Biallelic EPB41L3 variants underlie a developmental disorder with seizures and myelination defects.

Erythrocyte Membrane Protein Band 4.1 Like 3 (EPB41L3: NM_012307.5), also known as DAL-1, encodes the ubiquitously expressed, neuronally enriched 4.1B protein, part of the 4.1 superfamily of membrane-cytoskeleton adaptors. 4.1B plays key roles in cell spreading, migration, and cytoskeletal scaffolding that support oligodendrocyte axon adhesions essential for proper myelination. We herein describe six individuals from five unrelated families with global developmental delay, intellectual disability, seizures, hypotonia, neuroregression, and delayed myelination. Exome sequencing identified biallelic variants in EPB41L3 in all affected individuals: two nonsense (c.466C>T, p.(R156*); c.2776C>T, p.(R926*)) and three frameshift (c.666delT, p.(F222Lfs*46); c.2289dupC, p.(V764Rfs*19); c.948_949delTG, p.(A317Kfs*33)). Quantitative-real time PCR and Western blot analysis in human fibroblasts harbouring EPB41L3:c.666delT, p.(F222Lfs*46) indicate ablation of EPB41L3 mRNA and 4.1B protein expression. Inhibition of the nonsense mediated decay (NMD) pathway led to an upregulation of EPB41L3:c.666delT transcripts, supporting NMD as a pathogenic mechanism. Epb41l3-deficient mouse oligodendroglia cells showed significant reduction in mRNA expression of key myelin genes, reduced branching, and increased apoptosis. Our report provides the first clinical description of an autosomal recessive disorder associated with variants in EPB41L3, which we refer to as EPB41L3-associated developmental disorder (EADD). Moreover, our functional studies substantiate the pathogenicity of EPB41L3 hypothesized loss-of-function variants.

Biallelic variants in CSMD1 are implicated in a neurodevelopmental disorder with intellectual disability and variable cortical malformations.

CSMD1 (Cub and Sushi Multiple Domains 1) is a well-recognized regulator of the complement cascade, an important component of the innate immune response. CSMD1 is highly expressed in the central nervous system (CNS) where emergent functions of the complement pathway modulate neural development and synaptic activity. While a genetic risk factor for neuropsychiatric disorders, the role of CSMD1 in neurodevelopmental disorders is unclear. Through international variant sharing, we identified inherited biallelic CSMD1 variants in eight individuals from six families of diverse ancestry who present with global developmental delay, intellectual disability, microcephaly, and polymicrogyria. We modeled CSMD1 loss-of-function (LOF) pathogenesis in early-stage forebrain organoids differentiated from CSMD1 knockout human embryonic stem cells (hESCs). We show that CSMD1 is necessary for neuroepithelial cytoarchitecture and synchronous differentiation. In summary, we identified a critical role for CSMD1 in brain development and biallelic CSMD1 variants as the molecular basis of a previously undefined neurodevelopmental disorder.

A de novo variant in PAK2 detected in an individual with Knobloch type 2 syndrome.

P21-activated kinase 2 (PAK2) is a serine/threonine kinase essential for a variety of cellular processes including signal transduction, cellular survival, proliferation, and migration. A recent report proposed monoallelic PAK2 variants cause Knobloch syndrome type 2 (KNO2)-a developmental disorder primarily characterized by ocular anomalies. Here, we identified a novel de novo heterozygous missense variant in PAK2, NM_002577.4:c.1273G>A, p.(D425N), by whole genome sequencing in an individual with features consistent with KNO2. Notable clinical phenotypes include global developmental delay, congenital retinal detachment, mild cerebral ventriculomegaly, hypotonia, FTT, pyloric stenosis, feeding intolerance, patent ductus arteriosus, and mild facial dysmorphism. The p.(D425N) variant lies within the protein kinase domain and is predicted to be functionally damaging by in silico analysis. Previous clinical genetic testing did not report this variant due to unknown relevance of PAK2 variants at the time of testing, highlighting the importance of reanalysis. Our findings also substantiate the candidacy of PAK2 variants in KNO2 and expand the KNO2 clinical spectrum.

