A novel IKZF1 variant in a family with autosomal dominant CVID: A case for expanding exon coverage in inborn errors of immunity.

Common variable immune deficiency (CVID) is a heterogenous group of disorders characterized by varying degrees of hypogammaglobulinemia, recurrent infections, and autoimmunity. Currently, pathogenic variants are identified in approximately 20-30% of CVID cases. Here we report a 3-generation family with autosomal dominant Common Variable Immunodeficiency (CVID) diagnosed in 9 affected individuals. Although primary immune deficiency panels and exome sequencing were non-diagnostic, whole genome sequencing revealed a novel, pathogenic c.499C > T: p.His167Tyr variant in IKZF1, a critical regulator of B cell development. Functional testing done through pericentromeric heterochromatin localization and light shift chemiluminescent electrophoretic mobility shift assay confirmed the variant's deleterious effect via a haploinsufficiency mechanism. Our findings expand the spectrum of known IKZF1 mutations and contribute to a more comprehensive understanding of CVID's genetic heterogeneity. Furthermore, this case underscores the importance of considering whole genome sequencing for comprehensive genetic diagnosis when concern for a monogenic inborn errors of immunity is high.

Clinically significant findings in a decade-long retrospective study of prenatal chromosomal microarray testing.

BACKGROUND: Chromosomal microarray (CMA) is commonly utilized in the obstetrics setting. CMA is recommended when one or more fetal structural abnormalities is identified. CMA is also commonly used to determine genetic etiologies for miscarriages, fetal demise, and confirming positive prenatal cell-free DNA screening results. METHODS: In this study, we retrospectively examined 523 prenatal and 319 products-of-conception (POC) CMA cases tested at Nationwide Children's Hospital from 2011 to 2020. We reviewed the referral indications, the diagnostic yield, and the reported copy number variants (CNV) findings. RESULTS: In our cohort, the diagnostic yield of clinically significant CNV findings for prenatal testing was 7.8% (n = 41/523) compared to POC testing (16.3%, n = 52/319). Abnormal ultrasound findings were the most common indication present in 81% of prenatal samples. Intrauterine fetal demise was the common indication identified in POC samples. The most common pathogenic finding observed in all samples was isolated trisomy 21, detected in seven samples. CONCLUSION: Our CMA study supports the clinical utility of prenatal CMA for clinical management and identifying genetic etiology in POC arrays. In addition, it provides insight to the spectrum of prenatal and POC CMA results as detected in an academic hospital clinical laboratory setting that serves as a reference laboratory.

Significant roles in RNA-binding for the amino-terminal domains of Drosophila Pumilio and Nanos.

The Drosophila Pumilio (Pum) and Nanos (Nos) RNA-binding proteins govern abdominal segmentation in the early embryo, as well as a variety of other events during development. They bind together to a compound Nanos Response Element (NRE) present in thousands of maternal mRNAs in the ovary and embryo, including hunchback ( hb ) mRNA, thereby regulating poly-adenylation, translation, and stability. Many studies support a model in which mRNA recognition and effector recruitment are achieved by distinct regions of each protein. The well-ordered Pum and Nos RNA-binding domains (RBDs) are sufficient to specifically recognize NREs; the relatively larger low-complexity N-terminal domains (NTDs) of each protein have been thought to act by recruiting mRNA regulators. Here we use yeast interaction assays to show that the NTDs also play a significant role in recognition of the NRE, acting via two mechanisms. First, the Pum and Nos NTDs interact in trans to promote assembly of the Pum/Nos/NRE ternary complex. Second, the Pum NTD acts via an unknown mechanism in cis, modifying base recognition by its RBD. These activities of the Pum NTD are important for its regulation of maternal hb mRNA in vivo.

Post-zygotic rescue of meiotic errors causes brain mosaicism and focal epilepsy.

