A Mild PUM1 Mutation Is Associated with Adult-Onset Ataxia, whereas Haploinsufficiency Causes Developmental Delay and Seizures.

Certain mutations can cause proteins to accumulate in neurons, leading to neurodegeneration. We recently showed, however, that upregulation of a wild-type protein, Ataxin1, caused by haploinsufficiency of its repressor, the RNA-binding protein Pumilio1 (PUM1), also causes neurodegeneration in mice. We therefore searched for human patients with PUM1 mutations. We identified eleven individuals with either PUM1 deletions or de novo missense variants who suffer a developmental syndrome (Pumilio1-associated developmental disability, ataxia, and seizure; PADDAS). We also identified a milder missense mutation in a family with adult-onset ataxia with incomplete penetrance (Pumilio1-related cerebellar ataxia, PRCA). Studies in patient-derived cells revealed that the missense mutations reduced PUM1 protein levels by ∼25% in the adult-onset cases and by ∼50% in the infantile-onset cases; levels of known PUM1 targets increased accordingly. Changes in protein levels thus track with phenotypic severity, and identifying posttranscriptional modulators of protein expression should identify new candidate disease genes.

An Organismal CNV Mutator Phenotype Restricted to Early Human Development.

De novo copy number variants (dnCNVs) arising at multiple loci in a personal genome have usually been considered to reflect cancer somatic genomic instabilities. We describe a multiple dnCNV (MdnCNV) phenomenon in which individuals with genomic disorders carry five to ten constitutional dnCNVs. These CNVs originate from independent formation incidences, are predominantly tandem duplications or complex gains, exhibit breakpoint junction features reminiscent of replicative repair, and show increased de novo point mutations flanking the rearrangement junctions. The active CNV mutation shower appears to be restricted to a transient perizygotic period. We propose that a defect in the CNV formation process is responsible for the "CNV-mutator state," and this state is dampened after early embryogenesis. The constitutional MdnCNV phenomenon resembles chromosomal instability in various cancers. Investigations of this phenomenon may provide unique access to understanding genomic disorders, structural variant mutagenesis, human evolution, and cancer biology.

Noninvasive single-cell-based prenatal genetic testing: A proof of concept clinical study.

OBJECTIVE: To clinically assess a cell-based noninvasive prenatal genetic test using sequence-based copy number analysis of single trophoblasts from maternal blood. METHODS: Blood was obtained from 401 (243 + 158) individuals (8-22 weeks) and shipped overnight. Red cells were lysed, and nucleated cells stained for cytokeratin (CK) and CD45 and enriched for positive CK staining. Automated scanning was used to identify and pick single CK+ /CD45- trophoblasts which were subjected to next-generation sequencing. RESULTS: Blood was obtained from 243 pregnancies scheduled for CVS or amniocentesis. Luna results were normal for 160 singletons while 15 cases were abnormal (14 aneuploidy and one monozygotic twin with Williams syndrome deletion). The deletion was confirmed in both fetuses. Placental mosaicism occurred in 7 of 236 (3.0%) Luna cases and in 3 of 188 (1.6%) CVS cases (total 4.6%). No scorable trophoblasts were recovered in 32 of 236 usable samples. Additionally, 158 low-risk pregnancies not undergoing CVS/amniocentesis showed normal results in 133 cases. Seven had aneuploidy results, and there were three likely pathogenic deletions/duplications, including one15q11-q13 deletion. CONCLUSION: Although the sample size is modest and statistically accurate measures of test performance are not possible, the Luna test detected aneuploidy and deletions/duplications based on concordance with CVS/amniocentesis.

Factors that influence genetic counselors' participation in research.

Genetic counselors have skills and expertise in genetics and patient care that make them an asset to research and research teams. However, the National Society of Genetic Counselors (NSGC) found in 2020 that more than half of practicing genetic counselors do not participate in research activities. Information describing factors that influence their research participation is lacking in the literature. This study ascertained genetic counselors' workplace and graduate training experiences to provide insight into factors that increase or decrease participation in research activities. A survey was distributed through the NSGC Student Research Survey Program. Practicing genetic counselors that graduated in or before 2020 were eligible to participate. The survey included questions about demographics, implementation of student research projects, research-specific resources in their graduate programs, perceived barriers and motivations, and current research activities. Interestingly, the majority of respondents participated in research activities between 2017 and 2021; the most common activities included: recognizing a gap(s) in knowledge (68%) and presenting an abstract or poster (64%). Factors that most significantly influenced genetic counselors' research participation included their interest in research (p = 0.0037), their motivation to do research (p = 0.0014), and their perceived intimidation by the research process (p < 0.001). These results provide insight into solutions for the workplace and graduate programs that could increase genetic counselors' research participation.

