Novel protein-truncating variants of a chromatin-modifying gene MSL2 in syndromic neurodevelopmental disorders.

Numerous large scale genomic studies have uncovered rare but recurrent pathogenetic variants in a significant number of genes encoding epigenetic machinery in cases with neurodevelopmental disorders (NDD) especially autism spectrum disorder (ASD). These findings provide strong support for the functional importance of epigenetic regulators in neurodevelopment. After the clinical genomics evaluation of the patients using exome sequencing, we have identified, three novel protein-truncating variants (PTVs) in the MSL2 gene (OMIM: 614802) which encodes a chromatin modifying enzyme. MSL2 modifies chromatin through both mono-ubiquitination of histone 2B on lysine 34 (K34) and acetylation of histone H4 on lysine 16 (K16). We reported first time the detailed clinical features associated with 3 MSL2 PTVs. There are 15 PTVs (13 de novo) reported from the large genomics studies (12 cases) or ClinVar (3 cases) of NDD, ASD, and developmental disorders (DD) but the specific clinical features for these cases are not described. Taken together, our descriptions of dysmorphic face and other features support the causal role of MSL2 in a likely syndromic neurodevelopmental disorder and add MSL2 to a growing list of epigenetic genes implicated in ASD.

Diagnostic yield of exome and genome sequencing after non-diagnostic multi-gene panels in patients with single-system diseases.

BACKGROUND: Though next-generation sequencing (NGS) tests like exome sequencing (ES), genome sequencing (GS), and panels derived from exome and genome data (EGBP) are effective for rare diseases, the ideal diagnostic approach is debated. Limited research has explored reanalyzing raw ES and GS data post-negative EGBP results for diagnostics. RESULTS: We analyzed complete ES/GS raw sequencing data from Mayo Clinic's Program for Rare and Undiagnosed Diseases (PRaUD) patients to assess whether supplementary findings could augment diagnostic yield. ES data from 80 patients (59 adults) and GS data from 20 patients (10 adults), averaging 43 years in age, were analyzed. Most patients had renal (n=44) and auto-inflammatory (n=29) phenotypes. Ninety-six cases had negative findings and in four cases additional genetic variants were found, including a variant related to a recently described disease (RRAGD-related hypomagnesemia), a variant missed due to discordant inheritance pattern (COL4A3), a variant with high allelic frequency (NPHS2) in the general population, and a variant associated with an initially untargeted phenotype (HNF1A). CONCLUSION: ES and GS show diagnostic yields comparable to EGBP for single-system diseases. However, EGBP's limitations in detecting new disease-associated genes underscore the necessity for periodic updates.

Identification of skewed X chromosome inactivation using exome and transcriptome sequencing in patients with suspected rare genetic disease.

BACKGROUND: X-chromosome inactivation (XCI) is an epigenetic process that occurs during early development in mammalian females by randomly silencing one of two copies of the X chromosome in each cell. The preferential inactivation of either the maternal or paternal copy of the X chromosome in a majority of cells results in a skewed or non-random pattern of X inactivation and is observed in over 25% of adult females. Identifying skewed X inactivation is of clinical significance in patients with suspected rare genetic diseases due to the possibility of biased expression of disease-causing genes present on the active X chromosome. The current clinical test for the detection of skewed XCI relies on the methylation status of the methylation-sensitive restriction enzyme (Hpall) binding site present in proximity of short tandem polymorphic repeats on the androgen receptor (AR) gene. This approach using one locus results in uninformative or inconclusive data for 10-20% of tests. Further, recent studies have shown inconsistency between methylation of the AR locus and the state of inactivation of the X chromosome. Herein, we develop a method for estimating X inactivation status, using exome and transcriptome sequencing data derived from blood in 227 female samples. We built a reference model for evaluation of XCI in 135 females from the GTEx consortium. We tested and validated the model on 11 female individuals with different types of undiagnosed rare genetic disorders who were clinically tested for X-skew using the AR gene assay and compared results to our outlier-based analysis technique. RESULTS: In comparison to the AR clinical test for identification of X inactivation, our method was concordant with the AR method in 9 samples, discordant in 1, and provided a measure of X inactivation in 1 sample with uninformative clinical results. We applied this method on an additional 81 females presenting to the clinic with phenotypes consistent with different hereditary disorders without a known genetic diagnosis. CONCLUSIONS: This study presents the use of transcriptome and exome sequencing data to provide an accurate and complete estimation of X-inactivation and skew status in a cohort of female patients with different types of suspected rare genetic disease.

