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Attention-deficit/hyperactivity disorder (ADHD) belongs to a phenotypically broad class of mental health disorders impacting social and cognitive functioning. Despite heritability estimates of 77%-88% and a global prevalence of up to 1 in 20 children, most of the underlying genetic etiology of the disorder remains undiscovered, making it challenging to obtain a clinical molecular genetic diagnosis and to develop new treatments (Biological Psychiatry, 2005, 57, 1313; Psychological Bulletin, 2009, 135, 608; Psychological Medicine, 2014, 44, 2223). Here we report the identification of a novel ultra-rare heterozygous loss-of-function (p.Q1625*) variant in a child with complex ADHD (i.e., comorbid mild intellectual disability [ID]) and a missense (p.G1748R) variant (allele frequency of 4.7 × 10 -5 ) in a child with primary ADHD (i.e., absence of comorbid autism spectrum disorder [ASD], ID, or syndromic features) both in the SPTBN1 gene. Missense variants in SPTBN1 have been reported in individuals with developmental disorders, language and communication disorders, and motor delays in recent publications (Nature Genetics, 2021, 53, 1006; American Journal of Medical Genetics Part A, 2021, 185, 2037) and ClinVar, though most variants in ClinVar have uncertain disease associations. The functional impact of these 135 variants, including from the current study, were further assessed using prediction scores from the recently developed AlphaMissense tool and benchmarked against published functional studies on a subset of the variants. While heterozygous SPTBN1 variants have recently been associated with neurodevelopmental disorders characterized by global developmental delay, intellectual disability, and behavioral abnormalities, the two patients in the current study expand the phenotypic spectrum to include ADHD in the absence of more severe neurodevelopmental disorders, such as ASD and moderate to severe ID. Furthermore, the culmination of these data with existing reported cases suggests that variation including loss of function and missense events underlie a broader clinical spectrum than previously understood., (© 2024 Wiley Periodicals LLC.)