Validation Studies for Single Circulating Trophoblast Genetic Testing as a Form of Noninvasive Prenatal Diagnosis.

It has long been appreciated that genetic analysis of fetal or trophoblast cells in maternal blood could revolutionize prenatal diagnosis. We implemented a protocol for single circulating trophoblast (SCT) testing using positive selection by magnetic-activated cell sorting and single-cell low-coverage whole-genome sequencing to detect fetal aneuploidies and copy-number variants (CNVs) at ∼1 Mb resolution. In 95 validation cases, we identified on average 0.20 putative trophoblasts/mL, of which 55% were of high quality and scorable for both aneuploidy and CNVs. We emphasize the importance of analyzing individual cells because some cells are apoptotic, in S-phase, or otherwise of poor quality. When two or more high-quality trophoblast cells were available for singleton pregnancies, there was complete concordance between all trophoblasts unless there was evidence of confined placental mosaicism. SCT results were highly concordant with available clinical data from chorionic villus sampling (CVS) or amniocentesis procedures. Although determining the exact sensitivity and specificity will require more data, this study further supports the potential for SCT testing to become a diagnostic prenatal test.

Girl-Boy Twins with Developmental Delay from 16p11.2 Triplication due to Biparental Inheritance from Two Parents with 16p11.2 Duplication.

The 16p11.2 duplication is a well-known cause of developmental delay and autism, but there are only 2 previously reported cases of 16p11.2 triplication. Both of the previously reported cases exhibited tandem triplication on a 16p11.2 duplication inherited from 1 parent. We report fraternal twins presenting with developmental delay and 16p11.2 triplication resulting from inheritance of a 16p11.2 duplicated homolog from each parent. This report also reviews the overlapping features in previously published cases of 16p11.2 triplication, and possible implications are discussed.

Management of amended variant classification laboratory reports by genetic counselors in the United States and Canada: An exploratory study.

For the past two decades, the guidelines put forth by the American College of Medical Genetics and Genomics (ACMG) detailing providers' clinical responsibility to recontact patients have remained mostly unchanged, despite evolving variant interpretation practices which have yielded substantial rates of reclassification and amended reports. In fact, there is little information regarding genetic counselors' roles in informing patients of reclassified variants, or the process by which these amended reports are currently being handled. In this study, we developed a survey to measure current experiences with amended variant reports and preferences for ideal management, which was completed by 96 genetic counselors from the United States and Canada. All respondents indicated they were the individuals responsible for disclosing initial positive genetic testing results and any clinically actionable reclassified variant reports, and over half (56%) received at least a few amended variant reports each year. Nearly a quarter (20/87) of respondents reported having a standard operating procedure (SOP) for managing amended reports and all were very satisfied (12/20) or satisfied (8/20) with the SOP. Of those without a protocol, 76% (51/67) would prefer to have an SOP implemented. Respondents reported a preference for (1) laboratories to send amended variant reports directly to the genetic counselor or ordering physician through email or an online portal, and (2) notification to patients ideally occurring through a phone call. In the event that the original genetic counselor is inaccessible, respondents reported a preference for reports to be sent directly to another genetic counselor (36%) on the team or the clinic in general (27%). Information from this study provides insight into the current practices of genetic counselors as applied to amended reports and what improvements may increase the efficiency of the reporting process. Moreover, these results suggest a need for an updated statement addressing duty to recontact, specifically as it applies to amended variant reports.

Predicting human genes susceptible to genomic instability associated with Alu/Alu-mediated rearrangements.

