Enabling the clinical application of artificial intelligence in genomics: a perspective of the AMIA Genomics and Translational Bioinformatics Workgroup.

OBJECTIVE: Given the importance AI in genomics and its potential impact on human health, the American Medical Informatics Association-Genomics and Translational Biomedical Informatics (GenTBI) Workgroup developed this assessment of factors that can further enable the clinical application of AI in this space. PROCESS: A list of relevant factors was developed through GenTBI workgroup discussions in multiple in-person and online meetings, along with review of pertinent publications. This list was then summarized and reviewed to achieve consensus among the group members. CONCLUSIONS: Substantial informatics research and development are needed to fully realize the clinical potential of such technologies. The development of larger datasets is crucial to emulating the success AI is achieving in other domains. It is important that AI methods do not exacerbate existing socio-economic, racial, and ethnic disparities. Genomic data standards are critical to effectively scale such technologies across institutions. With so much uncertainty, complexity and novelty in genomics and medicine, and with an evolving regulatory environment, the current focus should be on using these technologies in an interface with clinicians that emphasizes the value each brings to clinical decision-making.

Large Chromosome 2p Duplication-Associated Mechanisms and Clinical Presentations.

Chromosome 2p (chr2p) duplication, also known as trisomy 2p, is a rare chromosome abnormality associated with developmental delay, intellectual disability, behavioral problems, and distinctive facial features. Most of the reported cases involving trisomy 2p include additional copy number variants (CNVs) in other regions of the genome and are usually small in size. Little is known about the clinical outcomes of large duplications of chr2p as the sole cytogenetic abnormality. In this study, 193 samples at the Greenwood Genetic Center (GGC) with CNVs involving chr2p were evaluated, out of which 86 had chr2p duplications. Among them, 8 patients were identified with large chr2p duplications ranging in size from 9.3 Mb to 89 Mb, and no deletions or duplications involving other chromosomes were identified in those patients. These duplications were associated with inverted duplication, tandem duplication, and duplication as the result of translocation, with no additional CNVs identified by microarray analysis. Confirmation by conventional cytogenetics was performed in 7 of the 8 patients, and the translocations were confirmed by fluorescence in situ hybridization. Interestingly, 1 patient was found to have mosaic complete trisomy 2p as the result of an unbalanced de novo (X;2) chromosomal translocation. X-inactivation was skewed toward the derivative X chromosome, yet it did not appear to extend into the chromosome 2 material. Various shared clinical manifestations were observed in the individuals in this study, including developmental delay, hemifacial hypoplasia, cleft palate, and short stature, and they also have distinct features such as hypotonia, cerebellar hypogenesis, and corpus callosum agenesis, which might result from a gene dosage effect of the duplication. In conclusion, single-event large chr2p duplications can result from different mechanisms, including inverted or tandem duplications within chromosome 2, or translocations involving chromosome 2 and other chromosomes. Partial or complete trisomy 2p is commonly associated with developmental delay, and additional clinical features may be related to gene dosage effects.

The clinical and molecular spectrum of the KDM6B-related neurodevelopmental disorder.

De novo variants are a leading cause of neurodevelopmental disorders (NDDs), but because every monogenic NDD is different and usually extremely rare, it remains a major challenge to understand the complete phenotype and genotype spectrum of any morbid gene. According to OMIM, heterozygous variants in KDM6B cause "neurodevelopmental disorder with coarse facies and mild distal skeletal abnormalities." Here, by examining the molecular and clinical spectrum of 85 reported individuals with mostly de novo (likely) pathogenic KDM6B variants, we demonstrate that this description is inaccurate and potentially misleading. Cognitive deficits are seen consistently in all individuals, but the overall phenotype is highly variable. Notably, coarse facies and distal skeletal anomalies, as defined by OMIM, are rare in this expanded cohort while other features are unexpectedly common (e.g., hypotonia, psychosis, etc.). Using 3D protein structure analysis and an innovative dual Drosophila gain-of-function assay, we demonstrated a disruptive effect of 11 missense/in-frame indels located in or near the enzymatic JmJC or Zn-containing domain of KDM6B. Consistent with the role of KDM6B in human cognition, we demonstrated a role for the Drosophila KDM6B ortholog in memory and behavior. Taken together, we accurately define the broad clinical spectrum of the KDM6B-related NDD, introduce an innovative functional testing paradigm for the assessment of KDM6B variants, and demonstrate a conserved role for KDM6B in cognition and behavior. Our study demonstrates the critical importance of international collaboration, sharing of clinical data, and rigorous functional analysis of genetic variants to ensure correct disease diagnosis for rare disorders.