Little is known about the role of non-coding regions in the etiology of autism spectrum disorder (ASD). We examined three classes of non-coding regions: human accelerated regions (HARs), which show signatures of positive selection in humans; experimentally validated neural VISTA enhancers (VEs); and conserved regions predicted to act as neural enhancers (CNEs). Targeted and whole-genome analysis of >16,600 samples and >4,900 ASD probands revealed that likely recessive, rare, inherited variants in HARs, VEs, and CNEs substantially contribute to ASD risk in probands whose parents share ancestry, which enriches for recessive contributions, but modestly contribute, if at all, in simplex family structures. We identified multiple patient variants in HARs near IL1RAPL1 and in VEs near OTX1 and SIM1 and showed that they change enhancer activity. Our results implicate both human-evolved and evolutionarily conserved non-coding regions in ASD risk and suggest potential mechanisms of how regulatory changes can modulate social behavior., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Creutzfeldt-Jakob Disease (CJD), the most common human prion disease, is associated with pathologic misfolding of the prion protein (PrP), encoded by the PRNP gene. Of human prion disease cases, < 1% were transmitted by misfolded PrP, ~ 15% are inherited, and ~ 85% are sporadic (sCJD). While familial cases are inherited through germline mutations in PRNP, the cause of sCJD is unknown. Somatic mutations have been hypothesized as a cause of sCJD, and recent studies have revealed that somatic mutations accumulate in neurons during aging. To investigate the hypothesis that somatic mutations in PRNP may underlie sCJD, we performed deep DNA sequencing of PRNP in 205 sCJD cases and 170 age-matched non-disease controls. We included 5 cases of Heidenhain variant sporadic CJD (H-sCJD), where visual symptomatology and neuropathology implicate localized initiation of prion formation, and examined multiple regions across the brain including in the affected occipital cortex. We employed Multiple Independent Primer PCR Sequencing (MIPP-Seq) with a median depth of > 5000× across the PRNP coding region and analyzed for variants using MosaicHunter. An allele mixing experiment showed positive detection of variants in bulk DNA at a variant allele fraction (VAF) as low as 0.2%. We observed multiple polymorphic germline variants among individuals in our cohort. However, we did not identify bona fide somatic variants in sCJD, including across multiple affected regions in H-sCJD, nor in control individuals. Beyond our stringent variant-identification pipeline, we also analyzed VAFs from raw sequencing data, and observed no evidence of prion disease enrichment for the known germline pathogenic variants P102L, D178N, and E200K. The lack of PRNP pathogenic somatic mutations in H-sCJD or the broader cohort of sCJD suggests that clonal somatic mutations may not play a major role in sporadic prion disease. With H-sCJD representing a localized presentation of neurodegeneration, this serves as a test of the potential role of clonal somatic mutations in genes known to cause familial neurodegeneration., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Thoracic aortic aneurysm (TAA) is associated with significant morbidity and mortality. Although individuals with family histories of TAA often undergo clinical molecular genetic testing, adults with nonsyndromic TAA are not typically evaluated for genetic causes. We sought to understand the genetic contribution of both germline and somatic mosaic variants in a cohort of adult individuals with nonsyndromic TAA at a single center., Methods and Results: One hundred eighty-one consecutive patients <60 years who presented with nonsyndromic TAA at the Massachusetts General Hospital underwent deep (>500×) targeted sequencing across 114 candidate genes associated with TAA and its related functional pathways. Samples from 354 age- and sex-matched individuals without TAA were also sequenced, with a 2:1 matching. We found significant enrichments for germline (odds ratio [OR], 2.44, P =4.6×10 -6 [95% CI, 1.67-3.58]) and also somatic mosaic variants (OR, 4.71, P =0.026 [95% CI, 1.20-18.43]) between individuals with and without TAA. Likely genetic causes were present in 24% with nonsyndromic TAA, of which 21% arose from germline variants and 3% from somatic mosaic alleles. The 3 most frequently mutated genes in our cohort were FLNA (encoding Filamin A), NOTCH3 (encoding Notch receptor 3), and FBN1 (encoding Fibrillin-1). There was increased frequency of both missense and loss of function variants in TAA individuals., Conclusions: Likely contributory dominant acting genetic variants were found in almost one quarter of nonsyndromic adults with TAA. Our findings suggest a more extensive genetic architecture to TAA than expected and that genetic testing may improve the care and clinical management of adults with nonsyndromic TAA.

Creutzfeldt-Jakob Disease (CJD), the most common human prion disease, is associated with pathologic misfolding of the prion protein (PrP), encoded by the PRNP gene. Of human prion disease cases, ~1% were transmitted by misfolded PrP, ~15% are inherited, and ~85% are sporadic (sCJD). While familial cases are inherited through germline mutations in PRNP , the cause of sCJD is unknown. Somatic mutations have been hypothesized as a cause of sCJD, and recent studies have revealed that somatic mutations accumulate in neurons during aging. To investigate the hypothesis that somatic mutations in PRNP may underlie sCJD, we performed deep DNA sequencing of PRNP in 205 sCJD cases and 170 age-matched non-disease controls. We included 5 cases of Heidenhain variant sporadic CJD (H-sCJD), where visual symptomatology and neuropathology implicate focal initiation of prion formation, and examined multiple regions across the brain including in the affected occipital cortex. We employed Multiple Independent Primer PCR Sequencing (MIPP-Seq) with a median depth of >5,000X across the PRNP coding region and analyzed for variants using MosaicHunter. An allele mixing experiment showed positive detection of variants in bulk DNA at a variant allele fraction (VAF) as low as 0.2%. We observed multiple polymorphic germline variants among individuals in our cohort. However, we did not identify bona fide somatic variants in sCJD, including across multiple affected regions in H-sCJD, nor in control individuals. Beyond our stringent variant-identification pipeline, we also analyzed VAFs from raw sequencing data, and observed no evidence of prion disease enrichment for the known germline pathogenic variants P102L, D178N, and E200K. The lack of PRNP pathogenic somatic mutations in H-sCJD or the broader cohort of sCJD suggests that clonal somatic mutations may not play a major role in sporadic prion disease. With H-sCJD representing a focal presentation of neurodegeneration, this serves as a test of the potential role of clonal somatic mutations in genes known to cause familial neurodegeneration., Competing Interests: Competing interests C.A.W. is a paid consultant (cash, no equity) to Third Rock Ventures and Flagship Pioneering (cash, no equity) and is on the Clinical Advisory Board (cash and equity) of Maze Therapeutics. No research support is received. These companies did not fund and had no role in the conception or performance of this research project. B.S.A. has received research funding from CDC, NIH, CJD Foundation, and Ionis. B.S.A. has provided consultation for Ionis, Gates Biosciences, and Sangamo and received royalties from Wolter-Kluwer. All other authors have no competing interests to declare.

