KIF11 Variants Associated With Novel Renal System Involvement-Two Cases That Expand the Phenotypic Spectrum of Microcephaly With or Without Chorioretinopathy, Lymphedema, or Impaired Intellectual Development.

Pathogenic variants in KIF11 are linked to microcephaly with or without chorioretinopathy, lymphedema, or impaired intellectual development (MCLMR). To our knowledge, renal phenotypes have not been described in the literature in association with KIF11-related disorders. This study is a case report of two probands with heterozygous pathogenic variants in KIF11 who presented with the common clinical features of MCLMR but also had additional renal involvement not previously reported as associated phenotypes of MCLMR, elucidating phenotypic expansion of this syndrome.

Reduction in ACE2 expression in peripheral blood mononuclear cells during COVID-19 - implications for post COVID-19 conditions.

BACKGROUND: Severe COVID-19 is uncommon, restricted to 19% of the total population. In response to the first virus wave (alpha variant of SARS-CoV-2), we investigated whether a biomarker indicated severity of disease and, in particular, if variable expression of angiotensin converting enzyme 2 (ACE2) in blood might clarify this difference in risk and of post COVID -19 conditions (PCC). METHODS: The IRB-approved study compared patients hospitalized with severe COVID-19 to healthy controls. Severe infection was defined requiring oxygen or increased oxygen need from baseline at admission with positive COVID-19 PCR. A single blood sample was obtained from patients within a day of admission. ACE2 RNA expression in blood cells was measured by an RT-PCR assay. Plasma ACE1 and ACE2 enzyme activities were quantified by fluorescent peptides. Plasma TIMP-1, PIIINP and MMP-9 antigens were quantified by ELISA. Data were entered into REDCap and analyzed using STATA v 14 and GraphPad Prism v 10. RESULTS: Forty-eight patients and 72 healthy controls were recruited during the pandemic. ACE2 RNA expression in peripheral blood mononuclear cells (PBMC) was rarely detected acutely during severe COVID-19 but common in controls (OR for undetected ACE2: 12.4 [95% CI: 2.62-76.1]). ACE2 RNA expression in PBMC did not determine plasma ACE1 and ACE2 activity, suggesting alternative cell-signaling pathways. Markers of fibrosis (TIMP-1 and PIIINP) and vasculopathy (MMP-9) were additionally elevated. ACE2 RNA expression during severe COVID-19 often responded within hours to convalescent plasma. Analogous to oncogenesis, we speculate that potent, persistent, cryptic processes following COVID-19 (the renin-angiotensin system (RAS), fibrosis and vasculopathy) initiate or promote post-COVID-19 conditions (PCC) in susceptible individuals. CONCLUSIONS: This work elucidates biological and temporal plausibility for ACE2, TIMP1, PIIINP and MMP-9 in the pathogenesis of PCC. Intersection of these independent systems is uncommon and may in part explain the rarity of PCC.

Prostate cancer risk locus at 8q24 as a regulatory hub by physical interactions with multiple genomic loci across the genome.

Chromosome 8q24 locus contains regulatory variants that modulate genetic risk to various cancers including prostate cancer (PC). However, the biological mechanism underlying this regulation is not well understood. Here, we developed a chromosome conformation capture (3C)-based multi-target sequencing technology and systematically examined three PC risk regions at the 8q24 locus and their potential regulatory targets across human genome in six cell lines. We observed frequent physical contacts of this risk locus with multiple genomic regions, in particular, inter-chromosomal interaction with CD96 at 3q13 and intra-chromosomal interaction with MYC at 8q24. We identified at least five interaction hot spots within the predicted functional regulatory elements at the 8q24 risk locus. We also found intra-chromosomal interaction genes PVT1, FAM84B and GSDMC and inter-chromosomal interaction gene CXorf36 in most of the six cell lines. Other gene regions appeared to be cell line-specific, such as RRP12 in LNCaP, USP14 in DU-145 and SMIN3 in lymphoblastoid cell line. We further found that the 8q24 functional domains more likely interacted with genomic regions containing genes enriched in critical pathways such as Wnt signaling and promoter motifs such as E2F1 and TCF3. This result suggests that the risk locus may function as a regulatory hub by physical interactions with multiple genes important for prostate carcinogenesis. Further understanding genetic effect and biological mechanism of these chromatin interactions will shed light on the newly discovered regulatory role of the risk locus in PC etiology and progression.

