Detailed Analysis of ITPR1 Missense Variants Guides Diagnostics and Therapeutic Design.

BACKGROUND: The ITPR1 gene encodes the inositol 1,4,5-trisphosphate (IP3 ) receptor type 1 (IP3 R1), a critical player in cerebellar intracellular calcium signaling. Pathogenic missense variants in ITPR1 cause congenital spinocerebellar ataxia type 29 (SCA29), Gillespie syndrome (GLSP), and severe pontine/cerebellar hypoplasia. The pathophysiological basis of the different phenotypes is poorly understood. OBJECTIVES: We aimed to identify novel SCA29 and GLSP cases to define core phenotypes, describe the spectrum of missense variation across ITPR1, standardize the ITPR1 variant nomenclature, and investigate disease progression in relation to cerebellar atrophy. METHODS: Cases were identified using next-generation sequencing through the Deciphering Developmental Disorders study, the 100,000 Genomes project, and clinical collaborations. ITPR1 alternative splicing in the human cerebellum was investigated by quantitative polymerase chain reaction. RESULTS: We report the largest, multinational case series of 46 patients with 28 unique ITPR1 missense variants. Variants clustered in functional domains of the protein, especially in the N-terminal IP3 -binding domain, the carbonic anhydrase 8 (CA8)-binding region, and the C-terminal transmembrane channel domain. Variants outside these domains were of questionable clinical significance. Standardized transcript annotation, based on our ITPR1 transcript expression data, greatly facilitated analysis. Genotype-phenotype associations were highly variable. Importantly, while cerebellar atrophy was common, cerebellar volume loss did not correlate with symptom progression. CONCLUSIONS: This dataset represents the largest cohort of patients with ITPR1 missense variants, expanding the clinical spectrum of SCA29 and GLSP. Standardized transcript annotation is essential for future reporting. Our findings will aid in diagnostic interpretation in the clinic and guide selection of variants for preclinical studies. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Primate-specific ZNF808 is essential for pancreatic development in humans.

Identifying genes linked to extreme phenotypes in humans has the potential to highlight biological processes not shared with all other mammals. Here, we report the identification of homozygous loss-of-function variants in the primate-specific gene ZNF808 as a cause of pancreatic agenesis. ZNF808 is a member of the KRAB zinc finger protein family, a large and rapidly evolving group of epigenetic silencers which target transposable elements. We show that loss of ZNF808 in vitro results in aberrant activation of regulatory potential contained in the primate-specific transposable elements it represses during early pancreas development. This leads to inappropriate specification of cell fate with induction of genes associated with liver identity. Our results highlight the essential role of ZNF808 in pancreatic development in humans and the contribution of primate-specific regions of the human genome to congenital developmental disease.

Neurodevelopmental and Epilepsy Phenotypes in Individuals With Missense Variants in the Voltage-Sensing and Pore Domains of KCNH5.

BACKGROUND AND OBJECTIVES: KCNH5 encodes the voltage-gated potassium channel EAG2/Kv10.2. We aimed to delineate the neurodevelopmental and epilepsy phenotypic spectrum associated with de novo KCNH5 variants. METHODS: We screened 893 individuals with developmental and epileptic encephalopathies for KCNH5 variants using targeted or exome sequencing. Additional individuals with KCNH5 variants were identified through an international collaboration. Clinical history, EEG, and imaging data were analyzed; seizure types and epilepsy syndromes were classified. We included 3 previously published individuals including additional phenotypic details. RESULTS: We report a cohort of 17 patients, including 9 with a recurrent de novo missense variant p.Arg327His, 4 with a recurrent missense variant p.Arg333His, and 4 additional novel missense variants. All variants were located in or near the functionally critical voltage-sensing or pore domains, absent in the general population, and classified as pathogenic or likely pathogenic using the American College of Medical Genetics and Genomics criteria. All individuals presented with epilepsy with a median seizure onset at 6 months. They had a wide range of seizure types, including focal and generalized seizures. Cognitive outcomes ranged from normal intellect to profound impairment. Individuals with the recurrent p.Arg333His variant had a self-limited drug-responsive focal or generalized epilepsy and normal intellect, whereas the recurrent p.Arg327His variant was associated with infantile-onset DEE. Two individuals with variants in the pore domain were more severely affected, with a neonatal-onset movement disorder, early-infantile DEE, profound disability, and childhood death. DISCUSSION: We describe a cohort of 17 individuals with pathogenic or likely pathogenic missense variants in the voltage-sensing and pore domains of Kv10.2, including 14 previously unreported individuals. We present evidence for a putative emerging genotype-phenotype correlation with a spectrum of epilepsy and cognitive outcomes. Overall, we expand the role of EAG proteins in human disease and establish KCNH5 as implicated in a spectrum of neurodevelopmental disorders and epilepsy.