TREX tetramer disruption alters RNA processing necessary for corticogenesis in THOC6 Intellectual Disability Syndrome.

THOC6 variants are the genetic basis of autosomal recessive THOC6 Intellectual Disability Syndrome (TIDS). THOC6 is critical for mammalian Transcription Export complex (TREX) tetramer formation, which is composed of four six-subunit THO monomers. The TREX tetramer facilitates mammalian RNA processing, in addition to the nuclear mRNA export functions of the TREX dimer conserved through yeast. Human and mouse TIDS model systems revealed novel THOC6-dependent, species-specific TREX tetramer functions. Germline biallelic Thoc6 loss-of-function (LOF) variants result in mouse embryonic lethality. Biallelic THOC6 LOF variants reduce the binding affinity of ALYREF to THOC5 without affecting the protein expression of TREX members, implicating impaired TREX tetramer formation. Defects in RNA nuclear export functions were not detected in biallelic THOC6 LOF human neural cells. Instead, mis-splicing was detected in human and mouse neural tissue, revealing novel THOC6-mediated TREX coordination of mRNA processing. We demonstrate that THOC6 is required for key signaling pathways known to regulate the transition from proliferative to neurogenic divisions during human corticogenesis. Together, these findings implicate altered RNA processing in the developmental biology of TIDS neuropathology.

Mechanisms of mRNA processing defects in inherited THOC6 intellectual disability syndrome.

THOC6 is the genetic basis of autosomal recessive THOC6 Intellectual Disability Syndrome (TIDS). THOC6 facilitates the formation of the Transcription Export complex (TREX) tetramer, composed of four THO monomers. The TREX tetramer supports mammalian mRNA processing that is distinct from yeast TREX dimer functions. Human and mouse TIDS model systems allow novel THOC6-dependent TREX tetramer functions to be investigated. Biallelic loss-of-functon(LOF) THOC6 variants do not influence the expression and localization of TREX members in human cells, but our data suggests reduced binding affinity of ALYREF. Impairment of TREX nuclear export functions were not detected in cells with biallelic THOC6 LOF. Instead, mRNA mis-splicing was observed in human and mouse neural tissue, revealing novel insights into THOC6-mediated TREX coordination of mRNA processing. We demonstrate that THOC6 is required for regulation of key signaling pathways in human corticogenesis that dictate the transition from proliferative to neurogenic divisions that may inform TIDS neuropathology.

A novel biallelic frameshift variant in C2orf69 causing developmental regression, seizures, microcephaly, autistic features, and hypertonia.

Combined oxidative phosphorylation deficiency type 53 (COXPD53) is an autosomal recessive neurodevelopmental disorder (NDD) caused by homozygous variants in the gene C2orf69. Here, we report a novel frameshift variant c.187_191dupGCCGA, p.D64Efs*56 identified in an individual with clinical presentation of COXPD53 with developmental regression and autistic features. The variant c.187_191dupGCCGA, p.D64Efs*56 represents the most N-terminal part of C2orf69. Notable clinical features of COXPD53of the proband include developmental delay, developmental regression, seizures, microcephaly, and hypertonia. Structural brain defects of cerebral atrophy, cerebellar atrophy, hypomyelination, and thin corpus callosum were also observed. While we observe strong phenotypic overlap among affected individuals with C2orf69 variants, developmental regression and autistic features have not been previously described in individuals with COXPD53. Together, this case expands the genetic and clinical phenotypic spectrum of C2orf69-associated COXPD53.

De novo variants in GATAD2A in individuals with a neurodevelopmental disorder: GATAD2A-related neurodevelopmental disorder.