RNF213 variants in a child with PHACE syndrome and moyamoya vasculopathy.

Segmental infantile hemangiomas (IH) can be associated with congenital anomalies in a regional distribution. PHACE refers to large cervicofacial segmental IH in association with congenital anomalies of the aortic arch and medium-sized arteries of the head and neck, as well as structural anomalies of the posterior fossa and eye. A subset of PHACE patients have arterial anomalies that progress to moyamoya vasculopathy (MMV). MMV is defined as stenosis of the supraclinoid segment of the internal carotid arteries and/or their major branches, with subsequent development of a compensatory collateral vessel network. We describe a patient with MMV and segmental IH on the back and lower body who meets diagnostic criteria for PHACE based on a posterior segment eye anomaly and cerebral arterial anomalies. Whole exome sequencing demonstrated two inherited heterozygous variants in RNF213. Variants in RNF213 are associated with increased susceptibility to MMV. Our findings suggest that RNF213 variants may play a role in the development of MMV in patients with hemangioma syndromes associated with congenital cerebral arterial anomalies.

8q21.11 microdeletion in two patients with syndromic peters anomaly.

Peters anomaly is a form of anterior segment dysgenesis characterized by central ocular opacity and corneo-lenticular adhesions. Isolated and syndromic Peters anomaly can be observed and demonstrate significant genetic heterogeneity. We report the identification of overlapping 8q21.11 deletions in two patients with syndromic Peters anomaly via whole exome sequencing and chromosomal microarray analyses. Microdeletions of 8q21.11 were recently reported in 10 patients with highly variable phenotypes involving craniofacial features, ptosis, intellectual disability, abnormalities of the hands/feet and other defects; sclerocornea and/or microphthalmia were reported in three cases. The two additional cases presented in this report expand the phenotypic spectrum of 8q21.11 microdeletions to include Peters anomaly (seen in both patients) and persistent primary dentition (seen in one patient with a larger deletion). The two novel deletions include the ZFHX4 and PEX2 genes, which were also affected in all three previous cases involving ocular anomalies. Screening of the remaining alleles of ZFHX4 and PEX2 did not identify any additional likely pathogenic variants in either patient, suggesting a dominant mechanism (haploinsufficiency) for the identified deletion. This report provides further insight into the phenotypes associated with 8q21.11 deletions and, for the first time, reports Peters anomaly as an additional ocular feature; screening for copy number variations of the 8q21.11 region should be considered in patients with Peters anomaly and related syndromic features. © 2016 Wiley Periodicals, Inc.

Implementation of a Customized Tertiary Analysis Platform for the Reporting of Somatic Variants.

Precision medicine for oncology requires the evaluation of variants identified in molecular profiling of solid tumors and hematologic malignancies. This includes evaluation of pre-analytical and postanalytical quality metrics, variant interpretation, classification, and tiering as outlined in established guidelines, association with clinical significance such as FDA approved drugs and clinical trials, and finally comprehensive reporting. This study documents our experience with the customization and implementation of a software platform that facilitates these requirements for effective reporting of somatic variants.

Evaluating direct amplification from viral transport medium for SARS-CoV-2 detection, strain typing, and angiotensin-converting enzyme genotyping and expression assays.

OBJECTIVE: The aim of this study was to compare the performance of direct amplification of viral nucleic acid from transport medium to extracted nucleic acid for polymerase chain reaction (PCR), sequencing, and genotyping applications. METHODS: XpressAmp lysate and extracted total nucleic acid from viral transport medium containing nasopharyngeal specimens were evaluated across different molecular applications to determine performance characteristics. RESULTS: SARS-CoV-2 quantitative PCR and angiotensin-converting enzyme (ACE) genotyping assays worked well with XpressAmp lysate, almost equal with or better than extracted nucleic acid in some specimens. However, XpressAmp completely failed to perform in next-generation sequencing for strain typing. Both protocols failed to detect ACE2 expression in viral transport medium. CONCLUSION: Direct amplification of viral nucleic acid from viral transport medium containing nasopharyngeal specimen works well for molecular assays with low thresholds of quality; however, it does have limitations with assays that require high quality nucleic acid for input. Use of the XpressAmp protocol significantly improves turnaround time and allows for easy ramp-up of PCR and genotyping assays.