Variants in CLDN5 cause a syndrome characterized by seizures, microcephaly and brain calcifications.

The blood-brain barrier ensures CNS homeostasis and protection from injury. Claudin-5 (CLDN5), an important component of tight junctions, is critical for the integrity of the blood-brain barrier. We have identified de novo heterozygous missense variants in CLDN5 in 15 unrelated patients who presented with a shared constellation of features including developmental delay, seizures (primarily infantile onset focal epilepsy), microcephaly and a recognizable pattern of pontine atrophy and brain calcifications. All variants clustered in one subregion/domain of the CLDN5 gene and the recurrent variants demonstrate genotype-phenotype correlations. We modelled both patient variants and loss of function alleles in the zebrafish to show that the variants analogous to those in patients probably result in a novel aberrant function in CLDN5. In total, human patient and zebrafish data provide parallel evidence that pathogenic sequence variants in CLDN5 cause a novel neurodevelopmental disorder involving disruption of the blood-brain barrier and impaired neuronal function.

Germline homozygous missense DEPDC5 variants cause severe refractory early-onset epilepsy, macrocephaly and bilateral polymicrogyria.

DEPDC5 (DEP Domain-Containing Protein 5) encodes an inhibitory component of the mammalian target of rapamycin (mTOR) pathway and is commonly implicated in sporadic and familial focal epilepsies, both non-lesional and in association with focal cortical dysplasia. Germline pathogenic variants are typically heterozygous and inactivating. We describe a novel phenotype caused by germline biallelic missense variants in DEPDC5. Cases were identified clinically. Available records, including magnetic resonance imaging and electroencephalography, were reviewed. Genetic testing was performed by whole exome and whole-genome sequencing and cascade screening. In addition, immunohistochemistry was performed on skin biopsy. The phenotype was identified in nine children, eight of which are described in detail herein. Six of the children were of Irish Traveller, two of Tunisian and one of Lebanese origin. The Irish Traveller children shared the same DEPDC5 germline homozygous missense variant (p.Thr337Arg), whereas the Lebanese and Tunisian children shared a different germline homozygous variant (p.Arg806Cys). Consistent phenotypic features included extensive bilateral polymicrogyria, congenital macrocephaly and early-onset refractory epilepsy, in keeping with other mTOR-opathies. Eye and cardiac involvement and severe neutropenia were also observed in one or more patients. Five of the children died in infancy or childhood; the other four are currently aged between 5 months and 6 years. Skin biopsy immunohistochemistry was supportive of hyperactivation of the mTOR pathway. The clinical, histopathological and genetic evidence supports a causal role for the homozygous DEPDC5 variants, expanding our understanding of the biology of this gene.

EMILIN1 deficiency causes arterial tortuosity with osteopenia and connects impaired elastogenesis with defective collagen fibrillogenesis.

EMILIN1 (elastin-microfibril-interface-located-protein-1) is a structural component of the elastic fiber network and localizes to the interface between the fibrillin microfibril scaffold and the elastin core. How EMILIN1 contributes to connective tissue integrity is not fully understood. Here, we report bi-allelic EMILIN1 loss-of-function variants causative for an entity combining cutis laxa, arterial tortuosity, aneurysm formation, and bone fragility, resembling autosomal-recessive cutis laxa type 1B, due to EFEMP2 (FBLN4) deficiency. In both humans and mice, absence of EMILIN1 impairs EFEMP2 extracellular matrix deposition and LOX activity resulting in impaired elastogenesis, reduced collagen crosslinking, and aberrant growth factor signaling. Collagen fiber ultrastructure and histopathology in EMILIN1- or EFEMP2-deficient skin and aorta corroborate these findings and murine Emilin1-/- femora show abnormal trabecular bone formation and strength. Altogether, EMILIN1 connects elastic fiber network with collagen fibril formation, relevant for both bone and vascular tissue homeostasis.

Non-coding variants disrupting a tissue-specific regulatory element in HK1 cause congenital hyperinsulinism.