OmicsFootPrint: a framework to integrate and interpret multi-omics data using circular images and deep neural networks.

The OmicsFootPrint framework addresses the need for advanced multi-omics data analysis methodologies by transforming data into intuitive two-dimensional circular images and facilitating the interpretation of complex diseases. Utilizing Deep Neural Networks and incorporating the SHapley Additive exPlanations (SHAP) algorithm, the framework enhances model interpretability. Tested with The Cancer Genome Atlas (TCGA) data, OmicsFootPrint effectively classified lung and breast cancer subtypes, achieving high Area Under Curve (AUC) scores - 0.98±0.02 for lung cancer subtype differentiation, 0.83±0.07 for breast cancer PAM50 subtypes, and successfully distinguishe between invasive lobular and ductal carcinomas in breast cancer, showcasing its robustness. It also demonstrated notable performance in predicting drug responses in cancer cell lines, with a median AUC of 0.74, surpassing existing algorithms. Furthermore, its effectiveness persists even with reduced training sample sizes. OmicsFootPrint marks an enhancement in multi-omics research, offering a novel, efficient, and interpretable approach that contributes to a deeper understanding of disease mechanisms.

Bi-allelic variants in CELSR3 are implicated in central nervous system and urinary tract anomalies.

CELSR3 codes for a planar cell polarity protein. We describe twelve affected individuals from eleven independent families with bi-allelic variants in CELSR3. Affected individuals presented with an overlapping phenotypic spectrum comprising central nervous system (CNS) anomalies (7/12), combined CNS anomalies and congenital anomalies of the kidneys and urinary tract (CAKUT) (3/12) and CAKUT only (2/12). Computational simulation of the 3D protein structure suggests the position of the identified variants to be implicated in penetrance and phenotype expression. CELSR3 immunolocalization in human embryonic urinary tract and transient suppression and rescue experiments of Celsr3 in fluorescent zebrafish reporter lines further support an embryonic role of CELSR3 in CNS and urinary tract formation.

Semiautomated approach focused on new genomic information results in time and effort-efficient reannotation of negative exome data.

Most rare disease patients (75-50%) undergoing genomic sequencing remain unsolved, often due to lack of information about variants identified. Data review over time can leverage novel information regarding disease-causing variants and genes, increasing this diagnostic yield. However, time and resource constraints have limited reanalysis of genetic data in clinical laboratories setting. We developed RENEW, (REannotation of NEgative WES/WGS) an automated reannotation procedure that uses relevant new information in on-line genomic databases to enable rapid review of genomic findings. We tested RENEW in an unselected cohort of 1066 undiagnosed cases with a broad spectrum of phenotypes from the Mayo Clinic Center for Individualized Medicine using new information in ClinVar, HGMD and OMIM between the date of previous analysis/testing and April of 2022. 5741 variants prioritized by RENEW were rapidly reviewed by variant interpretation specialists. Mean analysis time was approximately 20 s per variant (32 h total time). Reviewed cases were classified as: 879 (93.0%) undiagnosed, 63 (6.6%) putatively diagnosed, and 4 (0.4%) definitively diagnosed. New strategies are needed to enable efficient review of genomic findings in unsolved cases. We report on a fast and practical approach to address this need and improve overall diagnostic success in patient testing through a recurrent reannotation process.

Complex balanced intrachromosomal rearrangement involving PITX2 identified as a cause of Axenfeld-Rieger Syndrome.