Alu elements, the short interspersed element numbering more than 1 million copies per human genome, can mediate the formation of copy number variants (CNVs) between substrate pairs. These Alu/Alu-mediated rearrangements (AAMRs) can result in pathogenic variants that cause diseases. To investigate the impact of AAMR on gene variation and human health, we first characterized Alus that are involved in mediating CNVs (CNV-Alus) and observed that these Alus tend to be evolutionarily younger. We then computationally generated, with the assistance of a supercomputer, a test data set consisting of 78 million Alu pairs and predicted ∼18% of them are potentially susceptible to AAMR. We further determined the relative risk of AAMR in 12,074 OMIM genes using the count of predicted CNV-Alu pairs and experimentally validated the predictions with 89 samples selected by correlating predicted hotspots with a database of CNVs identified by clinical chromosomal microarrays (CMAs) on the genomes of approximately 54,000 subjects. We fine-mapped 47 duplications, 40 deletions, and two complex rearrangements and examined a total of 52 breakpoint junctions of simple CNVs. Overall, 94% of the candidate breakpoints were at least partially Alu mediated. We successfully predicted all (100%) of Alu pairs that mediated deletions (n = 21) and achieved an 87% positive predictive value overall when including AAMR-generated deletions and duplications. We provided a tool, AluAluCNVpredictor, for assessing AAMR hotspots and their role in human disease. These results demonstrate the utility of our predictive model and provide insights into the genomic features and molecular mechanisms underlying AAMR.

Parental somatic mosaicism for CNV deletions - A need for more sensitive and precise detection methods in clinical diagnostics settings.

To further assess the scale and level of parental somatic mosaicism, we queried the CMA database at Baylor Genetics. We selected 50 unrelated families where clinically relevant apparent de novo CNV-deletions were found in the affected probands. Parental blood samples screening using deletion junction-specific PCR revealed four parents with somatic mosaicism. Droplet digital PCR (ddPCR), qPCR, and amplicon-based next-generation sequencing (NGS) were applied to validate these findings. Using ddPCR levels of mosaicism ranged from undetectable to 18.5%. Amplicon-based NGS and qPCR for the father with undetectable mosaicism was able to detect mosaicism at 0.39%. In one mother, ddPCR analysis revealed 15.6%, 10.6%, 8.2%, and undetectable levels of mosaicism in her blood, buccal cells, saliva, and urine samples, respectively. Our data suggest that more sensitive and precise methods, e.g. CNV junction-specific LR-PCR, ddPCR, or qPCR may allow for a more refined assessment of the potential disease recurrence risk for an identified variant.

Cytogenetically visible inversions are formed by multiple molecular mechanisms.

Cytogenetically detected inversions are generally assumed to be copy number and phenotypically neutral events. While nonallelic homologous recombination is thought to play a major role, recent data suggest the involvement of other molecular mechanisms in inversion formation. Using a combination of short-read whole-genome sequencing (WGS), 10X Genomics Chromium WGS, droplet digital polymerase chain reaction and array comparative genomic hybridization we investigated the genomic structure of 18 large unique cytogenetically detected chromosomal inversions and achieved nucleotide resolution of at least one chromosomal inversion junction for 13/18 (72%). Surprisingly, we observed that seemingly copy number neutral inversions can be accompanied by a copy-number gain of up to 350 kb and local genomic complexities (3/18, 17%). In the resolved inversions, the mutational signatures are consistent with nonhomologous end-joining (8/13, 62%) or microhomology-mediated break-induced replication (5/13, 38%). Our study indicates that short-read 30x coverage WGS can detect a substantial fraction of chromosomal inversions. Moreover, replication-based mechanisms are responsible for approximately 38% of those events leading to a significant proportion of inversions that are actually accompanied by additional copy-number variation potentially contributing to the overall phenotypic presentation of those patients.

Human and mouse studies establish TBX6 in Mendelian CAKUT and as a potential driver of kidney defects associated with the 16p11.2 microdeletion syndrome.