Somatic mosaicism is a known cause of neurological disorders, including developmental brain malformations and epilepsy. Brain mosaicism is traditionally attributed to post-zygotic genetic alterations arising in fetal development. Here we describe post-zygotic rescue of meiotic errors as an alternate origin of brain mosaicism in patients with focal epilepsy who have mosaic chromosome 1q copy number gains. Genomic analysis showed evidence of an extra parentally derived chromosome 1q allele in the resected brain tissue from five of six patients. This copy number gain is observed only in patient brain tissue, but not in blood or buccal cells, and is strongly enriched in astrocytes. Astrocytes carrying chromosome 1q gains exhibit distinct gene expression signatures and hyaline inclusions, supporting a novel genetic association for astrocytic inclusions in epilepsy. Further, these data demonstrate an alternate mechanism of brain chromosomal mosaicism, with parentally derived copy number gain isolated to brain, reflecting rescue in other tissues during development.

Widespread regulation of the maternal transcriptome by Nanos in Drosophila.

The translational repressor Nanos (Nos) regulates a single target, maternal hunchback (hb) mRNA, to govern abdominal segmentation in the early Drosophila embryo. Nos is recruited specifically to sites in the 3'-UTR of hb mRNA in collaboration with the sequence-specific RNA-binding protein Pumilio (Pum); on its own, Nos has no binding specificity. Nos is expressed at other stages of development, but very few mRNA targets that might mediate its action at these stages have been described. Nor has it been clear whether Nos is targeted to other mRNAs in concert with Pum or via other mechanisms. In this report, we identify mRNAs targeted by Nos via two approaches. In the first method, we identify mRNAs depleted upon expression of a chimera bearing Nos fused to the nonsense mediated decay (NMD) factor Upf1. We find that, in addition to hb, Upf1-Nos depletes ~2600 mRNAs from the maternal transcriptome in early embryos. Virtually all of these appear to be targeted in a canonical, hb-like manner in concert with Pum. In a second, more conventional approach, we identify mRNAs that are stabilized during the maternal zygotic transition (MZT) in embryos from nos- females. Most (86%) of the 1185 mRNAs regulated by Nos are also targeted by Upf1-Nos, validating use of the chimera. Approximately 60% of mRNAs targeted by Upf1-Nos are not stabilized in the absence of Nos. However, Upf1-Nos mRNA targets are hypo-adenylated and inefficiently translated at the ovary-embryo transition, whether or not they suffer Nos-dependent degradation in the embryo. We suggest that the late ovarian burst of Nos represses a large fraction of the maternal transcriptome, priming it for later degradation by other factors during the MZT in the embryo.

Biallelic variants in HTRA2 cause 3-methylglutaconic aciduria mitochondrial disorder: case report and literature review.

Background: Leigh syndrome is a rare, genetic, and severe mitochondrial disorder characterized by neuromuscular issues (ataxia, seizure, hypotonia, developmental delay, dystonia) and ocular abnormalities (nystagmus, atrophy, strabismus, ptosis). It is caused by pathogenic variants in either mitochondrial or nuclear DNA genes, with an estimated incidence rate of 1 per 40,000 live births. Case presentation: Herein, we present an infant male with nystagmus, hypotonia, and developmental delay who carried a clinical diagnosis of Leigh-like syndrome. Cerebral magnetic resonance imaging changes further supported the clinical evidence of an underlying mitochondrial disorder, but extensive diagnostic testing was negative. Trio exome sequencing under a research protocol uncovered compound-heterozygous missense variants in the HTRA2 gene (MIM: #606441): NM_013247.5:c.1037A>T:(p.Glu346Val) (maternal) and NM_013247.5:c.1172T>A:(p.Val391Glu) (paternal). Both variants are absent from public databases, making them extremely rare in the population. The maternal variant is adjacent to an exon-intron boundary and predicted to disrupt splicing, while the paternal variant alters a highly conserved amino acid and is predicted to be damaging by nearly all in silico tools. Biallelic variants in HTRA2 cause 3-methylglutaconic aciduria, type VIII (MGCA8), an extremely rare autosomal recessive disorder with fewer than ten families reported to date. Variant interpretation is challenging given the paucity of known disease-causing variants, and indeed we assess both paternal and maternal variants as Variants of Uncertain Significance under current American College of Medical Genetics guidelines. However, based on the inheritance pattern, suggestive evidence of pathogenicity, and significant clinical correlation with other reported MGCA8 patients, the clinical care team considers this a diagnostic result. Conclusion: Our findings ended the diagnostic odyssey for this family and provide further insights into the genetic and clinical spectrum of this critically under-studied disorder.