Axenfeld-Rieger Syndrome (ARS) type 1 is a rare autosomal dominant condition characterized by anterior chamber anomalies, umbilical defects, dental hypoplasia, and craniofacial anomalies, with Meckel's diverticulum in some individuals. Here, we describe a clinically ascertained female of childbearing age with ARS for whom clinical targeted sequencing and deletion/duplication analysis followed by clinical exome and genome sequencing resulted in no pathogenic variants or variants of unknown significance in PITX2 or FOXC1. Advanced bioinformatic analysis of the genome data identified a complex, balanced rearrangement disrupting PITX2. This case is the first reported intrachromosomal rearrangement leading to ARS, illustrating that for patients with compelling clinical phenotypes but negative genomic testing, additional bioinformatic analysis are essential to identify subtle genomic abnormalities in target genes.

Nuclear Abnormalities in LMNA p.(Glu2Lys) Variant Segregating with LMNA-Associated Cardiocutaneous Progeria Syndrome.

The LMNA gene encodes lamin A and lamin C, which play important roles in nuclear organization. Pathogenic variants in LMNA cause laminopathies, a group of disorders with diverse phenotypes. There are two main groups of disease-causing variants: missense variants affecting dimerization and intermolecular interactions, and heterozygous substitutions activating cryptic splice sites. These variants lead to different disorders, such as dilated cardiomyopathy and Hutchinson-Gilford progeria (HGP). Among these, the phenotypic terms for LMNA-associated cardiocutaneous progeria syndrome (LCPS), which does not alter lamin A processing and has an older age of onset, have been described. Here, we present the workup of an LMNA variant of uncertain significance, NM_170707.2 c. 4G>A, p.(Glu2Lys), in a 36-year-old female with severe calcific aortic stenosis, a calcified mitral valve, premature aging, and a family history of similar symptoms. Due to the uncertainty of in silico predictions for this variant, an assessment of nuclear morphology was performed using the immunocytochemistry of stable cell lines to indicate whether the p.(Glu2Lys) had a similar pathogenic mechanism as a previously described pathogenic variant associated with LCPS, p.Asp300Gly. Indirect immunofluorescence analysis of nuclei from stable cell lines showed abnormal morphology, including lobulation and occasional ringed nuclei. Relative to the controls, p.Glu2Lys and p.Asp300Gly nuclei had significantly (p < 0.001) smaller average nuclear areas than controls (mean = 0.10 units, SD = 0.06 for p.Glu2Lys; and mean = 0.09 units, SD = 0.05 for p.Asp300Gly versus mean = 0.12, SD = 0.05 for WT). After functional studies and segregation studies, this variant was upgraded to likely pathogenic. In summary, our findings suggest that p.Glu2Lys impacts nuclear morphology in a manner comparable to what was observed in p.Asp300Gly cells, indicating that the variant is the likely cause of the LCPS segregating within this family.

Genomic epidemiology reveals the dominance of Hennepin County in the transmission of SARS-CoV-2 in Minnesota from 2020 to 2022.

IMPORTANCE: We analyzed over 22,000 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes of patient samples tested at Mayo Clinic Laboratories during a 2-year period in the COVID-19 pandemic, which included Alpha, Delta, and Omicron variants of concern to examine the roles and relationships of Minnesota virus transmission. We found that Hennepin County, the most populous county, drove the transmission of SARS-CoV-2 viruses in the state after including the formation of earlier clades including 20A, 20C, and 20G, as well as variants of concern Alpha and Delta. We also found that Hennepin County was the source for most of the county-to-county introductions after an initial predicted introduction with the virus in early 2020 from an international source, while other counties acted as transmission "sinks." In addition, major policies, such as the end of the lockdown period in 2020 or the end of all restrictions in 2021, did not appear to have an impact on virus diversity across individual counties.

Use of next-generation sequencing to detect mutations associated with antiviral drug resistance in cytomegalovirus.