Congenital anomalies of the kidney and urinary tract (CAKUTs) are the most common cause of chronic kidney disease in children. Human 16p11.2 deletions have been associated with CAKUT, but the responsible molecular mechanism remains to be illuminated. To explore this, we investigated 102 carriers of 16p11.2 deletion from multi-center cohorts, among which we retrospectively ascertained kidney morphologic and functional data from 37 individuals (12 Chinese and 25 Caucasian/Hispanic). Significantly higher CAKUT rates were observed in 16p11.2 deletion carriers (about 25% in Chinese and 16% in Caucasian/Hispanic) than those found in the non-clinically ascertained general populations (about 1/1000 found at autopsy). Furthermore, we identified seven additional individuals with heterozygous loss-of-function variants in TBX6, a gene that maps to the 16p11.2 region. Four of these seven cases showed obvious CAKUT. To further investigate the role of TBX6 in kidney development, we engineered mice with mutated Tbx6 alleles. The Tbx6 heterozygous null (i.e., loss-of-function) mutant (Tbx6+/‒) resulted in 13% solitary kidneys. Remarkably, this incidence increased to 29% in a compound heterozygous model (Tbx6mh/‒) that reduced Tbx6 gene dosage to below haploinsufficiency, by combining the null allele with a novel mild hypomorphic allele (mh). Renal hypoplasia was also frequently observed in these Tbx6-mutated mouse models. Thus, our findings in patients and mice establish TBX6 as a novel gene involved in CAKUT and its gene dosage insufficiency as a potential driver for kidney defects observed in the 16p11.2 microdeletion syndrome.

Haploinsufficiency of the Chromatin Remodeler BPTF Causes Syndromic Developmental and Speech Delay, Postnatal Microcephaly, and Dysmorphic Features.

Bromodomain PHD finger transcription factor (BPTF) is the largest subunit of nucleosome remodeling factor (NURF), a member of the ISWI chromatin-remodeling complex. However, the clinical consequences of disruption of this complex remain largely uncharacterized. BPTF is required for anterior-posterior axis formation of the mouse embryo and was shown to promote posterior neuroectodermal fate by enhancing Smad2-activated wnt8 expression in zebrafish. Here, we report eight loss-of-function and two missense variants (eight de novo and two of unknown origin) in BPTF on 17q24.2. The BPTF variants were found in unrelated individuals aged between 2.1 and 13 years, who manifest variable degrees of developmental delay/intellectual disability (10/10), speech delay (10/10), postnatal microcephaly (7/9), and dysmorphic features (9/10). Using CRISPR-Cas9 genome editing of bptf in zebrafish to induce a loss of gene function, we observed a significant reduction in head size of F0 mutants compared to control larvae. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and phospho-histone H3 (PH3) staining to assess apoptosis and cell proliferation, respectively, showed a significant increase in cell death in F0 mutants compared to controls. Additionally, we observed a substantial increase of the ceratohyal angle of the craniofacial skeleton in bptf F0 mutants, indicating abnormal craniofacial patterning. Taken together, our data demonstrate the pathogenic role of BPTF haploinsufficiency in syndromic neurodevelopmental anomalies and extend the clinical spectrum of human disorders caused by ablation of chromatin remodeling complexes.

Two Patients with Complex Rearrangements Suggestive of Germline Chromoanagenesis.

Chromoanagenesis, a phenomenon characterized by complex chromosomal rearrangement and reorganization events localized to a limited number of genomic regions, includes the subcategories chromothripsis, chromoanasynthesis, and chromoplexy. Although definitions of these terms are evolving, constitutional chromoanagenesis events have been reported in a limited number of patients with variable phenotypes. We report on 2 cases with complex genomic events characterized by multiple copy number gains and losses confined to a single chromosome region, which are suggestive of constitutional chromoanagenesis. Case 1 is a 43-year-old male with intellectual disability and recently developed generalized tonic-clonic seizures. Chromosomal microarray analysis identified a complex rearrangement involving chromosome region 14q31.1q32.2, consisting of 16 breakpoints ranging in size from 0.2 to 6.2 Mb, with 5 segments of normal copy number present between these alterations. Interestingly, this case represents the oldest known patient with a complex rearrangement indicative of constitutional chromoanagenesis. Case 2 is a 2-year-old female with developmental delay, speech delay, low muscle tone, and seizures. Chromosomal microarray analysis identified a complex rearrangement consisting of 28 breakpoints localized to 18q21.32q23. The size of the copy number alterations ranged from 0.042 to 5.1 Mb, flanked by 12 small segments of normal copy number. These cases add to a growing body of literature demonstrating complex chromosomal rearrangements as a disease mechanism for congenital anomalies.