Importance: Polymicrogyria is the most commonly diagnosed cortical malformation and is associated with neurodevelopmental sequelae including epilepsy, motor abnormalities, and cognitive deficits. Polymicrogyria frequently co-occurs with other brain malformations or as part of syndromic diseases. Past studies of polymicrogyria have defined heterogeneous genetic and nongenetic causes but have explained only a small fraction of cases., Objective: To survey germline genetic causes of polymicrogyria in a large cohort and to consider novel polymicrogyria gene associations., Design, Setting, and Participants: This genetic association study analyzed panel sequencing and exome sequencing of accrued DNA samples from a retrospective cohort of families with members with polymicrogyria. Samples were accrued over more than 20 years (1994 to 2020), and sequencing occurred in 2 stages: panel sequencing (June 2015 to January 2016) and whole-exome sequencing (September 2019 to March 2020). Individuals seen at multiple clinical sites for neurological complaints found to have polymicrogyria on neuroimaging, then referred to the research team by evaluating clinicians, were included in the study. Targeted next-generation sequencing and/or exome sequencing were performed on probands (and available parents and siblings) from 284 families with individuals who had isolated polymicrogyria or polymicrogyria as part of a clinical syndrome and no genetic diagnosis at time of referral from clinic, with sequencing from 275 families passing quality control., Main Outcomes and Measures: The number of families in whom genetic sequencing yielded a molecular diagnosis that explained the polymicrogyria in the family. Secondarily, the relative frequency of different genetic causes of polymicrogyria and whether specific genetic causes were associated with co-occurring head size changes were also analyzed., Results: In 32.7% (90 of 275) of polymicrogyria-affected families, genetic variants were identified that provided satisfactory molecular explanations. Known genes most frequently implicated by polymicrogyria-associated variants in this cohort were PIK3R2, TUBB2B, COL4A1, and SCN3A. Six candidate novel polymicrogyria genes were identified or confirmed: de novo missense variants in PANX1, QRICH1, and SCN2A and compound heterozygous variants in TMEM161B, KIF26A, and MAN2C1, each with consistent genotype-phenotype relationships in multiple families., Conclusions and Relevance: This study's findings reveal a higher than previously recognized rate of identifiable genetic causes, specifically of channelopathies, in individuals with polymicrogyria and support the utility of exome sequencing for families affected with polymicrogyria.

Although oncogenic mutations have been found in nondiseased, proliferative nonneural tissues, their prevalence in the human brain is unknown. Targeted sequencing of genes implicated in brain tumors in 418 samples derived from 110 individuals of varying ages, without tumor diagnoses, detected oncogenic somatic single-nucleotide variants (sSNV) in 5.4% of the brains, including IDH1 R132H . These mutations were largely present in subcortical white matter and enriched in glial cells and, surprisingly, were less common in older individuals. A depletion of high-allele frequency sSNVs representing macroscopic clones with age was replicated by analysis of bulk RNA sequencing data from 1,816 nondiseased brain samples ranging from fetal to old age. We also describe large clonal copy number variants and that sSNVs show mutational signatures resembling those found in gliomas, suggesting that mutational processes of the normal brain drive early glial oncogenesis. This study helps understand the origin and early evolution of brain tumors. SIGNIFICANCE: In the nondiseased brain, clonal oncogenic mutations are enriched in white matter and are less common in older individuals. We revealed early steps in acquiring oncogenic variants, which are essential to understanding brain tumor origins and building new mutational baselines for diagnostics. This article is highlighted in the In This Issue feature, p. 1 ., (©2021 The Authors; Published by the American Association for Cancer Research.)