Cytomegalovirus (CMV) is a significant cause of morbidity and mortality among immunocompromised hosts, including transplant recipients. Antiviral prophylaxis or treatment is used to reduce the incidence of CMV disease in this patient population; however, there is concern about increasing antiviral resistance. Detection of antiviral resistance in CMV was traditionally accomplished using Sanger sequencing of UL54 and UL97 genes, in which specific mutations may result in reduced antiviral activity. In this study, a novel next-generation sequencing (NGS) method was developed and validated to detect mutations in UL54/UL97 associated with antiviral resistance. Plasma samples (n = 27) submitted for antiviral resistance testing by Sanger sequencing were also analyzed using the NGS method. When compared to Sanger sequencing, the NGS assay demonstrated 100% (27/27) overall agreement for determining antiviral resistance/susceptibility and 88% (22/25) agreement at the level of resistance-associated mutations. The limit of detection of the NGS method was determined to be 500 IU/mL, and the lower threshold for detecting mutations associated with resistance was established at 15%. The NGS assay represents a novel laboratory tool that assists healthcare providers in treating patients who are infected with CMV harboring resistance-associated mutations and who may benefit from tailored antiviral therapy.

A somatic splice-site variant in PIK3R1 in a patient with vascular overgrowth and low immunoglobulin levels: A case report.

BACKGROUND: The PI3K/AKT pathway, extensively studied in cancer, is vital for regulating cell metabolism, differentiation, and proliferation. Pathogenic variants in the PIK3R1 gene, which encodes three regulatory units of class IA PI3Ks, have been found in affected tissue of individuals with vascular lesions. These variants predominantly occur in the iSH2 domain, disrupting inhibitory contacts with the catalytic unit and leading to PI3K activation. Germline variants in this gene are also linked to an immunological condition called Activated PI3K delta syndrome type 2 (APDS2). METHODS: This is a case report and literature review. Clinical data were retrieved from medical records. RESULTS: A male patient presented with extensive vascular malformation covering over 90% of his body, along with complete 2-3 toe syndactyly, suggesting a vascular malformation syndrome called PROS. Low levels of IgA and IgG were detected. The patient achieved his developmental milestones and had above-average weight, height, and head circumference. Exome sequencing of skin and blood DNA revealed a de novo variant in PIK3R1 (c.1746-2A>G, p.?) in 9% of the patient's blood cells and 25% of cultured fibroblasts. Initially, classified as a variant of uncertain significance, this variant was later confirmed to be the cause. CONCLUSIONS: This is the first intronic SNV in a canonical splice site within iSH2 described, highlighting the importance of iSH2 in the regulation of the PI3K/AKT pathway and its involvement in the development of vascular overgrowth and antibody deficiency.

Dominant-negative heterozygous mutations in AIRE confer diverse autoimmune phenotypes.

Autoimmune polyendocrine syndrome type 1 (APS-1) is an autosomal recessive disease characterized by severe and childhood onset organ-specific autoimmunity caused by mutations in the autoimmune regulator (AIRE) gene. More recently, dominant-negative mutations within the PHD1, PHD2, and SAND domains have been associated with an incompletely penetrant milder phenotype with later onset familial clustering, often masquerading as organ-specific autoimmunity. Patients with immunodeficiencies or autoimmunity where genetic analyses revealed heterozygous AIRE mutations were included in the study and the dominant-negative effects of the AIRE mutations were functionally assessed in vitro. We here report additional families with phenotypes ranging from immunodeficiency, enteropathy, and vitiligo to asymptomatic carrier status. APS-1-specific autoantibodies can hint to the presence of these pathogenic AIRE variants although their absence does not rule out their presence. Our findings suggest functional studies of heterozygous AIRE variants and close follow-up of identified individuals and their families.

Bi-allelic variants in HMGCR cause an autosomal-recessive progressive limb-girdle muscular dystrophy.

