Variants in ZFX are associated with an X-linked neurodevelopmental disorder with recurrent facial gestalt.

Pathogenic variants in multiple genes on the X chromosome have been implicated in syndromic and non-syndromic intellectual disability disorders. ZFX on Xp22.11 encodes a transcription factor that has been linked to diverse processes including oncogenesis and development, but germline variants have not been characterized in association with disease. Here, we present clinical and molecular characterization of 18 individuals with germline ZFX variants. Exome or genome sequencing revealed 11 variants in 18 subjects (14 males and 4 females) from 16 unrelated families. Four missense variants were identified in 11 subjects, with seven truncation variants in the remaining individuals. Clinical findings included developmental delay/intellectual disability, behavioral abnormalities, hypotonia, and congenital anomalies. Overlapping and recurrent facial features were identified in all subjects, including thickening and medial broadening of eyebrows, variations in the shape of the face, external eye abnormalities, smooth and/or long philtrum, and ear abnormalities. Hyperparathyroidism was found in four families with missense variants, and enrichment of different tumor types was observed. In molecular studies, DNA-binding domain variants elicited differential expression of a small set of target genes relative to wild-type ZFX in cultured cells, suggesting a gain or loss of transcriptional activity. Additionally, a zebrafish model of ZFX loss displayed an altered behavioral phenotype, providing additional evidence for the functional significance of ZFX. Our clinical and experimental data support that variants in ZFX are associated with an X-linked intellectual disability syndrome characterized by a recurrent facial gestalt, neurocognitive and behavioral abnormalities, and an increased risk for congenital anomalies and hyperparathyroidism.

Clinical case report: mosaic ANK3 pathogenic variant in a patient with autism spectrum disorder and neurodevelopmental delay.

Ankyrins are a family of proteins that link integral membrane proteins to the underlying spectrin-actin cytoskeleton and play a key role in activities such as cell motility, activation, proliferation, cell-cell contact, and the maintenance of specialized membrane domains. Ankyrin 3 (ANK3) is one of the three major subtypes of the ankyrin protein family. Ankryin genes are ubiquitously expressed, but their expression is highest in the brain. In the central nervous system, ankyrins have critical roles at the axonal initial segment, the nodes of Ranvier, and at synapses. To date, pathogenic variants in ANK3 have been reported in individuals with neuropsychiatric, cognitive, and neurodevelopmental disorders. The clinical severity is variable in these individuals with both autosomal recessive and autosomal dominant patterns of inheritance observed. These findings have suggested genotype-phenotype correlations and even isoform-specific implications for individuals with ANK3 pathogenic variants. Here, we report a patient with speech delay, autism spectrum disorder, and a language disorder in which a de novo nonsense ANK3 alteration was discovered by exome sequencing. Interestingly, the next-generation sequencing data suggested the change was mosaic in the affected child, and it was confirmed by digital polymerase chain reaction (dPCR) at 22% allelic fraction. To our knowledge, this is the first case of an individual with a pathogenic mosaic ANK3 variant. This finding expands upon the existing genotype-phenotype information available for the ANK3 gene while also highlighting potential gene expression correlations with phenotype.

RNA analysis of intronic variants in the LAMA2 gene detected by whole genome sequencing confirms a rare dual diagnosis of incontinentia pigmenti with limb-girdle muscular dystrophy.

We see that a multiple methods approach to diagnosis remains necessary in the era of whole genome sequencing. We also observe that reproductive risk genetic counseling can be a motivating factor for further testing along the diagnostic odyssey.

De novo missense variants in the E3 ubiquitin ligase adaptor KLHL20 cause a developmental disorder with intellectual disability, epilepsy, and autism spectrum disorder.

PURPOSE: KLHL20 is part of a CUL3-RING E3 ubiquitin ligase involved in protein ubiquitination. KLHL20 functions as the substrate adaptor that recognizes substrates and mediates the transfer of ubiquitin to the substrates. Although KLHL20 regulates neurite outgrowth and synaptic development in animal models, a role in human neurodevelopment has not yet been described. We report on a neurodevelopmental disorder caused by de novo missense variants in KLHL20. METHODS: Patients were ascertained by the investigators through Matchmaker Exchange. Phenotyping of patients with de novo missense variants in KLHL20 was performed. RESULTS: We studied 14 patients with de novo missense variants in KLHL20, delineating a genetic syndrome with patients having mild to severe intellectual disability, febrile seizures or epilepsy, autism spectrum disorder, hyperactivity, and subtle dysmorphic facial features. We observed a recurrent de novo missense variant in 11 patients (NM_014458.4:c.1069G>A p.[Gly357Arg]). The recurrent missense and the 3 other missense variants all clustered in the Kelch-type ß-propeller domain of the KLHL20 protein, which shapes the substrate binding surface. CONCLUSION: Our findings implicate KLHL20 in a neurodevelopmental disorder characterized by intellectual disability, febrile seizures or epilepsy, autism spectrum disorder, and hyperactivity.