Canine multiple system degeneration (CMSD) is a progressive hereditary neurodegenerative disorder commonly characterized by neuronal degeneration and loss in the cerebellum, olivary nuclei, substantia nigra, and caudate nuclei. In this article, we describe 3 cases of CMSD in Ibizan hounds. All patients exhibited marked cerebellar ataxia and had cerebellar atrophy on magnetic resonance imaging. At necropsy, all cases showed varying degrees of cerebellar atrophy, and 2 cases had gross cavitation of the caudate nuclei. Histologic findings included severe degeneration and loss of all layers of the cerebellum and neuronal loss and degeneration within the olivary nuclei, substantia nigra, and caudate nuclei. Pedigree analysis indicated an autosomal recessive mode of inheritance, but the causative gene in this breed is yet to be identified. CMSD resembles human multiple system atrophy and warrants further investigation.

Human accelerated regions (HARs) are the fastest-evolving regions of the human genome, and many are hypothesized to function as regulatory elements that drive human-specific gene regulatory programs. We interrogate the in vitro enhancer activity and in vivo epigenetic landscape of more than 3,100 HARs during human neurodevelopment, demonstrating that many HARs appear to act as neurodevelopmental enhancers and that sequence divergence at HARs has largely augmented their neuronal enhancer activity. Furthermore, we demonstrate PPP1R17 to be a putative HAR-regulated gene that has undergone remarkable rewiring of its cell type and developmental expression patterns between non-primates and primates and between non-human primates and humans. Finally, we show that PPP1R17 slows neural progenitor cell cycle progression, paralleling the cell cycle length increase seen predominantly in primate and especially human neurodevelopment. Our findings establish HARs as key components in rewiring human-specific neurodevelopmental gene regulatory programs and provide an integrated resource to study enhancer activity of specific HARs., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Objective: Focal cortical dysplasia (FCD) is a major cause of difficult-to-treat epilepsy in children and young adults, and the diagnosis is currently based on microscopic review of surgical brain tissue using the International League Against Epilepsy classification scheme of 2011. We developed an iterative histopathological agreement trial with genetic testing to identify areas of diagnostic challenges in this widely used classification scheme., Methods: Four web-based digital pathology trials were completed by 20 neuropathologists from 15 countries using a consecutive series of 196 surgical tissue blocks obtained from 22 epilepsy patients at a single center. Five independent genetic laboratories performed screening or validation sequencing of FCD-relevant genes in paired brain and blood samples from the same 22 epilepsy patients., Results: Histopathology agreement based solely on hematoxylin and eosin stainings was low in Round 1, and gradually increased by adding a panel of immunostainings in Round 2 and the Delphi consensus method in Round 3. Interobserver agreement was good in Round 4 (kappa = .65), when the results of genetic tests were disclosed, namely, MTOR, AKT3, and SLC35A2 brain somatic mutations in five cases and germline mutations in DEPDC5 and NPRL3 in two cases., Significance: The diagnoses of FCD 1 and 3 subtypes remained most challenging and were often difficult to differentiate from a normal homotypic or heterotypic cortical architecture. Immunohistochemistry was helpful, however, to confirm the diagnosis of FCD or no lesion. We observed a genotype-phenotype association for brain somatic mutations in SLC35A2 in two cases with mild malformation of cortical development with oligodendroglial hyperplasia in epilepsy. Our results suggest that the current FCD classification should recognize a panel of immunohistochemical stainings for a better histopathological workup and definition of FCD subtypes. We also propose adding the level of genetic findings to obtain a comprehensive, reliable, and integrative genotype-phenotype diagnosis in the near future., (© 2021 The Authors. Epilepsia published by Wiley Periodicals LLC on behalf of International League Against Epilepsy.)