Gene expression is tightly regulated, with many genes exhibiting cell-specific silencing when their protein product would disrupt normal cellular function1. This silencing is largely controlled by non-coding elements, and their disruption might cause human disease2. We performed gene-agnostic screening of the non-coding regions to discover new molecular causes of congenital hyperinsulinism. This identified 14 non-coding de novo variants affecting a 42-bp conserved region encompassed by a regulatory element in intron 2 of the hexokinase 1 gene (HK1). HK1 is widely expressed across all tissues except in the liver and pancreatic beta cells and is thus termed a 'disallowed gene' in these specific tissues. We demonstrated that the variants result in a loss of repression of HK1 in pancreatic beta cells, thereby causing insulin secretion and congenital hyperinsulinism. Using epigenomic data accessed from public repositories, we demonstrated that these variants reside within a regulatory region that we determine to be critical for cell-specific silencing. Importantly, this has revealed a disease mechanism for non-coding variants that cause inappropriate expression of a disallowed gene.

Hereditary spastic paraparesis presenting as cerebral palsy due to ADD3 variant with mechanistic insight provided by a Drosophila γ-adducin model.

Cerebral palsy (CP) causes neurological disability in early childhood. Hypoxic-ischaemic injury plays a major role in its aetiology, nevertheless, genetic and epigenetic factors may contribute to the clinical presentation. Mutations in ADD3 (encoding γ-adducin) gene have been described in a monogenic form of spastic quadriplegic cerebral palsy (OMIM 601568). We studied a 16-year-old male with spastic diplegia. Several investigations including neurometabolic testing, brain and spine magnetic resonance imaging (MRI) and CGH-Array were normal. Further, clinical genetics assessment and whole exome sequencing (WES) gave the diagnosis. We generated an animal model using Drosophila to study the effects of γ-adducin loss and gain of function. WES revealed a biallelic variant in the ADD3 gene, NM_016824.5(ADD3): c.1100G > A, p.(Gly367Asp). Mutations in this gene have been described as an ultra-rare autosomal recessive, which is a known form of inherited cerebral palsy. Molecular modelling suggests that this mutation leads to a loss of structural integrity of γ-adducin and is therefore expected to result in a decreased level of functional protein. Pan-neuronal over-expression or knock-down of the Drosophila ortholog of ADD3 called hts caused a reduction of life span and impaired locomotion thereby phenocopying aspects of the human disease. Our animal experiments present a starting point to understand the biological processes underpinning the clinical phenotype and pathogenic mechanisms, to gain insights into potential future methods for treating or preventing ADD3 related spastic quadriplegic cerebral palsy.

De Novo ZMYND8 variants result in an autosomal dominant neurodevelopmental disorder with cardiac malformations.

PURPOSE: ZMYND8 encodes a multidomain protein that serves as a central interactive hub for coordinating critical roles in transcription regulation, chromatin remodeling, regulation of super-enhancers, DNA damage response and tumor suppression. We delineate a novel neurocognitive disorder caused by variants in the ZMYND8 gene. METHODS: An international collaboration, exome sequencing, molecular modeling, yeast two-hybrid assays, analysis of available transcriptomic data and a knockdown Drosophila model were used to characterize the ZMYND8 variants. RESULTS: ZMYND8 variants were identified in 11 unrelated individuals; 10 occurred de novo and one suspected de novo; 2 were truncating, 9 were missense, of which one was recurrent. The disorder is characterized by intellectual disability with variable cardiovascular, ophthalmologic and minor skeletal anomalies. Missense variants in the PWWP domain of ZMYND8 abolish the interaction with Drebrin and missense variants in the MYND domain disrupt the interaction with GATAD2A. ZMYND8 is broadly expressed across cell types in all brain regions and shows highest expression in the early stages of brain development. Neuronal knockdown of the DrosophilaZMYND8 ortholog results in decreased habituation learning, consistent with a role in cognitive function. CONCLUSION: We present genomic and functional evidence for disruption of ZMYND8 as a novel etiology of syndromic intellectual disability.

Recommendations for clinical interpretation of variants found in non-coding regions of the genome.