CNVs cause autosomal recessive genetic diseases with or without involvement of SNV/indels.

PURPOSE: Improved resolution of molecular diagnostic technologies enabled detection of smaller sized exonic level copy-number variants (CNVs). The contribution of CNVs to autosomal recessive (AR) conditions may be better recognized using a large clinical cohort. METHODS: We retrospectively investigated the CNVs' contribution to AR conditions in cases subjected to chromosomal microarray analysis (CMA, N = ~70,000) and/or clinical exome sequencing (ES, N = ~12,000) at Baylor Genetics; most had pediatric onset neurodevelopmental disorders. RESULTS: CNVs contributed to biallelic variations in 87 cases, including 81 singletons and three affected sibling pairs. Seventy cases had CNVs affecting both alleles, and 17 had a CNV and a single-nucleotide variant (SNV)/indel in trans. In total, 94.3% of AR-CNVs affected one gene; among these 41.4% were single-exon and 35.0% were multiexon partial-gene events. Sixty-nine percent of homozygous AR-CNVs were embedded in homozygous genomic intervals. Five cases had large deletions unmasking an SNV/indel on the intact allele for a recessive condition, resulting in multiple molecular diagnoses. CONCLUSIONS: AR-CNVs are often smaller in size, transmitted through generations, and underrecognized due to limitations in clinical CNV detection methods. Our findings from a large clinical cohort emphasized integrated CNV and SNV/indel analyses for precise clinical and molecular diagnosis especially in the context of genomic disorders.

Clinical exome sequencing reveals locus heterogeneity and phenotypic variability of cohesinopathies.

PURPOSE: Defects in the cohesin pathway are associated with cohesinopathies, notably Cornelia de Lange syndrome (CdLS). We aimed to delineate pathogenic variants in known and candidate cohesinopathy genes from a clinical exome perspective. METHODS: We retrospectively studied patients referred for clinical exome sequencing (CES, N = 10,698). Patients with causative variants in novel or recently described cohesinopathy genes were enrolled for phenotypic characterization. RESULTS: Pathogenic or likely pathogenic single-nucleotide and insertion/deletion variants (SNVs/indels) were identified in established disease genes including NIPBL (N = 5), SMC1A (N = 14), SMC3 (N = 4), RAD21 (N = 2), and HDAC8 (N = 8). The phenotypes in this genetically defined cohort skew towards the mild end of CdLS spectrum as compared with phenotype-driven cohorts. Candidate or recently reported cohesinopathy genes were supported by de novo SNVs/indels in STAG1 (N = 3), STAG2 (N = 5), PDS5A (N = 1), and WAPL (N = 1), and one inherited SNV in PDS5A. We also identified copy-number deletions affecting STAG1 (two de novo, one of unknown inheritance) and STAG2 (one of unknown inheritance). Patients with STAG1 and STAG2 variants presented with overlapping features yet without characteristic facial features of CdLS. CONCLUSION: CES effectively identified disease-causing alleles at the mild end of the cohensinopathy spectrum and enabled characterization of candidate disease genes.

An unusual cause for Coffin-Lowry syndrome: Three brothers with a novel microduplication in RPS6KA3.

Coffin-Lowry syndrome (CLS) is a rare X-linked disorder characterized by moderate to severe intellectual disability, hypotonia, craniofacial features, tapering digits, short stature, and skeletal deformities. Using whole exome sequencing and high-resolution targeted comparative genomic hybridization array analysis, we identified a novel microduplication encompassing exons five through nine of RPS6KA3 in three full brothers. Each brother presented with intellectual disability and clinical and radiographic features consistent with CLS. qRT-PCR analyses performed on mRNA from the peripheral blood of the three siblings revealed a marked reduction of RPS6KA3 levels suggesting a loss-of-function mechanism. PCR analysis of the patients' cDNA detected a band greater than expected for an exon 4-10 amplicon, suggesting this was likely a direct duplication that lies between exons 4 through 10, which was later confirmed by Sanger sequencing. This microduplication is only the third intragenic duplication of RPS6KA3, and the second and smallest reported to date thought to cause CLS. Our study further supports the clinical utility of methods such as next-generation sequencing and high-resolution genomic arrays to detect small intragenic duplications. These methods, coupled with expression studies and cDNA structural analysis have the capacity to confirm the diagnosis of CLS in these rare cases.