Inherited and de novo variants extend the etiology of TAOK1-associated neurodevelopmental disorder.

Alterations in the TAOK1 gene have recently emerged as the cause of developmental delay with or without intellectual impairment or behavioral abnormalities (MIM # 619575). The 32 cases currently described in the literature have predominantly de novo alterations in TAOK1 and a wide spectrum of neurodevelopmental abnormalities. Here, we report four patients with novel pathogenic TAOK1 variants identified by research genome sequencing, clinical exome sequencing, and international matchmaking. The overlapping clinical features of our patients are consistent with the emerging core phenotype of TAOK1-associated syndrome: facial dysmorphism, feeding difficulties, global developmental delay, joint laxity, and hypotonia. However, behavioral abnormalities and gastrointestinal issues are more common in our cohort than previously reported. Two patients have de novo TAOK1 variants (one missense, one splice site) consistent with most known alterations in this gene. However, we also report the first sibling pair who both inherited a TAOK1 frameshift variant from a mildly affected mother. Our findings suggest that incomplete penetrance and variable expressivity are relatively common in TAOK1-associated syndrome, which holds important implications for clinical genetic testing.

Maternal mosaicism for a missense variant in the SMS gene that causes Snyder-Robinson syndrome.

There is increasing recognition for the contribution of genetic mosaicism to human disease, particularly as high-throughput sequencing has enabled detection of sequence variants at very low allele frequencies. Here, we describe an infant male who presented at 9 mo of age with hypotonia, dysmorphic features, congenital heart disease, hyperinsulinemic hypoglycemia, hypothyroidism, and bilateral sensorineural hearing loss. Whole-genome sequencing of the proband and the parents uncovered an apparent de novo mutation in the X-linked SMS gene. SMS encodes spermine synthase, which catalyzes the production of spermine from spermidine. Inactivation of the SMS gene disrupts the spermidine/spermine ratio, resulting in Snyder-Robinson syndrome. The variant in our patient is absent from the gnomAD and ExAC databases and causes a missense change (p.Arg130Cys) predicted to be damaging by most in silico tools. Although Sanger sequencing confirmed the de novo status in our proband, polymerase chain reaction (PCR) and deep targeted resequencing to ∼84,000×-175,000× depth revealed that the variant is present in blood from the unaffected mother at ∼3% variant allele frequency. Our findings thus provided a long-sought diagnosis for the family while highlighting the role of parental mosaicism in severe genetic disorders.

Correlation between protein secondary structure and mechanical performance for the ultra-tough dragline silk of Darwin's bark spider.

The spider major ampullate (MA) silk exhibits high tensile strength and extensibility and is typically a blend of MaSp1 and MaSp2 proteins with the latter comprising glycine-proline-glycine-glycine-X repeating motifs that promote extensibility and supercontraction. The MA silk from Darwin's bark spider (Caerostris darwini) is estimated to be two to three times tougher than the MA silk from other spider species. Previous research suggests that a unique MaSp4 protein incorporates proline into a novel glycine-proline-glycine-proline motif and may explain C. darwini MA silk's extraordinary toughness. However, no direct correlation has been made between the silk's molecular structure and its mechanical properties for C. darwini. Here, we correlate the relative protein secondary structure composition of MA silk from C. darwini and four other spider species with mechanical properties before and after supercontraction to understand the effect of the additional MaSp4 protein. Our results demonstrate that C. darwini MA silk possesses a unique protein composition with a lower ratio of helices (31%) and ß-sheets (20%) than other species. Before supercontraction, toughness, modulus and tensile strength correlate with percentages of ß-sheets, unordered or random coiled regions and ß-turns. However, after supercontraction, only modulus and strain at break correlate with percentages of ß-sheets and ß-turns. Our study highlights that additional information including crystal size and crystal and chain orientation is necessary to build a complete structure-property correlation model.

Protein composition correlates with the mechanical properties of spider ( Argiope trifasciata ) dragline silk.