Clinical and functional characterization of germline PIK3CA variants in patients with PIK3CA-related overgrowth spectrum disorders.

Mosaic variants in the PIK3CA gene, encoding the catalytic subunit of phosphoinositide 3-kinase (PI3K), produce constitutive PI3K activation, which causes PIK3CA-related overgrowth spectrum disorders. To date, fewer than 20 patients have been described with germline alterations in PIK3CA. In this study, we describe three unrelated individuals with overgrowth and germline PIK3CA variants. These variants were discovered through whole-exome sequencing and confirmed as germline by testing multiple tissue types, when available. Functional analysis using Patient 1's fibroblast cell line and two previously reported patients' cell lines showed increased phosphorylation of AKT during cellular starvation revealing constitutive activation of the phosphoinositide-3-kinase/protein kinase B/mechanistic target of rapamycin (PI3K/AKT/mTOR) pathway. Alternatively, stimulation of the cells by fetal bovine serum produced a reduced response, indicating an activated status of the PI3K complex reducing the pathway response to further external stimulation. Additional studies utilizing Biolog Phenotype Microarray technology indicated reduced energy production when cells were exposed to growth factors stimulating the PI3K/AKT/mTOR pathway, confirming the trend observed in the AKT phosphorylation test after stimulation. Furthermore, treatment with inhibitors of the PI3K/AKT/mTOR pathway rescued the normal energy response in the patients' cells. Collectively, these data demonstrate that disease-causing germline PIK3CA variants have a functional consequence, similar to mosaic variants in the PI3K/AKT/mTOR pathway.

DNA methylation episignature for Witteveen-Kolk syndrome due to SIN3A haploinsufficiency.

PURPOSE: Witteveen-Kolk syndrome (WITKOS) is a rare, autosomal dominant neurodevelopmental disorder caused by heterozygous loss-of-function alterations in the SIN3A gene. WITKOS has variable expressivity that commonly overlaps with other neurodevelopmental disorders. In this study, we characterized a distinct DNA methylation epigenetic signature (episignature) distinguishing WITKOS from unaffected individuals as well as individuals with other neurodevelopmental disorders with episignatures and described 9 previously unpublished individuals with SIN3A haploinsufficiency. METHODS: We studied the phenotypic characteristics and the genome-wide DNA methylation in the peripheral blood samples of 20 individuals with heterozygous alterations in SIN3A. A total of 14 samples were used for the identification of the episignature and building of a predictive diagnostic biomarker, whereas the diagnostic model was used to investigate the methylation pattern of the remaining 6 samples. RESULTS: A predominantly hypomethylated DNA methylation profile specific to WITKOS was identified, and the classifier model was able to diagnose a previously unresolved test case. The episignature was sensitive enough to detect individuals with varying degrees of phenotypic severity carrying SIN3A haploinsufficient variants. CONCLUSION: We identified a novel, robust episignature in WITKOS due to SIN3A haploinsufficiency. This episignature has the potential to aid identification and diagnosis of individuals with WITKOS.

Mosaicism of common pathogenic MECP2 variants identified in two males with a clinical diagnosis of Rett syndrome.

Rett (RTT) syndrome, a neurodevelopmental disorder caused by pathogenic variation in the MECP2 gene, is characterized by developmental regression, loss of purposeful hand movements, stereotypic hand movements, abnormal gait, and loss of spoken language. Due to the X-linked inheritance pattern, RTT is typically limited to females. Recent studies revealed somatic mosaicism in MECP2 in male patients with RTT-like phenotypes. While detecting mosaic variation using Sanger sequencing is theoretically possible for mosaicism over ~15%-20%, several variables, including efficiency of PCR, background noise, and/or human error, contribute to a low detection rate using this technology. Mosaic variants in two males were detected by next generation sequencing (NGS; Case 1) and by Sanger re-sequencing (Case 2). Both had targeted digital PCR (dPCR) to confirm the variants. In this report, we present two males with classic RTT syndrome in whom we identified pathogenic variation in the MECP2 gene in the mosaic state (c.730C > T (p.Gln244*) in Patient 1 and c.397C > T (p.Arg133Cys) in Patient 2). In addition, estimates and measures of mosaic variant fraction were surprisingly similar between Sanger sequencing, NGS, and dPCR. The mosaic state of these variants contributed to a lengthy diagnostic odyssey for these patients. While NGS and even Sanger sequencing may be viable methods of detecting mosaic variation in DNA or RNA samples, applying targeted dPCR to supplement these sequencing technologies would provide confirmation of somatic mosaicism and mosaic fraction.