SHANK3 overexpression causes manic-like behaviour with unique pharmacogenetic properties.

Mutations in SHANK3 and large duplications of the region spanning SHANK3 both cause a spectrum of neuropsychiatric disorders, indicating that proper SHANK3 dosage is critical for normal brain function. However, SHANK3 overexpression per se has not been established as a cause of human disorders because 22q13 duplications involve several genes. Here we report that Shank3 transgenic mice modelling a human SHANK3 duplication exhibit manic-like behaviour and seizures consistent with synaptic excitatory/inhibitory imbalance. We also identified two patients with hyperkinetic disorders carrying the smallest SHANK3-spanning duplications reported so far. These findings indicate that SHANK3 overexpression causes a hyperkinetic neuropsychiatric disorder. To probe the mechanism underlying the phenotype, we generated a Shank3 in vivo interactome and found that Shank3 directly interacts with the Arp2/3 complex to increase F-actin levels in Shank3 transgenic mice. The mood-stabilizing drug valproate, but not lithium, rescues the manic-like behaviour of Shank3 transgenic mice raising the possibility that this hyperkinetic disorder has a unique pharmacogenetic profile.

Mechanisms for Complex Chromosomal Insertions.

Chromosomal insertions are genomic rearrangements with a chromosome segment inserted into a non-homologous chromosome or a non-adjacent locus on the same chromosome or the other homologue, constituting ~2% of nonrecurrent copy-number gains. Little is known about the molecular mechanisms of their formation. We identified 16 individuals with complex insertions among 56,000 individuals tested at Baylor Genetics using clinical array comparative genomic hybridization (aCGH) and fluorescence in situ hybridization (FISH). Custom high-density aCGH was performed on 10 individuals with available DNA, and breakpoint junctions were fine-mapped at nucleotide resolution by long-range PCR and DNA sequencing in 6 individuals to glean insights into potential mechanisms of formation. We observed microhomologies and templated insertions at the breakpoint junctions, resembling the breakpoint junction signatures found in complex genomic rearrangements generated by replication-based mechanism(s) with iterative template switches. In addition, we analyzed 5 families with apparently balanced insertion in one parent detected by FISH analysis and found that 3 parents had additional small copy-number variants (CNVs) at one or both sides of the inserting fragments as well as at the inserted sites. We propose that replicative repair can result in interchromosomal complex insertions generated through chromothripsis-like chromoanasynthesis involving two or three chromosomes, and cause a significant fraction of apparently balanced insertions harboring small flanking CNVs.

Chromosomal microarray analysis on uncultured chorionic villus sampling can be complicated by confined placental mosaicism for aneuploidy and microdeletions.

OBJECTIVE: This study aims to establish the incidence and implications of confined placental mosaicism (CPM) in the context of prenatal chromosomal microarray analysis (CMA). METHODS: We retrospectively reviewed prenatal array data on 1382 consecutive chorionic villus sampling (CVS) specimens spanning the past 6 years, focusing on those for which whole CVS biopsy (both cytotrophoblast and mesenchymal cells) was used for CMA and cultured cells (primarily mesenchyme) was also analyzed or amniotic fluid (AF)/newborn blood was used for confirmation, to determine the frequency of mosaic abnormal findings that were the result of CPM. RESULTS: Out of a total of 1382 consecutive CVS cases, we identified 42 (42/1382 = 3.0%) cases with abnormal array findings suggestive of mosaicism. Among them, 10 cases were unequivocally interpreted as CPM based on a normal AF/newborn blood confirmatory result. In addition, another 10 cases were interpreted as provisional CPM based on normal results on cultured cells. Notably, 40% (8/20) of the cases revealed complex findings, including multiple mosaic aneuploidies, mosaic submicroscopic copy number variation (CNV), and mosaic aneuploidy plus mosaic CNV. CONCLUSION: Abnormal CMA results from CVS specimens should be interpreted with caution when mosaicism is evident or suspected. Furthermore, confirmatory testing on amniotic fluid, which contains cells derived from the fetus, is recommended in these cases.