Statins are a mainstay intervention for cardiovascular disease prevention, yet their use can cause rare severe myopathy. HMG-CoA reductase, an essential enzyme in the mevalonate pathway, is the target of statins. We identified nine individuals from five unrelated families with unexplained limb-girdle like muscular dystrophy and bi-allelic variants in HMGCR via clinical and research exome sequencing. The clinical features resembled other genetic causes of muscular dystrophy with incidental high CPK levels (>1,000 U/L), proximal muscle weakness, variable age of onset, and progression leading to impaired ambulation. Muscle biopsies in most affected individuals showed non-specific dystrophic changes with non-diagnostic immunohistochemistry. Molecular modeling analyses revealed variants to be destabilizing and affecting protein oligomerization. Protein activity studies using three variants (p.Asp623Asn, p.Tyr792Cys, and p.Arg443Gln) identified in affected individuals confirmed decreased enzymatic activity and reduced protein stability. In summary, we showed that individuals with bi-allelic amorphic (i.e., null and/or hypomorphic) variants in HMGCR display phenotypes that resemble non-genetic causes of myopathy involving this reductase. This study expands our knowledge regarding the mechanisms leading to muscular dystrophy through dysregulation of the mevalonate pathway, autoimmune myopathy, and statin-induced myopathy.

εγ-Thalassemia, a New Hemoglobinopathy Category.

BACKGROUND: Large ß-globin gene cluster deletions (hereditary persistence of fetal hemoglobin [Hb] or ß-, δß-, γδß-, and ϵγδß-thalassemia), are associated with widely disparate phenotypes, including variable degrees of microcytic anemia and Hb F levels. When present, increased Hb A2 is used as a surrogate marker for ß-thalassemia. Notably, ϵγδß-thalassemias lack the essential regulatory locus control region (LCR) and cause severe transient perinatal anemia but normal newborn screen (NBS) results and Hb A2 levels. Herein, we report a novel deletion of the ϵ, Aγ, Gγ, and ψß loci with intact LCR, δ-, and ß-regions in 2 women and newborn twins. METHODS: Capillary electrophoresis (CE), high-performance liquid chromatography (HPLC), DNA sequencing, multiplex ligation-dependent probe amplification (MLPA), gap-polymerase chain reaction (gap-PCR), and long-read sequencing (LRS) were performed. RESULTS: NBS showed an Hb A > Hb F pattern for both twins. At 20 months, Hb A2 was increased similarly to that in the mother and an unrelated woman. Unexplained microcytosis was absent and the twins lacked severe neonatal anemia. MLPA, LRS, and gap-PCR confirmed a 32 599 base pair deletion of ϵ (HBE1) through ψß (HBBP1) loci. CONCLUSIONS: This deletion represents a hemoglobinopathy category with a distinct phenotype that has not been previously described, an ϵγ-thalassemia. Both the NBS Hb A > F pattern and the subsequent increased Hb A2 without microcytosis are unusual. A similar deletion should be considered when this pattern is encountered and appropriate test methods selected for detection. Knowledge of the clinical impact of this new category will improve genetic counselling, with distinction from the severe transient anemia associated with ϵγδß-thalassemia.

Bi-allelic variants in INTS11 are associated with a complex neurological disorder.

The Integrator complex is a multi-subunit protein complex that regulates the processing of nascent RNAs transcribed by RNA polymerase II (RNAPII), including small nuclear RNAs, enhancer RNAs, telomeric RNAs, viral RNAs, and protein-coding mRNAs. Integrator subunit 11 (INTS11) is the catalytic subunit that cleaves nascent RNAs, but, to date, mutations in this subunit have not been linked to human disease. Here, we describe 15 individuals from 10 unrelated families with bi-allelic variants in INTS11 who present with global developmental and language delay, intellectual disability, impaired motor development, and brain atrophy. Consistent with human observations, we find that the fly ortholog of INTS11, dIntS11, is essential and expressed in the central nervous systems in a subset of neurons and most glia in larval and adult stages. Using Drosophila as a model, we investigated the effect of seven variants. We found that two (p.Arg17Leu and p.His414Tyr) fail to rescue the lethality of null mutants, indicating that they are strong loss-of-function variants. Furthermore, we found that five variants (p.Gly55Ser, p.Leu138Phe, p.Lys396Glu, p.Val517Met, and p.Ile553Glu) rescue lethality but cause a shortened lifespan and bang sensitivity and affect locomotor activity, indicating that they are partial loss-of-function variants. Altogether, our results provide compelling evidence that integrity of the Integrator RNA endonuclease is critical for brain development.