SEMA6B variants cause intellectual disability and alter dendritic spine density and axon guidance.

Intellectual disability (ID) is a neurodevelopmental disorder frequently caused by monogenic defects. In this study, we collected 14 SEMA6B heterozygous variants in 16 unrelated patients referred for ID to different centers. Whereas, until now, SEMA6B variants have mainly been reported in patients with progressive myoclonic epilepsy, our study indicates that the clinical spectrum is wider and also includes non-syndromic ID without epilepsy or myoclonus. To assess the pathogenicity of these variants, selected mutated forms of Sema6b were overexpressed in Human Embryonic Kidney 293T (HEK293T) cells and in primary neuronal cultures. shRNAs targeting Sema6b were also used in neuronal cultures to measure the impact of the decreased Sema6b expression on morphogenesis and synaptogenesis. The overexpression of some variants leads to a subcellular mislocalization of SEMA6B protein in HEK293T cells and to a reduced spine density owing to loss of mature spines in neuronal cultures. Sema6b knockdown also impairs spine density and spine maturation. In addition, we conducted in vivo rescue experiments in chicken embryos with the selected mutated forms of Sema6b expressed in commissural neurons after knockdown of endogenous SEMA6B. We observed that expression of these variants in commissural neurons fails to rescue the normal axon pathway. In conclusion, identification of SEMA6B variants in patients presenting with an overlapping phenotype with ID and functional studies highlight the important role of SEMA6B in neuronal development, notably in spine formation and maturation and in axon guidance. This study adds SEMA6B to the list of ID-related genes.

Structural model of human PORCN illuminates disease-associated variants and drug-binding sites.

Wnt signaling is essential for normal development and is a therapeutic target in cancer. The enzyme PORCN, or porcupine, is a membrane-bound O-acyltransferase (MBOAT) that is required for the post-translational modification of all Wnts, adding an essential mono-unsaturated palmitoleic acid to a serine on the tip of Wnt hairpin 2. Inherited mutations in PORCN cause focal dermal hypoplasia, and therapeutic inhibition of PORCN slows the growth of Wnt-dependent cancers. Based on homology to mammalian MBOAT proteins, we developed and validated a structural model of human PORCN. The model accommodates palmitoleoyl-CoA and Wnt hairpin 2 in two tunnels in the conserved catalytic core, shedding light on the catalytic mechanism. The model predicts how previously uncharacterized human variants of uncertain significance can alter PORCN function. Drugs including ETC-159, IWP-L6 and LGK-974 dock in the PORCN catalytic site, providing insights into PORCN pharmacologic inhibition. This structural model enhances our mechanistic understanding of PORCN substrate recognition and catalysis, as well as the inhibition of its enzymatic activity, and can facilitate the development of improved inhibitors and the understanding of disease-relevant PORCN mutants. This article has an associated First Person interview with the joint first authors of the paper.

DLG4-related synaptopathy: a new rare brain disorder.

PURPOSE: Postsynaptic density protein-95 (PSD-95), encoded by DLG4, regulates excitatory synaptic function in the brain. Here we present the clinical and genetic features of 53 patients (42 previously unpublished) with DLG4 variants. METHODS: The clinical and genetic information were collected through GeneMatcher collaboration. All the individuals were investigated by local clinicians and the gene variants were identified by clinical exome/genome sequencing. RESULTS: The clinical picture was predominated by early onset global developmental delay, intellectual disability, autism spectrum disorder, and attention deficit-hyperactivity disorder, all of which point to a brain disorder. Marfanoid habitus, which was previously suggested to be a characteristic feature of DLG4-related phenotypes, was found in only nine individuals and despite some overlapping features, a distinct facial dysmorphism could not be established. Of the 45 different DLG4 variants, 39 were predicted to lead to loss of protein function and the majority occurred de novo (four with unknown origin). The six missense variants identified were suggested to lead to structural or functional changes by protein modeling studies. CONCLUSION: The present study shows that clinical manifestations associated with DLG4 overlap with those found in other neurodevelopmental disorders of synaptic dysfunction; thus, we designate this group of disorders as DLG4-related synaptopathy.