GATA zinc finger domain containing 2A (GATAD2A) is a subunit of the nucleosome remodeling and deacetylase (NuRD) complex. NuRD is known to regulate gene expression during neural development and other processes. The NuRD complex modulates chromatin status through histone deacetylation and ATP-dependent chromatin remodeling activities. Several neurodevelopmental disorders (NDDs) have been previously linked to variants in other components of NuRD's chromatin remodeling subcomplex (NuRDopathies). We identified five individuals with features of an NDD that possessed de novo autosomal dominant variants in GATAD2A. Core features in affected individuals include global developmental delay, structural brain defects, and craniofacial dysmorphology. These GATAD2A variants are predicted to affect protein dosage and/or interactions with other NuRD chromatin remodeling subunits. We provide evidence that a GATAD2A missense variant disrupts interactions of GATAD2A with CHD3, CHD4, and CHD5. Our findings expand the list of NuRDopathies and provide evidence that GATAD2A variants are the genetic basis of a previously uncharacterized developmental disorder.

Identifying adaptive alleles in the human genome: from selection mapping to functional validation.

The suite of phenotypic diversity across geographically distributed human populations is the outcome of genetic drift, gene flow, and natural selection throughout human evolution. Human genetic variation underlying local biological adaptations to selective pressures is incompletely characterized. With the emergence of population genetics modeling of large-scale genomic data derived from diverse populations, scientists are able to map signatures of natural selection in the genome in a process known as selection mapping. Inferred selection signals further can be used to identify candidate functional alleles that underlie putative adaptive phenotypes. Phenotypic association, fine mapping, and functional experiments facilitate the identification of candidate adaptive alleles. Functional investigation of candidate adaptive variation using novel techniques in molecular biology is slowly beginning to unravel how selection signals translate to changes in biology that underlie the phenotypic spectrum of our species. In addition to informing evolutionary hypotheses of adaptation, the discovery and functional annotation of adaptive alleles also may be of clinical significance. While selection mapping efforts in non-European populations are growing, there remains a stark under-representation of diverse human populations in current public genomic databases, of both clinical and non-clinical cohorts. This lack of inclusion limits the study of human biological variation. Identifying and functionally validating candidate adaptive alleles in more global populations is necessary for understanding basic human biology and human disease.

Quantitative assessment of skin, hair, and iris variation in a diverse sample of individuals and associated genetic variation.

OBJECTIVES: The main goals of this study are to 1) quantitatively measure skin, hair, and iris pigmentation in a diverse sample of individuals, 2) describe variation within and between these samples, and 3) demonstrate how quantitative measures can facilitate genotype-phenotype association tests. MATERIALS AND METHODS: We quantitatively characterize skin, hair, and iris pigmentation using the Melanin (M) Index (skin) and CIELab values (hair) in 1,450 individuals who self-identify as African American, East Asian, European, Hispanic, or South Asian. We also quantify iris pigmentation in a subset of these individuals using CIELab values from high-resolution iris photographs. We compare mean skin M index and hair and iris CIELab values among populations using ANOVA and MANOVA respectively and test for genotype-phenotype associations in the European sample. RESULTS: All five populations are significantly different for skin (P <2 × 10(-16) ) and hair color (P <2 × 10(-16) ). Our quantitative analysis of iris and hair pigmentation reinforces the continuous, rather than discrete, nature of these traits. We confirm the association of three loci (rs16891982, rs12203592, and rs12913832) with skin pigmentation and four loci (rs12913832, rs12203592, rs12896399, and rs16891982) with hair pigmentation. Interestingly, the derived rs12203592 T allele located within the IRF4 gene is associated with lighter skin but darker hair color. DISCUSSION: The quantitative methods used here provide a fine-scale assessment of pigmentation phenotype and facilitate genotype-phenotype associations, even with relatively small sample sizes. This represents an important expansion of current investigations into pigmentation phenotype and associated genetic variation by including non-European and admixed populations. Am J Phys Anthropol 160:570-581, 2016. © 2015 Wiley Periodicals, Inc.

