, which encodes the protein cryopyrin, was identified to be responsible for CAPS in 2001, and since then several additional pathogenic mutations have been found. Moreover, other phenotypes have been identified based on severity and symptomatology, including familial cold autoinflammatory syndrome, Muckle-Wells syndrome, and neonatal-onset multisystem inflammatory disease/chronic neurologic cutaneous articular syndrome. Prompt diagnosis of CAPS remains challenging, however, due to unspecific, extensive clinical signs, and delayed diagnosis and treatment targeting IL-1 lead to multiorgan damage. Another factor complicating diagnosis is the existence of somatic mosaic mutations in the NLRP3 gene in some cases, resulting in symptoms and clinical courses that are atypical. The frequency of somatic mosaic mutations in CAPS was estimated to be 19% in a systematic review. Psoriasis is a chronic inflammatory skin disease that affects ∼3% of the global population. Although no reports have shown complication between CAPS and psoriasis, these diseases have several similarities and potential relationships, for instance activation of T helper 17 cells in the dermis and increased NLRP3 gene expression in psoriatic skin compared with normal skin. Here, we report a case of CAPS due to a somatic mosaic mutation with recurrent circinate erythematous psoriasis.

Asxl1-mutant Dominant Clones Have an Erythropoietic Lineage Deficiency through a Disturbed Epigenetic Landscape

Two hematologic features of red blood cells (RBCs) rank among the top three high risk factors associated with increased risk of malignant progression from clonal hematopoiesis (CH). These risk factors are macrocytosis as measured by elevated mean corpuscular volume (MCV) of 100 fL or greater and red cell distribution width (RDW) of 15% or greater. It is not known how these changes in RBC parameters are related to the leukemic transformation. Without major disturbance in the quantitative output of RBCs, these changes may reflect clone specific changes in erythropoiesis that are averaged in such measurements. We used an in vivo model of CH in zebrafish to study epigenetic and transcriptional changes in mutant clones, focusing on the erythropoietic program. In this model, mosaic somatic mutations in exon 12 of asxl1 with CRISPR-Cas9 mutagenesis are combined with color labeling of hematopoietic stem and progenitor cell (HSPC) clones. These mosaic asxl1 mutants exhibit an expansion of single-colored mutant clones to greater than 30% of hematopoietic output. No significant hematopoietic disturbance has been noted in these mutants by flow cytometric and morphologic analysis. To study clone specific effects of this epigenetic factor mutation, we sorted HSPCs from the dominant asxl1-mutant clone and compared it to HSPCs from control animals using transposase-accessible chromatin with sequencing (ATAC-seq) assay. Among over 22,000 peaks, we identified 998 (4.5%) that increased and 938 (4.2%) that decreased by 2-fold in asxl1-mutant clones compared to controls. Of the upregulated peaks, 73% were distal elements (+/- 100kb from transcriptional start sites, TSS) and 27% were proximal or promoter peaks (-2kb to +500bp from TSS), while in decreased peaks 67% were in distal elements and 33% in the proximal/promoter region. Pathway analysis of assigned genes related to decreased peaks identified a striking loss of the erythropoietic program and the heme biosynthesis process. HOMER analysis of sequences within the decreased peaks showed significant association with GATA ( q-value <0.001) and TRPS1 ( q-value <0.001) motifs. This was confirmed by individual assessment of gene loci for lineage-specifying transcriptional factors, such as tal1 and gata1a and genes in erythropoiesis, including alas2, cahz, tmod4 and the hemoglobin locus with known enhancers in nprl3 gene . Using an independently generated mutant zebrafish cohort, we performed bulk RNA-sequencing on sorted dominant clone and control HSPCs. Downregulated genes were consistent with a loss of erythroid differentiation program, with up to 16-fold downregulation of hbba1, hbaa1, alas2 and cahz although less pronounced or no significant changes were noted in erythroid transcription factors ( tal1, gata1a, lmo2) . Our data show that asxl1-mutant clones have an erythropoietic lineage defect with reduced epigenetic accessibility at lineage-specific loci with selective changes in gene expression. This ineffective erythropoiesis characterized by large MCV in mutant RBCs would increase the RDW due to the mixed contribution from mutant and wildtype clones in the peripheral blood. Our findings offer a biologic basis of the strong clinical association of abnormal RBC characteristics with higher risk CH for malignant transformation.

RAB1A haploinsufficiency phenocopies the 2p14–p15 microdeletion and is associated with impaired neuronal differentiation

Hereditary spastic parapareses (HSPs) are clinically heterogeneous motor neuron diseases with variable age of onset and severity. Although variants in dozens of genes are implicated in HSPs, much of the genetic basis for pediatric-onset HSP remains unexplained. Here, we re-analyzed clinical exome-sequencing data from siblings with HSP of unknown genetic etiology and identified an inherited nonsense mutation (c.523C>T [p.Arg175Ter]) in the highly conserved RAB1A. The mutation is predicted to produce a truncated protein with an intact RAB GTPase domain but without two C-terminal cysteine residues required for proper subcellular protein localization. Additional RAB1A mutations, including two frameshift mutations and a mosaic missense mutation (c.83T>C [p.Leu28Pro]), were identified in three individuals with similar neurodevelopmental presentations. In rescue experiments, production of the full-length, but not the truncated, RAB1a rescued Golgi structure and cell proliferation in Rab1-depleted cells. In contrast, the missense-variant RAB1a disrupted Golgi structure despite intact Rab1 expression, suggesting a dominant-negative function of the mosaic missense mutation. Knock-down of RAB1A in cultured human embryonic stem cell-derived neurons resulted in impaired neuronal arborization. Finally, RAB1A is located within the 2p14-p15 microdeletion syndrome locus. The similar clinical presentations of individuals with RAB1A loss-of-function mutations and the 2p14-p15 microdeletion syndrome implicate loss of RAB1A in the pathogenesis of neurodevelopmental manifestations of this microdeletion syndrome. Our study identifies a RAB1A-related neurocognitive disorder with speech and motor delay, demonstrates an essential role for RAB1a in neuronal differentiation, and implicates RAB1A in the etiology of the neurodevelopmental sequelae associated with the 2p14-p15 microdeletion syndrome.

Evaluating the clinical utility of a long-read sequencing-based approach in genetic testing of fragile-X syndrome

BackgroundFragile X syndrome (FXS) arises from the FMR1 CGG expansion. Comprehensive genetic testing for FMR1 CGG expansions, AGG interruptions, and microdeletions is essential to provide genetic counseling for females carrying premutation alleles. However, conventional PCR-based FMR1 assays mainly focus on CGG repeats, and could detect AGG interruption only in males. MethodsThe clinical utility of a long-read sequencing-based assay termed comprehensive analysis of FXS (CAFXS) was evaluated in 238 high-risk samples by comparing to conventional PCR assays. ResultsPCR assays identified five premuation and three full mutation categories alleles in all the samples, and CAFXS successfully called all the FMR1 CGG expansion. CAFXS identified 24-bp microdeletions upstream to the trinucleotide region with 30 CGG repeats, which was miscalled by the length-based PCR methods. CAFXS also identified a 187-bp deletion in about 1/7 of the sequencing reads in a male patient with mosaic full mutation alleles. CAFXS allowed for precise constructing the FMR1 CGG repeat and AGG interruption pattern in all the samples, and identified a novel and alternative CGA interruption in one normal female sample. ConclusionsCAFXS represents a more comprehensive and accurate approach for FXS genetic testing that potentially enables more informed genetic counseling compared to PCR-based methods.