De novo coding variants in the AGO1 gene cause a neurodevelopmental disorder with intellectual disability.

BACKGROUND: High-impact pathogenic variants in more than a thousand genes are involved in Mendelian forms of neurodevelopmental disorders (NDD). METHODS: This study describes the molecular and clinical characterisation of 28 probands with NDD harbouring heterozygous AGO1 coding variants, occurring de novo for all those whose transmission could have been verified (26/28). RESULTS: A total of 15 unique variants leading to amino acid changes or deletions were identified: 12 missense variants, two in-frame deletions of one codon, and one canonical splice variant leading to a deletion of two amino acid residues. Recurrently identified variants were present in several unrelated individuals: p.(Phe180del), p.(Leu190Pro), p.(Leu190Arg), p.(Gly199Ser), p.(Val254Ile) and p.(Glu376del). AGO1 encodes the Argonaute 1 protein, which functions in gene-silencing pathways mediated by small non-coding RNAs. Three-dimensional protein structure predictions suggest that these variants might alter the flexibility of the AGO1 linker domains, which likely would impair its function in mRNA processing. Affected individuals present with intellectual disability of varying severity, as well as speech and motor delay, autistic behaviour and additional behavioural manifestations. CONCLUSION: Our study establishes that de novo coding variants in AGO1 are involved in a novel monogenic form of NDD, highly similar to the recently reported AGO2-related NDD.

Expanding the Phenotypic Spectrum of GPI Anchoring Deficiency Due to Biallelic Variants in GPAA1.

BACKGROUND AND OBJECTIVES: To expand the clinical knowledge of GPAA1-related glycosylphosphatidylinositol (GPI) deficiency. METHODS: An international case series of 7 patients with biallelic GPAA1 variants were identified. Clinical, biochemical, and neuroimaging data were collected for comparison. Where possible, GPI-anchored proteins were assessed using flow cytometry. RESULTS: Ten novel variants were identified in 7 patients. Flow cytometry samples of 3 available patients confirmed deficiency of several GPI-anchored proteins on leukocytes. Extensive phenotypic information was available for each patient. The majority experienced developmental delay, seizures, and hypotonia. Neuroimaging revealed cerebellar anomalies in the majority of the patients. Alkaline phosphatase was within the normal range in 5 individuals and low in 1 individual, as has been noted in other transamidase defects. We notably describe individuals either less affected or older than the ones published previously. DISCUSSION: Clinical features of the cases reported broaden the spectrum of the known phenotype of GPAA1-related GPI deficiency, while outlining the importance of using functional studies such as flow cytometry to aid in variant classification.

Tenorio syndrome: Description of 14 novel cases and review of the clinical and molecular features.

Tenorio syndrome (TNORS) (OMIM #616260) is a relatively recent disorder with very few cases described so far. Clinical features included macrocephaly, intellectual disability, hypotonia, enlarged ventricles and autoimmune diseases. Molecular underlying mechanism demonstrated missense variants and a large deletion encompassing RNF125, a gene that encodes for an U3 ubiquitin ligase protein. Since the initial description of the disorder in six patients from four families, several new patients were diagnosed, adding more evidence to the clinical spectrum. In this article, we described 14 additional cases with deep phenotyping and make an overall review of all the cases with pathogenic variants in RNF125. Not all patients presented with overgrowth, but instead, most patients showed a common pattern of neurodevelopmental disease, macrocephaly and/or large forehead. Segregation analysis showed that, though the variant was inherited in some patients from an apparently asymptomatic parent, deep phenotyping suggested a mild form of the disease in some of them. The mechanism underlying the development of this disease is not well understood yet and the report of further cases will help to a better understanding and clinical characterization of the syndrome.

Loss of function mutations in GEMIN5 cause a neurodevelopmental disorder.