Analytical validation and implementation of a pan cancer next-generation sequencing panel, CANSeqTMKids for molecular profiling of childhood malignancies.

Next-Generation Sequencing (NGS) allows rapid analysis of multiple genes for the detection of clinically actionable variants. This study reports the analytical validation of a targeted pan cancer NGS panel CANSeqTMKids for molecular profiling of childhood malignancies. Analytical validation included DNA and RNA extracted from de-identified clinical specimens including formalin fixed paraffin embedded (FFPE) tissue, bone marrow and whole blood as well as commercially available reference materials. The DNA component of the panel evaluates 130 genes for the detection of single nucleotide variants (SNVs), Insertion and Deletions (INDELs), and 91 genes for fusion variants associated with childhood malignancies. Conditions were optimized to use as low as 20% neoplastic content with 5 ng of nucleic acid input. Evaluation of the data determined greater than 99% accuracy, sensitivity, repeatability, and reproducibility. The limit of detection was established to be 5% allele fraction for SNVs and INDELs, 5 copies for gene amplifications and 1,100 reads for gene fusions. Assay efficiency was improved by automation of library preparation. In conclusion, the CANSeqTMKids allows for the comprehensive molecular profiling of childhood malignancies from different specimen sources with high quality and fast turnaround time.

A novel variant of TNNC1 associated with severe dilated cardiomyopathy causing infant mortality and stillbirth: a case of germline mosaicism.

Pediatric cardiomyopathies (CM) are rare and challenging to diagnose due to the complex and mixed phenotypes. With the advent of next-generation sequencing (NGS), variants in several genes associated with CM have been identified, such as Troponin C (TnC), encoded by the TNNC1 gene. De novo variants in TNNC1 have been associated with different types of CM, including dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM). The American College of Medical Genetics and Genomics recently added TNNC1 to their recommended list of genes for reporting secondary findings. In this study, we report a de novo variant, c.100G>C (p.Gly34Arg) in the TNNC1 gene identified in three siblings with a diagnosis of severe DCM causing infant death for one of the siblings and stillbirth in the other two pregnancies. The identification of the same de novo variant in all affected siblings is suggestive of germline mosaicism in this family.

Implementation of an Active Screening Program for SARS-CoV2 - Experience at an Academic Medical Center.

BACKGROUND: This study documents the experience of an academic medical center implementing SARS-CoV2 screening of asymptomatic research personnel to support the "return-to-work" initiative and donor cadavers to support in-person student education. METHODS: Testing was performed on samples received June 1, 2020 (for the cadaver program) and July 20, 2020 (for the personnel screening program) through September 30, 2021. Data were evaluated to document the number of cases and the positivity rate. RESULTS: Approximately 3000 specimens were tested across both programs, with an overall positivity rate of 2.5% and 3.6% in the personnel and cadaver screening programs, respectively. DISCUSSION: This screening program serves as an example of institutional investment in the safety of its faculty, staff, and students alike to address specific needs of a global pandemic.

Long-term retinal imaging of a case of suspected congenital rubella infection.

PURPOSE: Many retinal disorders present with pigmentary retinopathy, most of which are progressive conditions. Here we present over nine years of follow up on a case of stable pigmentary retinopathy that is suspected to stem from a congenital rubella infection. Parafoveal cone photoreceptors were tracked through this period to gain insight into photoreceptor disruption in this pigmentary retinopathy. METHODS: The patient was examined at 8 visits spanning a total of 111 months. Examination at baseline included clinical fundus examination, full-field electroretinography (ERG), kinetic visual field assessment (Goldmann), and best corrected visual acuity; all of these except ERG were repeated at follow up visits. Imaging was performed with fundus photography, spectral-domain optical coherence tomography (SD-OCT) and confocal adaptive optics scanning light ophthalmoscopy (AOSLO). For the latter four time points AOSLO imaging also included split-detector imaging. RESULTS: There were no defects in hearing or cardiac health found in this patient. There were minimal visual deficits found at baseline, with mild rod suppression on ERG; best corrected visual acuity was 20/25 OD and 20/20 OS at baseline, which was stable throughout the follow-up period. Retinal thickness as measured by OCT was within the normal range, though foveal hypoplasia was present and outer nuclear layer thickness was slightly below the normal range at all time points. Cone density was relatively stable throughout the follow-up period. A number of cones were non-reflective when observed with confocal AOSLO imaging and density was markedly lower than expected values (foveal cone density was 43,782 cones/mm2 on average). Genetic analysis revealed no causative variations explaining the phenotype. CONCLUSIONS AND IMPORTANCE: This patient appears to have a stable pigmentary retinopathy. This case is likely due to a congenital insult, rather than progressive retinal disease. This finding of stability agrees with other reports of rubella pigmentary retinopathy. Imaging with AOSLO enabled observation of two notable phenotypic features. First is the observation of dark cones, which are seen in many retinal disorders including color vision defects and degenerative retinal disease. Second, the cone density is well below what is expected - this is especially interesting as this patient has near-normal visual acuity despite this greatly decreased number of normally-waveguiding cones in the fovea.