BACKGROUND: The majority of clinical genetic testing focuses almost exclusively on regions of the genome that directly encode proteins. The important role of variants in non-coding regions in penetrant disease is, however, increasingly being demonstrated, and the use of whole genome sequencing in clinical diagnostic settings is rising across a large range of genetic disorders. Despite this, there is no existing guidance on how current guidelines designed primarily for variants in protein-coding regions should be adapted for variants identified in other genomic contexts. METHODS: We convened a panel of nine clinical and research scientists with wide-ranging expertise in clinical variant interpretation, with specific experience in variants within non-coding regions. This panel discussed and refined an initial draft of the guidelines which were then extensively tested and reviewed by external groups. RESULTS: We discuss considerations specifically for variants in non-coding regions of the genome. We outline how to define candidate regulatory elements, highlight examples of mechanisms through which non-coding region variants can lead to penetrant monogenic disease, and outline how existing guidelines can be adapted for the interpretation of these variants. CONCLUSIONS: These recommendations aim to increase the number and range of non-coding region variants that can be clinically interpreted, which, together with a compatible phenotype, can lead to new diagnoses and catalyse the discovery of novel disease mechanisms.

Assessing the clinical utility of protein structural analysis in genomic variant classification: experiences from a diagnostic laboratory.

BACKGROUND: The widespread clinical application of genome-wide sequencing has resulted in many new diagnoses for rare genetic conditions, but testing regularly identifies variants of uncertain significance (VUS). The remarkable rise in the amount of genomic data has been paralleled by a rise in the number of protein structures that are now publicly available, which may have clinical utility for the interpretation of missense and in-frame insertions or deletions. METHODS: Within a UK National Health Service genomic medicine diagnostic laboratory, we investigated the number of VUS over a 5-year period that were evaluated using protein structural analysis and how often this analysis aided variant classification. RESULTS: We found 99 novel missense and in-frame variants across 67 genes that were initially classified as VUS by our diagnostic laboratory using standard variant classification guidelines and for which further analysis of protein structure was requested. Evidence from protein structural analysis was used in the re-assessment of 64 variants, of which 47 were subsequently reclassified as pathogenic or likely pathogenic and 17 remained as VUS. We identified several case studies where protein structural analysis aided variant interpretation by predicting disease mechanisms that were consistent with the observed phenotypes, including loss-of-function through thermodynamic destabilisation or disruption of ligand binding, and gain-of-function through de-repression or escape from proteasomal degradation. CONCLUSIONS: We have shown that using in silico protein structural analysis can aid classification of VUS and give insights into the mechanisms of pathogenicity. Based on our experience, we propose a generic evidence-based workflow for incorporating protein structural information into diagnostic practice to facilitate variant classification.

De novo putative loss-of-function variants in TAF4 are associated with a neuro-developmental disorder.

TATA-binding protein associated factor 4 (TAF4) is a subunit of the Transcription Factor IID (TFIID) complex, a central player in transcription initiation. Other members of this multimeric complex have been implicated previously as monogenic disease genes in human developmental disorders. TAF4 has not been described to date as a monogenic disease gene. We here present a cohort of eight individuals, each carrying de novo putative loss-of-function (pLoF) variants in TAF4 and expressing phenotypes consistent with a neuro-developmental disorder (NDD). Common features include intellectual disability, abnormal behavior, and facial dysmorphisms. We propose TAF4 as a novel dominant disease gene for NDD, and coin this novel disorder "TAF4-related NDD" (T4NDD). We place T4NDD in the context of other disorders related to TFIID subunits, revealing shared features of T4NDD with other TAF-opathies.

Sedaghatian spondylometaphyseal dysplasia in two siblings.

Sedaghatian type spondylometaphyseal dysplasia (SSMD) is a rare skeletal dysplasia with only 24 reported cases to date. Despite the limited literature available, evidence suggests this is a multi-system disorder, with neurological and cardiovascular abnormalities reported in addition to the skeletal features. We report a new family with two affected siblings and detailed phenotypic description of the affected proband. Diagnosis in the neonatal period led to retrospective genetic diagnosis of a previous affected pregnancy that was terminated due to severe ventriculomegaly. We suggest that a diagnosis of SSMD should be considered when shortened long bones are found in combination with significant brain abnormalities.

Reclassification of clinically-detected sequence variants: Framework for genetic clinicians and clinical scientists by CanVIG-UK (Cancer Variant Interpretation Group UK).