GEMIN5, an RNA-binding protein is essential for assembly of the survival motor neuron (SMN) protein complex and facilitates the formation of small nuclear ribonucleoproteins (snRNPs), the building blocks of spliceosomes. Here, we have identified 30 affected individuals from 22 unrelated families presenting with developmental delay, hypotonia, and cerebellar ataxia harboring biallelic variants in the GEMIN5 gene. Mutations in GEMIN5 perturb the subcellular distribution, stability, and expression of GEMIN5 protein and its interacting partners in patient iPSC-derived neurons, suggesting a potential loss-of-function mechanism. GEMIN5 mutations result in disruption of snRNP complex assembly formation in patient iPSC neurons. Furthermore, knock down of rigor mortis, the fly homolog of human GEMIN5, leads to developmental defects, motor dysfunction, and a reduced lifespan. Interestingly, we observed that GEMIN5 variants disrupt a distinct set of transcripts and pathways as compared to SMA patient neurons, suggesting different molecular pathomechanisms. These findings collectively provide evidence that pathogenic variants in GEMIN5 perturb physiological functions and result in a neurodevelopmental delay and ataxia syndrome.

BICRA, a SWI/SNF Complex Member, Is Associated with BAF-Disorder Related Phenotypes in Humans and Model Organisms.

SWI/SNF-related intellectual disability disorders (SSRIDDs) are rare neurodevelopmental disorders characterized by developmental disability, coarse facial features, and fifth digit/nail hypoplasia that are caused by pathogenic variants in genes that encode for members of the SWI/SNF (or BAF) family of chromatin remodeling complexes. We have identified 12 individuals with rare variants (10 loss-of-function, 2 missense) in the BICRA (BRD4 interacting chromatin remodeling complex-associated protein) gene, also known as GLTSCR1, which encodes a subunit of the non-canonical BAF (ncBAF) complex. These individuals exhibited neurodevelopmental phenotypes that include developmental delay, intellectual disability, autism spectrum disorder, and behavioral abnormalities as well as dysmorphic features. Notably, the majority of individuals lack the fifth digit/nail hypoplasia phenotype, a hallmark of most SSRIDDs. To confirm the role of BICRA in the development of these phenotypes, we performed functional characterization of the zebrafish and Drosophila orthologs of BICRA. In zebrafish, a mutation of bicra that mimics one of the loss-of-function variants leads to craniofacial defects possibly akin to the dysmorphic facial features seen in individuals harboring putatively pathogenic BICRA variants. We further show that Bicra physically binds to other non-canonical ncBAF complex members, including the BRD9/7 ortholog, CG7154, and is the defining member of the ncBAF complex in flies. Like other SWI/SNF complex members, loss of Bicra function in flies acts as a dominant enhancer of position effect variegation but in a more context-specific manner. We conclude that haploinsufficiency of BICRA leads to a unique SSRIDD in humans whose phenotypes overlap with those previously reported.

Genetic variants in the KDM6B gene are associated with neurodevelopmental delays and dysmorphic features.

Lysine-specific demethylase 6B (KDM6B) demethylates trimethylated lysine-27 on histone H3. The methylation and demethylation of histone proteins affects gene expression during development. Pathogenic alterations in histone lysine methylation and demethylation genes have been associated with multiple neurodevelopmental disorders. We have identified a number of de novo alterations in the KDM6B gene via whole exome sequencing (WES) in a cohort of 12 unrelated patients with developmental delay, intellectual disability, dysmorphic facial features, and other clinical findings. Our findings will allow for further investigation in to the role of the KDM6B gene in human neurodevelopmental disorders.

CHD8 intragenic deletion associated with autism spectrum disorder.

Autism spectrum disorders (ASDs) are a heterogeneous group of neurodevelopmental disorders that are highly heritable. De novo genomic alterations are considered an important cause of autism spectrum disorders. Recent research has shown that de novo loss-of-function mutations in the chromodomain helicase DNA-binding protein 8 (CHD8) gene are associated with an increased risk of ASD. We describe a single case of an intragenic deletion of exons 26-28 in the CHD8 gene in a patient with autism and global developmental delay. Our clinical case supports the hypothesis that CHD8 may play a central role in neuronal cell development and ASD risk.

The study to understand the genetics of the acute response to metformin and glipizide in humans (SUGAR-MGH): design of a pharmacogenetic resource for type 2 diabetes.