MEF2C Hypofunction in Neuronal and Neuroimmune Populations Produces MEF2C Haploinsufficiency Syndrome-like Behaviors in Mice.

BACKGROUND: Microdeletions of the MEF2C gene are linked to a syndromic form of autism termed MEF2C haploinsufficiency syndrome (MCHS). MEF2C hypofunction in neurons is presumed to underlie most of the symptoms of MCHS. However, it is unclear in which cell populations MEF2C functions to regulate neurotypical development. METHODS: Multiple biochemical, molecular, electrophysiological, behavioral, and transgenic mouse approaches were used to characterize MCHS-relevant synaptic, behavioral, and gene expression changes in mouse models of MCHS. RESULTS: We showed that MCHS-associated missense mutations cluster in the conserved DNA binding domain and disrupt MEF2C DNA binding. DNA binding-deficient global Mef2c heterozygous mice (Mef2c-Het) displayed numerous MCHS-related behaviors, including autism-related behaviors, changes in cortical gene expression, and deficits in cortical excitatory synaptic transmission. We detected hundreds of dysregulated genes in Mef2c-Het cortex, including significant enrichments of autism risk and excitatory neuron genes. In addition, we observed an enrichment of upregulated microglial genes, but this was not due to neuroinflammation in the Mef2c-Het cortex. Importantly, conditional Mef2c heterozygosity in forebrain excitatory neurons reproduced a subset of the Mef2c-Het phenotypes, while conditional Mef2c heterozygosity in microglia reproduced social deficits and repetitive behavior. CONCLUSIONS: Taken together, our findings show that mutations found in individuals with MCHS disrupt the DNA-binding function of MEF2C, and DNA binding-deficient Mef2c global heterozygous mice display numerous MCHS-related phenotypes, including excitatory neuron and microglia gene expression changes. Our findings suggest that MEF2C regulates typical brain development and function through multiple cell types, including excitatory neuronal and neuroimmune populations.

BAZ2B haploinsufficiency as a cause of developmental delay, intellectual disability, and autism spectrum disorder.

The bromodomain adjacent to zinc finger 2B gene (BAZ2B) encodes a protein involved in chromatin remodeling. Loss of BAZ2B function has been postulated to cause neurodevelopmental disorders. To determine whether BAZ2B deficiency is likely to contribute to the pathogenesis of these disorders, we performed bioinformatics analyses that demonstrated a high level of functional convergence during fetal cortical development between BAZ2B and genes known to cause autism spectrum disorder (ASD) and neurodevelopmental disorder. We also found an excess of de novo BAZ2B loss-of-function variants in exome sequencing data from previously published cohorts of individuals with neurodevelopmental disorders. We subsequently identified seven additional individuals with heterozygous deletions, stop-gain, or de novo missense variants affecting BAZ2B. All of these individuals have developmental delay (DD), intellectual disability (ID), and/or ASD. Taken together, our findings suggest that haploinsufficiency of BAZ2B causes a neurodevelopmental disorder, whose cardinal features include DD, ID, and ASD.

Redefining the Etiologic Landscape of Cerebellar Malformations.

Cerebellar malformations are diverse congenital anomalies frequently associated with developmental disability. Although genetic and prenatal non-genetic causes have been described, no systematic analysis has been performed. Here, we present a large-exome sequencing study of Dandy-Walker malformation (DWM) and cerebellar hypoplasia (CBLH). We performed exome sequencing in 282 individuals from 100 families with DWM or CBLH, and we established a molecular diagnosis in 36 of 100 families, with a significantly higher yield for CBLH (51%) than for DWM (16%). The 41 variants impact 27 neurodevelopmental-disorder-associated genes, thus demonstrating that CBLH and DWM are often features of monogenic neurodevelopmental disorders. Though only seven monogenic causes (19%) were identified in more than one individual, neuroimaging review of 131 additional individuals confirmed cerebellar abnormalities in 23 of 27 genetic disorders (85%). Prenatal risk factors were frequently found among individuals without a genetic diagnosis (30 of 64 individuals [47%]). Single-cell RNA sequencing of prenatal human cerebellar tissue revealed gene enrichment in neuronal and vascular cell types; this suggests that defective vasculogenesis may disrupt cerebellar development. Further, de novo gain-of-function variants in PDGFRB, a tyrosine kinase receptor essential for vascular progenitor signaling, were associated with CBLH, and this discovery links genetic and non-genetic etiologies. Our results suggest that genetic defects impact specific cerebellar cell types and implicate abnormal vascular development as a mechanism for cerebellar malformations. We also confirmed a major contribution for non-genetic prenatal factors in individuals with cerebellar abnormalities, substantially influencing diagnostic evaluation and counseling regarding recurrence risk and prognosis.