PURPOSE: Variant classifications may change over time, driven by emergence of fresh or contradictory evidence or evolution in weighing or combination of evidence items. For variant classifications above the actionability threshold, which is classification of likely pathogenic or pathogenic, clinical actions may be irreversible, such as risk-reducing surgery or prenatal interventions. Variant reclassification up or down across the actionability threshold can therefore have significant clinical consequences. Laboratory approaches to variant reinterpretation and reclassification vary widely. METHODS: Cancer Variant Interpretation Group UK is a multidisciplinary network of clinical scientists and genetic clinicians from across the 24 Molecular Diagnostic Laboratories and Clinical Genetics Services of the United Kingdom (NHS) and Republic of Ireland. We undertook surveys, polls, and national meetings of Cancer Variant Interpretation Group UK to evaluate opinions about clinical and laboratory management regarding variant reclassification. RESULTS: We generated a consensus framework on variant reclassification applicable to cancer susceptibility genes and other clinical areas, which provides explicit recommendations for clinical and laboratory management of variant reclassification scenarios on the basis of the nature of the new evidence, the magnitude of evidence shift, and the final classification score. CONCLUSION: In this framework, clinical and laboratory resources are targeted for maximal clinical effect and minimal patient harm, as appropriate to all resource-constrained health care settings.

Identification and functional evaluation of GRIA1 missense and truncation variants in individuals with ID: An emerging neurodevelopmental syndrome.

GRIA1 encodes the GluA1 subunit of α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors, which are ligand-gated ion channels that act as excitatory receptors for the neurotransmitter L-glutamate (Glu). AMPA receptors (AMPARs) are homo- or heteromeric protein complexes with four subunits, each encoded by different genes, GRIA1 to GRIA4. Although GluA1-containing AMPARs have a crucial role in brain function, the human phenotype associated with deleterious GRIA1 sequence variants has not been established. Subjects with de novo missense and nonsense GRIA1 variants were identified through international collaboration. Detailed phenotypic and genetic assessments of the subjects were carried out and the pathogenicity of the variants was evaluated in vitro to characterize changes in AMPAR function and expression. In addition, two Xenopus gria1 CRISPR-Cas9 F0 models were established to characterize the in vivo consequences. Seven unrelated individuals with rare GRIA1 variants were identified. One individual carried a homozygous nonsense variant (p.Arg377Ter), and six had heterozygous missense variations (p.Arg345Gln, p.Ala636Thr, p.Ile627Thr, and p.Gly745Asp), of which the p.Ala636Thr variant was recurrent in three individuals. The cohort revealed subjects to have a recurrent neurodevelopmental disorder mostly affecting cognition and speech. Functional evaluation of major GluA1-containing AMPAR subtypes carrying the GRIA1 variant mutations showed that three of the four missense variants profoundly perturb receptor function. The homozygous stop-gain variant completely destroys the expression of GluA1-containing AMPARs. The Xenopus gria1 models show transient motor deficits, an intermittent seizure phenotype, and a significant impairment to working memory in mutants. These data support a developmental disorder caused by both heterozygous and homozygous variants in GRIA1 affecting AMPAR function.

Congenital beta cell defects are not associated with markers of islet autoimmunity, even in the context of high genetic risk for type 1 diabetes.

AIMS/HYPOTHESIS: A key unanswered question in type 1 diabetes is whether beta cells initiate their own destruction or are victims of an aberrant immune response (beta cell suicide or homicide?). To investigate this, we assessed islet autoantibodies in individuals with congenital beta cell defects causing neonatal diabetes mellitus (NDM). METHODS: We measured autoantibodies to GAD (GADA), islet antigen-2 (IA-2A) and zinc transporter 8 (ZnT8A) in 242 individuals with NDM (median age diagnosed 1.8 months [IQR 0.39-2.9 months]; median age collected 4.6 months [IQR 1.8-27.6 months]; median diabetes duration 2 months [IQR 0.6-23 months]), including 75 whose NDM resulted from severe beta cell endoplasmic reticulum (ER) stress. As a control cohort we also tested samples from 69 diabetes-free individuals (median age collected 9.9 months [IQR 9.0-48.6 months]) for autoantibodies. RESULTS: We found low prevalence of islet autoantibodies in individuals with monogenic NDM; 13/242 (5.4% [95% CI 2.9, 9.0%]) had detectable GADA, IA-2A and/or ZnT8A. This was similar to the proportion in the control participants who did not have diabetes (1/69 positive [1.4%, 95% CI 0.03, 7.8%], p=0.3). Importantly, monogenic individuals with beta cell ER stress had a similar rate of GADA/IA-2A/ZnT8A positivity to non-ER stress aetiologies (2.7% [95% CI 0.3, 9.3%] vs 6.6% [95% CI 3.3, 11.5%] p=0.4). We observed no association between islet autoimmunity and genetic risk, age at testing (including 30 individuals >10 years at testing) or diabetes duration (p>0.4 for all). CONCLUSIONS/INTERPRETATION: Our data support the hypothesis that beta cell stress/dysfunction alone does not lead to the production of islet autoantibodies, even in the context of high-risk HLA types. This suggests that additional factors are required to trigger an autoimmune response towards beta cells.