Although cell lineage information is fundamental to understanding organismal development, very little direct information is available for humans. We performed high-depth (250×) whole-genome sequencing of multiple tissues from three individuals to identify hundreds of somatic single-nucleotide variants (sSNVs). Using these variants as "endogenous barcodes" in single cells, we reconstructed early embryonic cell divisions. Targeted sequencing of clonal sSNVs in different organs (about 25,000×) and in more than 1000 cortical single cells, as well as single-nucleus RNA sequencing and single-nucleus assay for transposase-accessible chromatin sequencing of ~100,000 cortical single cells, demonstrated asymmetric contributions of early progenitors to extraembryonic tissues, distinct germ layers, and organs. Our data suggest onset of gastrulation at an effective progenitor pool of about 170 cells and about 50 to 100 founders for the forebrain. Thus, mosaic mutations provide a permanent record of human embryonic development at very high resolution., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Background: Mosaic mutations contribute to numerous human disorders. As such, the identification and precise quantification of mosaic mutations is essential for a wide range of research applications, clinical diagnoses, and early detection of cancers. Currently, the low-throughput nature of single allele assays (e.g., allele-specific ddPCR) commonly used for genotyping known mutations at very low alternate allelic fractions (AAFs) have limited the integration of low-level mosaic analyses into clinical and research applications. The growing importance of mosaic mutations requires a more rapid, low-cost solution for mutation detection and validation., Methods: To overcome these limitations, we developed Multiple Independent Primer PCR Sequencing (MIPP-Seq) which combines the power of ultra-deep sequencing and truly independent assays. The accuracy of MIPP-seq to quantifiable detect and measure extremely low allelic fractions was assessed using a combination of SNVs, insertions, and deletions at known allelic fractions in blood and brain derived DNA samples., Results: The Independent amplicon analyses of MIPP-Seq markedly reduce the impact of allelic dropout, amplification bias, PCR-induced, and sequencing artifacts. Using low DNA inputs of either 25 ng or 50 ng of DNA, MIPP-Seq provides sensitive and quantitative assessments of AAFs as low as 0.025% for SNVs, insertion, and deletions., Conclusions: MIPP-Seq provides an ultra-sensitive, low-cost approach for detecting and validating known and novel mutations in a highly scalable system with broad utility spanning both research and clinical diagnostic testing applications. The scalability of MIPP-Seq allows for multiplexing mutations and samples, which dramatically reduce costs of variant validation when compared to methods like ddPCR. By leveraging the power of individual analyses of multiple unique and independent reactions, MIPP-Seq can validate and precisely quantitate extremely low AAFs across multiple tissues and mutational categories including both indels and SNVs. Furthermore, using Illumina sequencing technology, MIPP-seq provides a robust method for accurate detection of novel mutations at an extremely low AAF.

We characterize the landscape of somatic mutations-mutations occurring after fertilization-in the human brain using ultra-deep (~250×) whole-genome sequencing of prefrontal cortex from 59 donors with autism spectrum disorder (ASD) and 15 control donors. We observe a mean of 26 somatic single-nucleotide variants per brain present in ≥4% of cells, with enrichment of mutations in coding and putative regulatory regions. Our analysis reveals that the first cell division after fertilization produces ~3.4 mutations, followed by 2-3 mutations in subsequent generations. This suggests that a typical individual possesses ~80 somatic single-nucleotide variants present in ≥2% of cells-comparable to the number of de novo germline mutations per generation-with about half of individuals having at least one potentially function-altering somatic mutation somewhere in the cortex. ASD brains show an excess of somatic mutations in neural enhancer sequences compared with controls, suggesting that mosaic enhancer mutations may contribute to ASD risk.