Pathogenic WDFY3 variants cause neurodevelopmental disorders and opposing effects on brain size.

The underpinnings of mild to moderate neurodevelopmental delay remain elusive, often leading to late diagnosis and interventions. Here, we present data on exome and genome sequencing as well as array analysis of 13 individuals that point to pathogenic, heterozygous, mostly de novo variants in WDFY3 (significant de novo enrichment P = 0.003) as a monogenic cause of mild and non-specific neurodevelopmental delay. Nine variants were protein-truncating and four missense. Overlapping symptoms included neurodevelopmental delay, intellectual disability, macrocephaly, and psychiatric disorders (autism spectrum disorders/attention deficit hyperactivity disorder). One proband presented with an opposing phenotype of microcephaly and the only missense-variant located in the PH-domain of WDFY3. Findings of this case are supported by previously published data, demonstrating that pathogenic PH-domain variants can lead to microcephaly via canonical Wnt-pathway upregulation. In a separate study, we reported that the autophagy scaffolding protein WDFY3 is required for cerebral cortical size regulation in mice, by controlling proper division of neural progenitors. Here, we show that proliferating cortical neural progenitors of human embryonic brains highly express WDFY3, further supporting a role for this molecule in the regulation of prenatal neurogenesis. We present data on Wnt-pathway dysregulation in Wdfy3-haploinsufficient mice, which display macrocephaly and deficits in motor coordination and associative learning, recapitulating the human phenotype. Consequently, we propose that in humans WDFY3 loss-of-function variants lead to macrocephaly via downregulation of the Wnt pathway. In summary, we present WDFY3 as a novel gene linked to mild to moderate neurodevelopmental delay and intellectual disability and conclude that variants putatively causing haploinsufficiency lead to macrocephaly, while an opposing pathomechanism due to variants in the PH-domain of WDFY3 leads to microcephaly.

Gene domain-specific DNA methylation episignatures highlight distinct molecular entities of ADNP syndrome.

BACKGROUND: ADNP syndrome is a rare Mendelian disorder characterized by global developmental delay, intellectual disability, and autism. It is caused by truncating mutations in ADNP, which is involved in chromatin regulation. We hypothesized that the disruption of chromatin regulation might result in specific DNA methylation patterns that could be used in the molecular diagnosis of ADNP syndrome. RESULTS: We identified two distinct and partially opposing genomic DNA methylation episignatures in the peripheral blood samples from 22 patients with ADNP syndrome. The "epi-ADNP-1" episignature included ~ 6000 mostly hypomethylated CpGs, and the "epi-ADNP-2" episignature included ~ 1000 predominantly hypermethylated CpGs. The two signatures correlated with the locations of the ADNP mutations. Epi-ADNP-1 mutations occupy the N- and C-terminus, and epi-ADNP-2 mutations are centered on the nuclear localization signal. The episignatures were enriched for genes involved in neuronal system development and function. A classifier trained on these profiles yielded full sensitivity and specificity in detecting patients with either of the two episignatures. Applying this model to seven patients with uncertain clinical diagnosis enabled reclassification of genetic variants of uncertain significance and assigned new diagnosis when the primary clinical suspicion was not correct. When applied to a large cohort of unresolved patients with developmental delay (N = 1150), the model predicted three additional previously undiagnosed patients to have ADNP syndrome. DNA sequencing of these subjects, wherever available, identified pathogenic mutations within the gene domains predicted by the model. CONCLUSIONS: We describe the first Mendelian condition with two distinct episignatures caused by mutations in a single gene. These highly sensitive and specific DNA methylation episignatures enable diagnosis, screening, and genetic variant classifications in ADNP syndrome.

RIT1 oncoproteins escape LZTR1-mediated proteolysis.