Human PIK3R1 mutations disrupt lymphocyte differentiation to cause activated PI3Kδ syndrome 2.

Heterozygous loss-of-function (LOF) mutations in PIK3R1 (encoding phosphatidylinositol 3-kinase [PI3K] regulatory subunits) cause activated PI3Kδ syndrome 2 (APDS2), which has a similar clinical profile to APDS1, caused by heterozygous gain-of-function (GOF) mutations in PIK3CD (encoding the PI3K p110δ catalytic subunit). While several studies have established how PIK3CD GOF leads to immune dysregulation, less is known about how PIK3R1 LOF mutations alter cellular function. By studying a novel CRISPR/Cas9 mouse model and patients' immune cells, we determined how PIK3R1 LOF alters cellular function. We observed some overlap in cellular defects in APDS1 and APDS2, including decreased intrinsic B cell class switching and defective Tfh cell function. However, we also identified unique APDS2 phenotypes including defective expansion and affinity maturation of Pik3r1 LOF B cells following immunization, and decreased survival of Pik3r1 LOF pups. Further, we observed clear differences in the way Pik3r1 LOF and Pik3cd GOF altered signaling. Together these results demonstrate crucial differences between these two genetic etiologies.

Identification of AFG3L2 dominant optic atrophy following reanalysis of clinical exome sequencing.

Purpose: To highlight the importance of the utility of clinical exome sequencing, and show how it led to the diagnosis of nonsyndromic autosomal dominant optic atrophy arising from an autosomal dominant variant in AFG3L2. Observations: A healthy father and daughter of East African heritage experienced the onset of vision loss in the first decade of life due to optic atrophy. No additional neurologic or neuroimaging abnormalities were detected. Clinical exome sequencing was initially performed and provided a negative result. Reanalysis of the sequencing data revealed an autosomal dominant pathogenic variant in AFG3L2, c.1064C>T (p.Thr355Met), a gene that was recently identified to be associated with non-syndromic optic atrophy. This variant has previously been reported in a patient with optic atrophy, motor disturbances, and an abnormal brain MRI. Conclusions: As the causes of dominant optic atrophy continue to expand, accurate genetic diagnosis is aided by an iterative reanalysis process for individuals and families when initial exome and genome testing does not provide an answer.

Further clinical and molecular characterization of an XLID syndrome associated with BRWD3 variants, a gene implicated in the leukemia-related JAK-STAT pathway.

BACKGROUND: Since the first description of a BRWD3-associated nonsydromic intellectual disability (ID) disorder in 2007, 21 additional families have been reported in the literature. METHODS: Using exome sequencing (ES) and international data sharing, we identified 14 additional unrelated individuals with pathogenic BRWD3 variants (12 males and 2 females, including one with skewed X-inactivation). We reviewed the 31 previously published cases in the literature with clinical data available, and describe the collective phenotypes of 43 males and 2 females, with 33 different BRWD3 variants. RESULTS: The most common features in males (excluding one patient with a mosaic variant) included ID (39/39 males), speech delay (24/25 males), postnatal macrocephaly (28/35 males) with prominent forehead (18/25 males) and large ears (14/26 males), and obesity (12/27 males). Both females presented with macrocephaly, speech delay, and epilepsy, while epilepsy was only observed in 4/41 males. Among the 28 variants with available segregation reported, 19 were inherited from unaffected mothers and 9 were de novo. CONCLUSION: This study demonstrates that the BRWD3-related phenotypes are largely non-specific, leading to difficulty in clinical recognition of this disorder. A genotype-first approach, however, allows for the more efficient diagnosis of the BRWD3-related nonsyndromic ID. The refined clinical features presented here may provide additional diagnostic assistance for reverse phenotyping efforts.