Objective: Congenital structural brain malformations have been described in patients with pathogenic phosphatase and tensin homologue (PTEN) variants, but the frequency of cortical malformations in patients with PTEN variants and their impact on clinical phenotype are not well understood. Our goal was to systematically characterize brain malformations in patients with PTEN variants and assess the relevance of their brain malformations to clinical presentation., Methods: We systematically searched a local radiology database for patients with PTEN variants who had available brain magnetic resonance imaging (MRI). The MRI scans were reviewed systematically for cortical abnormalities. We reviewed electroencephalogram (EEG) data and evaluated the electronic medical record for evidence of epilepsy and developmental delay., Results: In total, we identified 22 patients with PTEN pathogenic variants for which brain MRIs were available (age range 0.4-17 years). Twelve among these 22 patients (54%) had polymicrogyria (PMG). Variants associated with PMG or atypical gyration encoded regions of the phosphatase or C2 domains of PTEN. Interestingly, epilepsy was present in only 2 of the 12 patients with PMG. We found a trend toward higher rates of global developmental delay (GDD), intellectual disability (ID), and motor delay in individuals with cortical abnormalities, although cohort size limited statistical significance., Interpretation: Malformations of cortical development, PMG in particular, represent an under-recognized phenotype associated with PTEN pathogenic variants and may have an association with cognitive and motor delays. Epilepsy was infrequent compared to the previously reported high risk of epilepsy in patients with PMG. ANN NEUROL 2020;88:1153-1164., (© 2020 American Neurological Association.)

More than 98% of the human genome is made up of non-coding DNA, but techniques to ascertain its contribution to human disease have lagged far behind our understanding of protein coding variations. Autism spectrum disorder (ASD) has been mostly associated with coding variations via de novo single nucleotide variants (SNVs), recessive/homozygous SNVs, or de novo copy number variants (CNVs); however, most ASD cases continue to lack a genetic diagnosis. We analyzed 187 consanguineous ASD families for biallelic CNVs. Recessive deletions were significantly enriched in affected individuals relative to their unaffected siblings (17% versus 4%, p < 0.001). Only a small subset of biallelic deletions were predicted to result in coding exon disruption. In contrast, biallelic deletions in individuals with ASD were enriched for overlap with regulatory regions, with 23/28 CNVs disrupting histone peaks in ENCODE (p < 0.009). Overlap with regulatory regions was further demonstrated by comparisons to the 127-epigenome dataset released by the Roadmap Epigenomics project, with enrichment for enhancers found in primary brain tissue and neuronal progenitor cells. Our results suggest a novel noncoding mechanism of ASD, describe a powerful method to identify important noncoding regions in the human genome, and emphasize the potential significance of gene activation and regulation in cognitive and social function.

Autism spectrum disorder (ASD) affects up to 1 in 59 individuals 1 . Genome-wide association and large-scale sequencing studies strongly implicate both common variants 2-4 and rare de novo variants 5-10 in ASD. Recessive mutations have also been implicated 11-14 but their contribution remains less well defined. Here we demonstrate an excess of biallelic loss-of-function and damaging missense mutations in a large ASD cohort, corresponding to approximately 5% of total cases, including 10% of females, consistent with a female protective effect. We document biallelic disruption of known or emerging recessive neurodevelopmental genes (CA2, DDHD1, NSUN2, PAH, RARB, ROGDI, SLC1A1, USH2A) as well as other genes not previously implicated in ASD including FEV (FEV transcription factor, ETS family member), which encodes a key regulator of the serotonergic circuitry. Our data refine estimates of the contribution of recessive mutation to ASD and suggest new paths for illuminating previously unknown biological pathways responsible for this condition.