RIT1 oncoproteins have emerged as an etiologic factor in Noonan syndrome and cancer. Despite the resemblance of RIT1 to other members of the Ras small guanosine triphosphatases (GTPases), mutations affecting RIT1 are not found in the classic hotspots but rather in a region near the switch II domain of the protein. We used an isogenic germline knock-in mouse model to study the effects of RIT1 mutation at the organismal level, which resulted in a phenotype resembling Noonan syndrome. By mass spectrometry, we detected a RIT1 interactor, leucine zipper-like transcription regulator 1 (LZTR1), that acts as an adaptor for protein degradation. Pathogenic mutations affecting either RIT1 or LZTR1 resulted in incomplete degradation of RIT1. This led to RIT1 accumulation and dysregulated growth factor signaling responses. Our results highlight a mechanism of pathogenesis that relies on impaired protein degradation of the Ras GTPase RIT1.

A neurodevelopmental disorder caused by mutations in the VPS51 subunit of the GARP and EARP complexes.

Golgi-associated retrograde protein (GARP) and endosome-associated recycling protein (EARP) are related heterotetrameric complexes that associate with the cytosolic face of the trans-Golgi network and recycling endosomes, respectively. At these locations, GARP and EARP function to promote the fusion of endosome-derived transport carriers with their corresponding compartments. GARP and EARP share three subunits, VPS51, VPS52 and VPS53, and each has an additional complex-specific subunit, VPS54 or VPS50, respectively. The role of these complexes in human physiology, however, remains poorly understood. By exome sequencing, we have identified compound heterozygous mutations in the gene encoding the shared GARP/EARP subunit VPS51 in a 6-year-old patient with severe global developmental delay, microcephaly, hypotonia, epilepsy, cortical vision impairment, pontocerebellar abnormalities, failure to thrive, liver dysfunction, lower extremity edema and dysmorphic features. The mutation in one allele causes a frameshift that produces a longer but highly unstable protein that is degraded by the proteasome. In contrast, the other mutant allele produces a protein with a single amino acid substitution that is stable but assembles less efficiently with the other GARP/EARP subunits. Consequently, skin fibroblasts from the patient have reduced levels of fully assembled GARP and EARP complexes. Likely because of this deficiency, the patient's fibroblasts display altered distribution of the cation-independent mannose 6-phosphate receptor, which normally sorts acid hydrolases to lysosomes. Furthermore, a fraction of the patient's fibroblasts exhibits swelling of lysosomes. These findings thus identify a novel genetic locus for a neurodevelopmental disorder and highlight the critical importance of GARP/EARP function in cellular and organismal physiology.

The intellectual disability-associated CAMK2G p.Arg292Pro mutation acts as a pathogenic gain-of-function.

The abundantly expressed calcium/calmodulin-dependent protein kinase II (CAMK2), alpha (CAMK2A), and beta (CAMK2B) isoforms are essential for learning and memory formation. Recently, a de novo candidate mutation (p.Arg292Pro) in the gamma isoform of CAMK2 (CAMK2G) was identified in a patient with severe intellectual disability (ID), but the mechanism(s) by which this mutation causes ID is unknown. Here, we identified a second, unrelated individual, with a de novo CAMK2G p.Arg292Pro mutation, and used in vivo and in vitro assays to assess the impact of this mutation on CAMK2G and neuronal function. We found that knockdown of CAMK2G results in inappropriate precocious neuronal maturation. We further found that the CAMK2G p.Arg292Pro mutation acts as a highly pathogenic gain-of-function mutation, leading to increased phosphotransferase activity and impaired neuronal maturation as well as impaired targeting of the nuclear CAMK2G isoform. Silencing the catalytic site of the CAMK2G p.Arg292Pro protein reversed the pathogenic effect of the p.Arg292Pro mutation on neuronal maturation, without rescuing its nuclear targeting. Taken together, our results reveal an indispensable function of CAMK2G in neurodevelopment and indicate that the CAMK2G p.Arg292Pro protein acts as a pathogenic gain-of-function mutation, through constitutive activity toward cytosolic targets, rather than impaired targeting to the nucleus.

Autosomal recessive Noonan syndrome associated with biallelic LZTR1 variants.