The rs387907171 SNP in TYRP1 is not associated with blond hair color on the Island of Bougainville.

OBJECTIVES: The aims of this study are to characterize the frequency of the derived allele at rs387907171 in populations from the islands of New Britain and Bougainville in Northern Island Melanesia, to confirm its association with lighter hair color, and to refine hypotheses regarding its evolutionary history. METHODS: rs387907171 was genotyped in 93 individuals from New Britain and 101 from Bougainville for whom quantitative assessments of skin and hair pigmentation were available. Combining these with existing data from other Melanesian islands we tested for differences in allele frequencies between islands and for associations with skin and hair pigmentation using ANOVA, including sex, age, and island affiliations as covariates. RESULTS: The derived allele at rs387907171 was observed in a single copy in the New Britain and Bougainville populations genotyped here. Its frequency differs significantly among islands in the region (χ(2) = 206.5, df = 3, P < 0.001). rs387907171 remains significantly, although weakly, associated with lighter hair pigmentation (F = 10.28, R(2) = 0.0125, P = 0.0014). This association increases when sex and age (F = 20.68, R(2) = 0.074, P < 7.92 × 10(-13) ) are included as covariates. CONCLUSIONS: The rs387907171 SNP exhibits strong allele frequency differences among islands in Northern Island Melanesia. Its absence from Bougainville, as well as the weak association with decreased hair color, indicates that additional alleles contribute to the blondism phenotype. Its geographic distribution suggests that a Lapita-mediated model for the dispersal of the derived allele at rs387907171 remains a viable evolutionary scenario. Am. J. Hum. Biol. 28:431-435, 2016. © 2015 Wiley Periodicals, Inc.

MC1R diversity in Northern Island Melanesia has not been constrained by strong purifying selection and cannot explain pigmentation phenotype variation in the region.

BACKGROUND: Variation in human skin pigmentation evolved in response to the selective pressure of ultra-violet radiation (UVR). Selection to maintain darker skin in high UVR environments is expected to constrain pigmentation phenotype and variation in pigmentation loci. Consistent with this hypothesis, the gene MC1R exhibits reduced diversity in African populations from high UVR regions compared to low-UVR non-African populations. However, MC1R diversity in non-African populations that have evolved under high-UVR conditions is not well characterized. METHODS: In order to test the hypothesis that MC1R variation has been constrained in Melanesians the coding region of the MC1R gene was sequenced in 188 individuals from Northern Island Melanesia. The role of purifying selection was assessed using a modified McDonald Kreitman's test. Pairwise FST was calculated between Melanesian populations and populations from the 1000 Genomes Project. The SNP rs2228479 was genotyped in a larger sample (n = 635) of Melanesians and tested for associations with skin and hair pigmentation. RESULTS: We observe three nonsynonymous and two synonymous mutations. A modified McDonald Kreitman's test failed to detect a significant signal of purifying selection. Pairwise FST values calculated between the four islands sampled here indicate little regional substructure in MC1R. When compared to African, European, East and South Asian populations, Melanesians do not exhibit reduced population divergence (measured as FST) or a high proportion of haplotype sharing with Africans, as one might expect if ancestral haplotypes were conserved across high UVR populations in and out of Africa. The only common nonsynonymous polymorphism observed, rs2228479, is not significantly associated with skin or hair pigmentation in a larger sample of Melanesians. CONCLUSIONS: The pattern of sequence diversity here does not support a model of strong selective constraint on MC1R in Northern Island Melanesia This absence of strong constraint, as well as the recent population history of the region, may explain the observed frequencies of the derived rs2228479 allele. These results emphasize the complex genetic architecture of pigmentation phenotypes, which are controlled by multiple, possibly interacting loci. They also highlight the role that population history can play in influencing phenotypic diversity in the absence of strong natural selection.