BMP4 loss-of-function mutations in developmental eye disorders including SHORT syndrome.

BMP4 loss-of-function mutations and deletions have been shown to be associated with ocular, digital, and brain anomalies, but due to the paucity of these reports, the full phenotypic spectrum of human BMP4 mutations is not clear. We screened 133 patients with a variety of ocular disorders for BMP4 coding region mutations or genomic deletions. BMP4 deletions were detected in two patients: a patient affected with SHORT syndrome and a patient with anterior segment anomalies along with craniofacial dysmorphism and cognitive impairment. In addition to this, three intragenic BMP4 mutations were identified. A patient with anophthalmia, microphthalmia with sclerocornea, right-sided diaphragmatic hernia, and hydrocephalus was found to have a c.592C >T (p.R198X) nonsense mutation in BMP4. A frameshift mutation, c.171dupC (p.E58RfsX17), was identified in two half-siblings with anophthalmia/microphthalmia, discordant developmental delay/postaxial polydactyly, and poor growth as well as their unaffected mother; one affected sibling carried an additional BMP4 mutation in the second allele, c.362A > G (p.H121R). This is the first report indicating a role for BMP4 in SHORT syndrome, Axenfeld-Rieger malformation, growth delay, macrocephaly, and diaphragmatic hernia. These results significantly expand the number of reported loss-of-function mutations, further support the critical role of BMP4 in ocular development, and provide additional evidence of variable expression/non-penetrance of BMP4 mutations.

Analyzing the Genetic Spectrum of Vascular Anomalies with Overgrowth via Cancer Genomics.

Vascular anomalies are variably associated with overgrowth, skeletal anomalies, and abnormalities of the brain, leptomeninges, and eye. We assembled a 16-institution network to determine the range of genetic variants associated with a spectrum of vascular anomalies with overgrowth, ranging from mild to severe. Because of the overlap between cancer-associated variants and previously described somatic variants in vascular overgrowth syndromes, we employed tumor genetic profiling via high-depth next-generation sequencing using a panel to assay affected tissue from a diverse cohort of subjects with vascular anomalies with overgrowth. Seventy-five percent (43/57) harbored pathogenic or likely pathogenic variants in 10 genes. We identified two genes (mTOR, PIK3R1) and several variants previously described in the setting of cancer but that, to our knowledge, have not been described in vascular malformations. All were identified at low variant allele frequency consistent with somatic mosaic etiology. By leveraging somatic variant detection technology typically applied to cancer in a cohort inclusive of broad phenotypic severity, we demonstrated that most vascular anomalies with overgrowth harbor postzygotic gain-of-function mutations in oncogenes. Furthermore, continued interrogation of oncogenes in benign developmental disorders could provide insight into fundamental mechanisms regulating cell growth.

Identification of an Alu-repeat-mediated deletion of OPTN upstream region in a patient with a complex ocular phenotype.