Refinements and considerations for trio whole-genome sequence analysis when investigating Mendelian diseases presenting in early childhood.

To facilitate early deployment of whole-genome sequencing (WGS) for severely ill children, a standardized pipeline for WGS analysis with timely turnaround and primary care pediatric uptake is needed. We developed a bioinformatics pipeline for comprehensive gene-agnostic trio WGS analysis of children suspected of having an undiagnosed monogenic disease that included detection and interpretation of primary genetic mechanisms of disease, including SNVs/indels, CNVs/SVs, uniparental disomy (UPD), imprinted genes, short tandem repeat expansions, mobile element insertions, SMN1/2 copy number calling, and mitochondrial genome variants. We assessed primary care practitioner experience and competence in a large cohort of 521 families (comprising 90% WGS trios). Children were identified by primary practitioners for recruitment, and we used the UK index of multiple deprivation to confirm lack of patient socio-economic status ascertainment bias. Of the 521 children sequenced, 176 (34%) received molecular diagnoses, with rates as high as 45% for neurology clinics. Twenty-three of the diagnosed cases (13%) required bespoke methods beyond routine SNV/CNV analysis. In our multidisciplinary clinician user experience assessment, both pediatricians and clinical geneticists expressed strong support for rapid WGS early in the care pathway, but requested further training in determining patient selection, consenting, and variant interpretation. Rapid trio WGS provides an efficacious single-pass screening test for children when deployed by primary practitioners in clinical settings that carry high a priori risk for rare pediatric disease presentations.

SavvyCNV: Genome-wide CNV calling from off-target reads.

Identifying copy number variants (CNVs) can provide diagnoses to patients and provide important biological insights into human health and disease. Current exome and targeted sequencing approaches cannot detect clinically and biologically-relevant CNVs outside their target area. We present SavvyCNV, a tool which uses off-target read data from exome and targeted sequencing data to call germline CNVs genome-wide. Up to 70% of sequencing reads from exome and targeted sequencing fall outside the targeted regions. We have developed a new tool, SavvyCNV, to exploit this 'free data' to call CNVs across the genome. We benchmarked SavvyCNV against five state-of-the-art CNV callers using truth sets generated from genome sequencing data and Multiplex Ligation-dependent Probe Amplification assays. SavvyCNV called CNVs with high precision and recall, outperforming the five other tools at calling CNVs genome-wide, using off-target or on-target reads from targeted panel and exome sequencing. We then applied SavvyCNV to clinical samples sequenced using a targeted panel and were able to call previously undetected clinically-relevant CNVs, highlighting the utility of this tool within the diagnostic setting. SavvyCNV outperforms existing tools for calling CNVs from off-target reads. It can call CNVs genome-wide from targeted panel and exome data, increasing the utility and diagnostic yield of these tests. SavvyCNV is freely available at https://github.com/rdemolgen/SavvySuite.

Evaluation of Evidence for Pathogenicity Demonstrates That BLK, KLF11, and PAX4 Should Not Be Included in Diagnostic Testing for MODY.

Maturity-onset diabetes of the young (MODY) is an autosomal dominant form of monogenic diabetes, reported to be caused by variants in 16 genes. Concern has been raised about whether variants in BLK (MODY11), KLF11 (MODY7), and PAX4 (MODY9) cause MODY. We examined variant-level genetic evidence (cosegregation with diabetes and frequency in population) for published putative pathogenic variants in these genes and used burden testing to test gene-level evidence in a MODY cohort (n = 1,227) compared with a control population (UK Biobank [n = 185,898]). For comparison we analyzed well-established causes of MODY, HNF1A, and HNF4A. The published variants in BLK, KLF11, and PAX4 showed poor cosegregation with diabetes (combined logarithm of the odds [LOD] scores ≤1.2), compared with HNF1A and HNF4A (LOD scores >9), and are all too common to cause MODY (minor allele frequency >4.95 × 10-5). Ultra-rare missense and protein-truncating variants (PTV) were not enriched in a MODY cohort compared with the UK Biobank population (PTV P > 0.05, missense P > 0.1 for all three genes) while HNF1A and HNF4A were enriched (P < 10-6). Findings of sensitivity analyses with different population cohorts supported our results. Variant and gene-level genetic evidence does not support BLK, KLF11, or PAX4 as a cause of MODY. They should not be included in MODY diagnostic genetic testing.