We investigated the natural variation in silk composition and mechanical performance of the orb-weaving spider Argiope trifasciata at multiple spatial and temporal scales in order to assess how protein composition contributes to the remarkable material properties of spider dragline silk. Major ampullate silk in orb-weaving spiders consists predominantly of two proteins (MaSp1 and MaSp2) with divergent amino acid compositions and functionally different microstructures. Adjusting the expression of these two proteins therefore provides spiders with a simple mechanism to alter the material properties of their silk. We first assessed the reliability and precision of the Waters AccQ-Tag amino acid composition analysis kit for determining the amino acid composition of small quantities of spider silk. We then tested how protein composition varied within single draglines, across draglines spun by the same spider on different days, and finally between spiders. Then, we correlated chemical composition with the material properties of dragline silk. Overall, we found that the chemical composition of major ampullate silk was in general homogeneous among individuals of the same population. Variation in chemical composition was not detectable within silk spun by a single spider on a single day. However, we found that variation within a single spider's silk across different days could, in rare instances, be greater than variation among individual spiders. Most of the variation in silk composition in our investigation resulted from a small number of outliers (three out of sixteen individuals) with a recent history of stress, suggesting stress affects silk production process in orb web spiders. Based on reported sequences for MaSp genes, we developed a gene expression model showing the covariation of the most abundant amino acids in major ampullate silk. Our gene expression model supports that dragline silk composition was mostly determined by the relative abundance of MaSp1 and MaSp2. Finally, we showed that silk composition (especially proline content) strongly correlated with some measures of mechanical performance, particularly how much fibers shrunk during supercontraction as well as their breaking strains. Our findings suggest that spiders are able to change the relative expression rates of different MaSp genes to produce silk fibers with different chemical compositions, and hence, different material properties.

Biomaterial evolution parallels behavioral innovation in the origin of orb-like spider webs.

Correlated evolution of traits can act synergistically to facilitate organism function. But, what happens when constraints exist on the evolvability of some traits, but not others? The orb web was a key innovation in the origin of >12,000 species of spiders. Orb evolution hinged upon the origin of novel spinning behaviors and innovations in silk material properties. In particular, a new major ampullate spidroin protein (MaSp2) increased silk extensibility and toughness, playing a critical role in how orb webs stop flying insects. Here, we show convergence between pseudo-orb-weaving Fecenia and true orb spiders. As in the origin of true orbs, Fecenia dragline silk improved significantly compared to relatives. But, Fecenia silk lacks the high compliance and extensibility found in true orb spiders, likely due in part to the absence of MaSp2. Our results suggest how constraints limit convergent evolution and provide insight into the evolution of nature's toughest fibers.

The velvet spiders: an atlas of the Eresidae (Arachnida, Araneae).

The family Eresidae C. L. Koch, 1850 is reviewed at the genus level. The family comprises nine genera including one new genus. They are: Adonea Simon, 1873, Dorceus C. L. Koch, 1846, Dresserus Simon, 1876, Eresus Walckenaer, 1805, Gandanameno Lehtinen, 1967, Loureediagen. n., ParadoneaLawrence, 1968, Seothyra Purcell, 1903, and Stegodyphus Simon, 1873. A key to all genera and major lineages is provided along with corresponding diagnoses, as well as descriptions of selected species. These are documented with collections of photographs, scanning electron micrographs, and illustrations. A new phylogeny of Eresidae based on molecular sequence data expands on a previously published analysis. A species of the genus Paradonea Lawrence, 1968 is sequenced and placed phylogenetically for the first time. New sequences from twenty Gandanameno Lehtinen, 1967 specimens were added to investigate species limits within the genus. The genus Loureediagen. n. is proposed to accommodate Eresus annulipes Lucas, 1857. Two species, Eresus semicanus Simon, 1908 and Eresus jerbae El-Hennawy, 2005, are synonymized with Loureedia annulipescomb. n. One new species, Paradonea presleyisp. n. is described. Eresus algericus El-Hennawy, 2004 is transferred to Adonea Simon, 1873. The female of Dorceus fastuosus C. L. Koch, 1846 is described for the first time. The first figures depicting Paradonea splendens (Lawrence, 1936) are presented.