Neonates of all species, including foals, are highly susceptible to infection, and neutrophils play a crucial role in innate immunity to infection. Evidence exists that neutrophils of neonatal foals are functionally deficient during the first weeks of life, including expression of cytokine genes such as IFNG. We hypothesized that postnatal epigenetic changes were likely to regulate the observed age-related changes in foal neutrophils. Using ChIP-Seq, we identified significant differences in trimethylated histone H3 lysine 4, an epigenetic modification associated with active promoters and enhancers, in neutrophils in foals at 30 days of age relative to 1 day of age. These chromatin changes were associated with genes implicated in immune responses and were consistent with age-related changes in neutrophil functional responses including ROS generation and IFN expression. Postnatal changes in epigenetic modifications suggest that environmentally-mediated cues help to promote maturation of neutrophil functional responses. Elucidating the environmental triggers and their signaling pathways could provide a means for improving innate immune responses of neonates to improve their ability to combat infectious diseases., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Channelopathies are disorders caused by abnormal ion channel function in differentiated excitable tissues. We discovered a unique neurodevelopmental channelopathy resulting from pathogenic variants in SCN3A, a gene encoding the voltage-gated sodium channel Na V 1.3. Pathogenic Na V 1.3 channels showed altered biophysical properties including increased persistent current. Remarkably, affected individuals showed disrupted folding (polymicrogyria) of the perisylvian cortex of the brain but did not typically exhibit epilepsy; they presented with prominent speech and oral motor dysfunction, implicating SCN3A in prenatal development of human cortical language areas. The development of this disorder parallels SCN3A expression, which we observed to be highest early in fetal cortical development in progenitor cells of the outer subventricular zone and cortical plate neurons and decreased postnatally, when SCN1A (Na V 1.1) expression increased. Disrupted cerebral cortical folding and neuronal migration were recapitulated in ferrets expressing the mutant channel, underscoring the unexpected role of SCN3A in progenitor cells and migrating neurons., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Understanding the biological basis for human-specific cognitive traits presents both immense challenges and unique opportunities. Although the question of what makes us human has been investigated with several different methods, the rise of comparative genomics, epigenomics, and medical genetics has provided tools to help narrow down and functionally assess the regions of the genome that seem evolutionarily relevant along the human lineage. In this review, we focus on how medical genetic cases have provided compelling functional evidence for genes and loci that appear to have interesting evolutionary signatures in humans. Furthermore, we examine a special class of noncoding regions, human accelerated regions (HARs), that have been suggested to show human-lineage-specific divergence, and how the use of clinical and population data has started to provide functional information to examine these regions. Finally, we outline methods that provide new insights into functional noncoding sequences in evolution.

During meiosis I, homologous chromosomes join together to form bivalents. Through trial and error, bivalents achieve stable bipolar orientations (attachments) on the spindle that eventually allow the segregation of homologous chromosomes to opposite poles. Bipolar orientations are stable through tension generated by poleward forces to opposite poles. Unipolar orientations lack tension and are stereotypically not stable. The behavior of sex chromosomes during meiosis I in the male black widow spider Latrodectus mactans (Araneae, Theridiidae) challenges the principles governing such a scenario. We found that male L. mactans has two distinct X chromosomes, X 1 and X 2 . The X chromosomes join together to form a connection that is present in prometaphase I but is lost during metaphase I, before the autosomes disjoin at anaphase I. We found that both X chromosomes form stable unipolar orientations to the same pole that assure their co-segregation at anaphase I. Using micromanipulation, immunofluorescence microscopy, and electron microscopy, we studied this unusual chromosome behavior to explain how it may fit the current dogma of chromosome distribution during cell division.