OBJECTIVE: Genome-wide association studies have uncovered a large number of genetic variants associated with type 2 diabetes or related phenotypes. In many cases the causal gene or polymorphism has not been identified, and its impact on response to anti-hyperglycemic medications is unknown. The Study to Understand the Genetics of the Acute Response to Metformin and Glipizide in Humans (SUGAR-MGH, NCT01762046) is a novel resource of genetic and biochemical data following glipizide and metformin administration. We describe recruitment, enrollment, and phenotyping procedures and preliminary results for the first 668 of our planned 1,000 participants enriched for individuals at risk of requiring anti-diabetic therapy in the future. METHODS: All individuals are challenged with 5 mg glipizide × 1; twice daily 500 mg metformin × 2 days; and 75-g oral glucose tolerance test following metformin. Genetic variants associated with glycemic traits and blood glucose, insulin, and other hormones at baseline and following each intervention are measured. RESULTS: Approximately 50% of the cohort is female and 30% belong to an ethnic minority group. Following glipizide administration, peak insulin occurred at 60 minutes and trough glucose at 120 minutes. Thirty percent of participants experienced non-severe symptomatic hypoglycemia and required rescue with oral glucose. Following metformin administration, fasting glucose and insulin were reduced. Common genetic variants were associated with fasting glucose levels. CONCLUSIONS: SUGAR-MGH represents a viable pharmacogenetic resource which, when completed, will serve to characterize genetic influences on pharmacological perturbations, and help establish the functional relevance of newly discovered genetic loci to therapy of type 2 diabetes. TRIAL REGISTRATION: ClinicalTrials.gov NCT01762046.

Branched chain and aromatic amino acids change acutely following two medical therapies for type 2 diabetes mellitus.

OBJECTIVE: Elevated circulating levels of branched chain and aromatic amino acids (BCAA/AAAs) are associated with insulin resistance and incident type 2 diabetes (T2D). BCAA/AAAs decrease acutely during an oral glucose tolerance test (OGTT), a diagnostic test for T2D. It is unknown whether changes in BCAA/AAAs also signal an early response to commonly used medical therapies for T2D. MATERIALS AND METHODS: A liquid chromatography-mass spectrometry approach was used to measure BCAA/AAAs in 30 insulin sensitive (IS) and 30 insulin resistant (IR) subjects before and after: (1) one dose of a sulfonylurea medication, glipizide, 5 mg orally; (2) two days of twice daily metformin 500 mg orally; and (3) a 75-g OGTT. Percent change in BCAA/AAAs was determined after each intervention. RESULTS: Following glipizide, which increased insulin and decreased glucose in both subject groups, BCAA/AAAs decreased in the IS subjects only (all P<0.05). Following metformin, which decreased glucose and insulin in only the IR subjects, 4 BCAA/AAAs increased in the IR subjects at or below P=0.05, and none changed in the IS subjects. Following OGTT, which increased glucose and insulin in all subjects, BCAA/AAAs decreased in all subjects (P<0.05). CONCLUSIONS: BCAA/AAAs changed acutely during glipizide and metformin administration, and the magnitude and direction of change differed by the insulin resistance status of the individual and the intervention. These results indicate that BCAA/AAAs may be useful biomarkers for monitoring the early response to therapeutic interventions for T2D.

Common variants at 10 genomic loci influence hemoglobin A1(C) levels via glycemic and nonglycemic pathways.