Genetic causes of ocular conditions remain largely unknown. To reveal the molecular basis for a congenital ocular phenotype associated with glaucoma we performed whole-exome sequencing (WES) and whole-genome copy number analyses of patient DNA. WES did not identify a causative variant. Copy number variation analysis identified a deletion of 10p13 in the patient and his unaffected father; the deletion breakpoint contained a single 37-bp sequence that is normally present in two distinct Alu repeats separated by ~181 kb. The deletion removed part of the upstream region of optineurin (OPTN) as well as the upstream sequence and two coding exons of coiled-coil domain containing 3 (CCDC3); analysis of the patient's second allele showed normal OPTN and CCDC3 sequences. Studies of zebrafish orthologs identified expression in the developing eye for both genes. OPTN is a known factor in dominant adult-onset glaucoma and Amyotrophic Lateral Sclerosis (ALS). The deletion eliminates 98 kb of the OPTN upstream sequence leaving only ~1 kb of the proximal promoter region. Comparison of transcriptional activation capability of the 3 kb normal and the rearranged del(10)(p13) OPTN promoter sequences demonstrated a statistically significant decrease for the deleted allele; sequence analysis of the entire deleted region identified multiple conserved elements with possible cis-regulatory activity. Additional screening of CCDC3 indicated that heterozygous loss-of-function alleles are unlikely to cause congenital ocular disease. In summary, we report the first regulatory region deletion involving OPTN, caused by Alu-mediated nonallelic homologous recombination and possibly contributing to the patient's ocular phenotype. In addition, our data indicate that Alu-mediated rearrangements of the OPTN upstream region may represent a new source of affected alleles in human conditions. Evaluation of the upstream OPTN sequences in additional ocular and ALS patients may help to determine the role of this region, if any, in human disease.

PITX2 and FOXC1 spectrum of mutations in ocular syndromes.

Anterior segment dysgenesis (ASD) encompasses a broad spectrum of developmental conditions affecting anterior ocular structures and associated with an increased risk for glaucoma. Various systemic anomalies are often observed in ASD conditions such as Axenfeld-Rieger syndrome (ARS) and De Hauwere syndrome. We report DNA sequencing and copy number analysis of PITX2 and FOXC1 in 76 patients with syndromic or isolated ASD and related conditions. PITX2 mutations and deletions were found in 24 patients with dental and/or umbilical anomalies seen in all. Seven PITX2-mutant alleles were novel including c.708_730del, the most C-terminal mutation reported to date. A second case of deletion of the distant upstream but not coding region of PITX2 was identified, highlighting the importance of this recently discovered mechanism for ARS. FOXC1 deletions were observed in four cases, three of which demonstrated hearing and/or heart defects, including a patient with De Hauwere syndrome; no nucleotide mutations in FOXC1 were identified. Review of the literature identified several other patients with 6p25 deletions and features of De Hauwere syndrome. The 1.3-Mb deletion of 6p25 presented here defines the critical region for this phenotype and includes the FOXC1, FOXF2, and FOXQ1 genes. In summary, PITX2 or FOXC1 disruptions explained 63% of ARS and 6% of other ASD in our cohort; all affected patients demonstrated additional systemic defects with PITX2 mutations showing a strong association with dental and/or umbilical anomalies and FOXC1 with heart and hearing defects. FOXC1 deletion was also found to be associated with De Hauwere syndrome.

Potential novel mechanism for Axenfeld-Rieger syndrome: deletion of a distant region containing regulatory elements of PITX2.

PURPOSE: Mutations in PITX2 are associated with Axenfeld-Rieger syndrome (ARS), which involves ocular, dental, and umbilical abnormalities. Identification of cis-regulatory elements of PITX2 is important to better understand the mechanisms of disease. METHODS: Conserved noncoding elements surrounding PITX2/pitx2 were identified and examined through transgenic analysis in zebrafish; expression pattern was studied by in situ hybridization. Patient samples were screened for deletion/duplication of the PITX2 upstream region using arrays and probes. RESULTS: Zebrafish pitx2 demonstrates conserved expression during ocular and craniofacial development. Thirteen conserved noncoding sequences positioned within a gene desert as far as 1.1 Mb upstream of the human PITX2 gene were identified; 11 have enhancer activities consistent with pitx2 expression. Ten elements mediated expression in the developing brain, four regions were active during eye formation, and two sequences were associated with craniofacial expression. One region, CE4, located approximately 111 kb upstream of PITX2, directed a complex pattern including expression in the developing eye and craniofacial region, the classic sites affected in ARS. Screening of ARS patients identified an approximately 7600-kb deletion that began 106 to 108 kb upstream of the PITX2 gene, leaving PITX2 intact while removing regulatory elements CE4 to CE13. CONCLUSIONS: These data suggest the presence of a complex distant regulatory matrix within the gene desert located upstream of PITX2 with an essential role in its activity and provides a possible mechanism for the previous reports of ARS in patients with balanced translocations involving the 4q25 region upstream of PITX2 and the current patient with an upstream deletion.