PURPOSE: To characterize the molecular genetics of autosomal recessive Noonan syndrome. METHODS: Families underwent phenotyping for features of Noonan syndrome in children and their parents. Two multiplex families underwent linkage analysis. Exome, genome, or multigene panel sequencing was used to identify variants. The molecular consequences of observed splice variants were evaluated by reverse-transcription polymerase chain reaction. RESULTS: Twelve families with a total of 23 affected children with features of Noonan syndrome were evaluated. The phenotypic range included mildly affected patients, but it was lethal in some, with cardiac disease and leukemia. All of the parents were unaffected. Linkage analysis using a recessive model supported a candidate region in chromosome 22q11, which includes LZTR1, previously shown to harbor mutations in patients with Noonan syndrome inherited in a dominant pattern. Sequencing analyses of 21 live-born patients and a stillbirth identified biallelic pathogenic variants in LZTR1, including putative loss-of-function, missense, and canonical and noncanonical splicing variants in the affected children, with heterozygous, clinically unaffected parents and heterozygous or normal genotypes in unaffected siblings. CONCLUSION: These clinical and genetic data confirm the existence of a form of Noonan syndrome that is inherited in an autosomal recessive pattern and identify biallelic mutations in LZTR1.

Expanding the mutational spectrum in Johanson-Blizzard syndrome: identification of whole exon deletions and duplications in the UBR1 gene by multiplex ligation-dependent probe amplification analysis.

BACKGROUND: Johanson-Blizzard syndrome (JBS, MIM #243800) is a very rare autosomal recessive disorder characterized by exocrine pancreatic insufficiency, nasal wing hypoplasia, hypodontia, and other abnormalities. JBS is caused by mutations of the UBR1 gene (MIM *605981), encoding a ubiquitin ligase of the N-end rule pathway. METHODS: Molecular findings in a total of 65 unrelated patients with a clinical diagnosis of JBS who were previously screened for UBR1 mutations by Sanger sequencing were reviewed and cases lacking a disease-causing UBR1 mutation on either one or both alleles were included in this study. In order to discover mutations that are not detectable by Sanger sequencing, we designed a probe set for multiplex ligation-dependent probe amplification (MLPA) analysis of the UBR1 gene and analyzed the copy number status of all 47 UBR1 exons. RESULTS: Our previous studies using Sanger sequencing could detect mutations in 93.1% of 130 disease-associated UBR1 alleles. Six patients with a highly suggestive clinical diagnosis of JBS and unsolved genotype were included in this study. MLPA analysis detected six alleles harboring exon deletions/duplications, thereby raising the mutation detection rate in the entire cohort to 97.7% (127/130 alleles). CONCLUSION: We conclude that single or multi-exon deletions or duplications account for a substantial proportion of JBS-associated UBR1 mutations.

Spinocerebellar ataxia type 29 due to mutations in ITPR1: a case series and review of this emerging congenital ataxia.

BACKGROUND: Spinocerebellar ataxia type 29 (SCA29) is an autosomal dominant, non-progressive cerebellar ataxia characterized by infantile-onset hypotonia, gross motor delay and cognitive impairment. Affected individuals exhibit cerebellar dysfunction and often have cerebellar atrophy on neuroimaging. Recently, missense mutations in ITPR1 were determined to be responsible. RESULTS: Clinical information on 21 individuals from 15 unrelated families with ITPR1 mutations was retrospectively collected using standardized questionnaires, including 11 previously unreported singletons and 2 new patients from a previously reported family. We describe the genetic, clinical and neuroimaging features of these patients to further characterize the clinical features of this rare condition and assess for any genotype-phenotype correlation for this disorder. Our cohort consisted of 9 males and 12 females, with ages ranging from 28 months to 49 years. Disease course was non-progressive with infantile-onset hypotonia and delays in motor and speech development. Gait ataxia was present in all individuals and 10 (48%) were not ambulating independently between the ages of 3-12 years of age. Mild-to-moderate cognitive impairment was present in 17 individuals (85%). Cerebellar atrophy developed after initial symptom presentation in 13 individuals (72%) and was not associated with disease progression or worsening functional impairment. We identified 12 different mutations including 6 novel mutations; 10 mutations were missense (with 4 present in >1 individual), 1 a splice site mutation leading to an in-frame insertion and 1 an in-frame deletion. No specific genotype-phenotype correlations were observed within our cohort. CONCLUSIONS: Our findings document significant clinical heterogeneity between individuals with SCA29 in a large cohort of molecularly confirmed cases. Based on the retrospective observed clinical features and disease course, we provide recommendations for management. Further research into the natural history of SCA29 through prospective studies is an important next step in better understanding the condition.