Comparative analyses have identified genomic regions potentially involved in human evolution but do not directly assess function. Human accelerated regions (HARs) represent conserved genomic loci with elevated divergence in humans. If some HARs regulate human-specific social and behavioral traits, then mutations would likely impact cognitive and social disorders. Strikingly, rare biallelic point mutations-identified by whole-genome and targeted "HAR-ome" sequencing-showed a significant excess in individuals with ASD whose parents share common ancestry compared to familial controls, suggesting a contribution in 5% of consanguineous ASD cases. Using chromatin interaction sequencing, massively parallel reporter assays (MPRA), and transgenic mice, we identified disease-linked, biallelic HAR mutations in active enhancers for CUX1, PTBP2, GPC4, CDKL5, and other genes implicated in neural function, ASD, or both. Our data provide genetic evidence that specific HARs are essential for normal development, consistent with suggestions that their evolutionary changes may have altered social and/or cognitive behavior. PAPERCLIP., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The human cerebral cortex depends for its normal development and size on a precisely controlled balance between self-renewal and differentiation of diverse neural progenitor cells. Specialized progenitors that are common in humans but virtually absent in rodents, called outer radial glia (ORG), have been suggested to be crucial to the evolutionary expansion of the human cortex. We combined progenitor subtype-specific sorting with transcriptome-wide RNA sequencing to identify genes enriched in human ORG, which included targets of the transcription factor neurogenin and previously uncharacterized, evolutionarily dynamic long noncoding RNAs. Activating the neurogenin pathway in ferret progenitors promoted delamination and outward migration. Finally, single-cell transcriptional profiling in human, ferret and mouse revealed more cells coexpressing proneural neurogenin targets in human than in other species, suggesting greater neuronal lineage commitment and differentiation of self-renewing progenitors. Thus, we find that the abundance of human ORG is paralleled by increased transcriptional heterogeneity of cortical progenitors.

Karyotypes can be difficult to create due to odd chromosome behaviors and high levels of adhesion between sex chromosomes. We have used live-cell imaging and micromanipulation to determine precisely the karyotype of the orb-weaving spider Neoscona arabesca (Walckenaer). We have found that N. arabesca has a sex determination mechanism of XO (male)-XX (female) with 2n = 20 autosomes. Staining of female tissues revealed a chromosome number of 22 in mitotic cells.

Iron status, assessed by ferritin (ng/ml), transferrin saturation (%), and hemoglobin (g/100 ml), was examined in a sample of 1398 women from blood samples drawn at their first prenatal visit and in 712 of these women later at their 32-week prenatal visit. Self-reported alcohol use (average ounces of absolute alcohol consumed per day around the time of conception) was not systematically related to iron status. Furthermore, the proportion of iron-depleted or anemic women did not vary according to different levels of alcohol use. An exception is noted in a small group of nine very heavy drinkers, reporting eight or more drinks per day, who, at 32 weeks gestation, had a higher proportion of iron depletion but not anemia. It was concluded that at least in a sample of primarily middle class women receiving good prenatal care, iron depletion and/or anemia probably do not explain the alcohol-related adverse pregnancy outcomes that have been reported previously. Nevertheless, results from several very heavy drinkers suggest that the iron status of pregnant alcoholic women should be investigated further.

The protoporphyrinemia of iron deficiency is well recognized. Clinically, information on the protoporphyrin/heme molar ratio in whole blood offers certain advantages over protoporphyrin measurement alone. A procedure for determining this ratio is reported. Protoporphyrin is extracted, solubilized, and measured fluorometrically. Heme (as hemin chloride) is precipitated with the blood proteins, the precipitate is dissolved in an alkaline/pyridine solvent, and the resulting bispyridine ferriprotoporphyrin is measured spectrophotometrically. The molar ratio of these two metabolites correlates well with values for plasma ferritin, plasma iron, transferrin saturation, hemoglobin, and hematocrit. In some cases the ratio increases detectably while the other variables, especially hematocrit and hemoglobin, remain normal. Evidently it is a more sensitive index to iron status. For healthy men and women, the mean ratio is 16.0 (SD, 5.3). The mean + 3 SD, or a ratio of 32, is distinctly abnormal, as shown by a confirmatory test. We validated the test by surveying routine blood specimens obtained from several population groups.