OBJECTIVE: Glycated hemoglobin (HbA1(c)), used to monitor and diagnose diabetes, is influenced by average glycemia over a 2- to 3-month period. Genetic factors affecting expression, turnover, and abnormal glycation of hemoglobin could also be associated with increased levels of HbA1(c). We aimed to identify such genetic factors and investigate the extent to which they influence diabetes classification based on HbA1(c) levels. RESEARCH DESIGN AND METHODS: We studied associations with HbA1(c) in up to 46,368 nondiabetic adults of European descent from 23 genome-wide association studies (GWAS) and 8 cohorts with de novo genotyped single nucleotide polymorphisms (SNPs). We combined studies using inverse-variance meta-analysis and tested mediation by glycemia using conditional analyses. We estimated the global effect of HbA1(c) loci using a multilocus risk score, and used net reclassification to estimate genetic effects on diabetes screening. RESULTS: Ten loci reached genome-wide significant association with HbA(1c), including six new loci near FN3K (lead SNP/P value, rs1046896/P = 1.6 × 10⁻²6), HFE (rs1800562/P = 2.6 × 10⁻²°), TMPRSS6 (rs855791/P = 2.7 × 10⁻¹4), ANK1 (rs4737009/P = 6.1 × 10⁻¹²), SPTA1 (rs2779116/P = 2.8 × 10⁻9) and ATP11A/TUBGCP3 (rs7998202/P = 5.2 × 10⁻9), and four known HbA1(c) loci: HK1 (rs16926246/P = 3.1 × 10⁻54), MTNR1B (rs1387153/P = 4.0 × 10⁻¹¹), GCK (rs1799884/P = 1.5 × 10⁻²°) and G6PC2/ABCB11 (rs552976/P = 8.2 × 10⁻¹8). We show that associations with HbA1(c) are partly a function of hyperglycemia associated with 3 of the 10 loci (GCK, G6PC2 and MTNR1B). The seven nonglycemic loci accounted for a 0.19 (% HbA1(c)) difference between the extreme 10% tails of the risk score, and would reclassify ∼2% of a general white population screened for diabetes with HbA1(c). CONCLUSIONS: GWAS identified 10 genetic loci reproducibly associated with HbA1(c). Six are novel and seven map to loci where rarer variants cause hereditary anemias and iron storage disorders. Common variants at these loci likely influence HbA1(c) levels via erythrocyte biology, and confer a small but detectable reclassification of diabetes diagnosis by HbA1(c).

Genomics of type 2 diabetes mellitus: implications for the clinician.

Our understanding of the genetics of type 2 diabetes mellitus (T2DM) has changed, in part owing to implementation of genome-wide association studies as a method for unraveling the genetic architecture of complex traits. These studies enable a global search throughout the nuclear genome for variants that are associated with specific phenotypes. Currently, single nucleotide polymorphisms in about 24 different genetic loci have been associated with T2DM. Most of these genetic loci are associated with the insulin secretion pathway rather than insulin resistance. Study design, heritability differences and the intrinsic properties of in vivo insulin resistance measures might partially explain why only a few loci associated with insulin resistance have been detected through genome-wide association approaches. Despite the success of these approaches at detecting loci associated with T2DM, currently known associations explain only a small amount of the genetic variance involved in the disease. Compared with previous studies, larger cohorts might be needed to identify variants of smaller effect sizes and lower allele frequencies. Finally, the current list of genetic loci that are related to T2DM does not seem to offer greater predictive value in determining diabetes risk than do commonly used phenotypic risk factors and family history.

Haplotype structure of the ENPP1 Gene and Nominal Association of the K121Q missense single nucleotide polymorphism with glycemic traits in the Framingham Heart Study.

OBJECTIVE: A recent meta-analysis demonstrated a nominal association of the ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) K-->Q missense single nucleotide polymorphism (SNP) at position 121 with type 2 diabetes. We set out to confirm the association of ENPP1 K121Q with hyperglycemia, expand this association to insulin resistance traits, and determine whether the association stems from K121Q or another variant in linkage disequilibrium with it. RESEARCH DESIGN AND METHODS: We characterized the haplotype structure of ENPP1 and selected 39 tag SNPs that captured 96% of common variation in the region (minor allele frequency > or =5%) with an r(2) value > or =0.80. We genotyped the SNPs in 2,511 Framingham Heart Study participants and used age- and sex-adjusted linear mixed effects (LME) models to test for association with quantitative metabolic traits. We also examined whether interaction between K121Q and BMI affected glycemic trait levels. RESULTS: The Q allele of K121Q (rs1044498) was associated with increased fasting plasma glucose (FPG), A1C, fasting insulin, and insulin resistance by homeostasis model assessment (HOMA-IR; all P = 0.01-0.006). Two noncoding SNPs (rs7775386 and rs7773477) demonstrated similar associations, but LME models indicated that their effects were not independent from K121Q. We found no association of K121Q with obesity, but interaction models suggested that the effect of the Q allele on FPG and HOMA-IR was stronger in those with a higher BMI (P = 0.008 and 0.01 for interaction, respectively). CONCLUSIONS: The Q allele of ENPP1 K121Q is associated with hyperglycemia and insulin resistance in whites. We found an adiposity-SNP interaction, with a stronger association of K121Q with diabetes-related quantitative traits in people with a higher BMI.