Challenges of variant reinterpretation: Opinions of stakeholders and need for guidelines.

PURPOSE: The knowledge used to classify genetic variants is continually evolving, and the classification can change on the basis of newly available data. Although up-to-date variant classification is essential for clinical management, reproductive planning, and identifying at-risk family members, there is no consistent practice across laboratories or clinicians on how or under what circumstances to perform variant reinterpretation. METHODS: We conducted exploratory focus groups (N = 142) and surveys (N = 1753) with stakeholders involved in the process of variant reinterpretation (laboratory directors, clinical geneticists, genetic counselors, nongenetic providers, and patients/parents) to assess opinions on key issues, including initiation of reinterpretation, variants to report, termination of the responsibility to reinterpret, and concerns about consent, cost, and liability. RESULTS: Stakeholders widely agreed that there should be no fixed termination point to the responsibility to reinterpret a previously reported genetic variant. There were significant concerns about liability and lack of agreement about many logistical aspects of variant reinterpretation. CONCLUSION: Our findings suggest a need to (1) develop consensus and (2) create transparency and awareness about the roles and responsibilities of parties involved in variant reinterpretation. These data provide a foundation for developing guidelines on variant reinterpretation that can aid in the development of a low-cost, scalable, and accessible approach.

Knowledge and attitudes about genetic counseling in patients at a major hospital in Addis Ababa, Ethiopia.

Previous work at St. Paul's Hospital Millennium Medical College (SPHMMC) in Addis Ababa, Ethiopia, demonstrated a need for genetic counseling (GC) services, with 4% of pediatric, neonatal intensive care, and prenatal patients identified as having indications for genetic evaluation (Quinonez et al, 2019). The aim of this study was to investigate SPHMMC patients' familiarity with, knowledge of, and attitudes toward GC services. Surveys were adapted from previous work in North America populations (Riesgraf et al, 2015 and Gemmell et al, 2017) and administered to 102 patients, and results were compared to North American populations using Student's t test. 30% of respondents reported at least some familiarity with GC, primarily via the media or healthcare providers. Patients had generally positive attitudes toward GC, reporting they would trust information provided by a genetic counselor and that GC is in line with their values. Knowledge of GC showed similar trends overall when compared to results from North American populations. Our work indicates limited exposure to GC in this population, but generally positive feelings toward GC. Patients' attitudes toward GC were comparable to rural North American populations surveyed using the same tool on most items; however, cultural differences including views on abortions and directiveness of healthcare providers could account for discrepancies and are important considerations when implementing genetic services globally.

Genotype-phenotype analysis of 523 patients by genetics evaluation and clinical exome sequencing.

BACKGROUND: As clinical exome sequencing (CES) becomes more common, understanding which patients are most likely to benefit and in what manner is critical for the general pediatrics community to appreciate. METHODS: Five hundred and twenty-three patients referred to the Pediatric Genetics clinic at Michigan Medicine were systematically phenotyped by the presence or absence of abnormalities for 13 body/organ systems by a Clinical Genetics team. All patients then underwent CES. RESULTS: Overall, 30% of patients who underwent CES had an identified pathogenic mutation. The most common phenotypes were developmental delay (83%), neuromuscular system abnormalities (81%), and multiple congenital anomalies (42%). In all, 67% of patients had a variant of uncertain significance (VUS) or gene of uncertain significance (GUS); 23% had no variants reported. There was a significant difference in the average number of body systems affected among these groups (pathogenic 5.89, VUS 6.0, GUS 6.12, and no variant 4.6; P < 0.00001). Representative cases highlight four ways in which CES is changing clinical pediatric practice. CONCLUSIONS: Patients with identified variants are enriched for multiple organ system involvement. Furthermore, our phenotyping provides broad insights into which patients are most likely to benefit from genetics referral and CES and how those results can help guide clinical practice more generally.

Additive effect of TAp63 deficiency on the adrenocortical dysplasia (acd) phenotype.

Adrenocortical dysplasia (acd) is a spontaneous autosomal recessive mouse mutation exhibiting caudal truncation, vertebral segmentation defects, hydronephrosis, limb hypoplasia, and perinatal lethality. Acd encodes TPP1, a component of the shelterin complex that maintains telomere integrity, and consequently acd mutant mice have telomere dysfunction and genomic instability. We previously showed that apoptosis is the primary mechanism causing the acd skeletal phenotype, and that p53 deficiency rescues the skeletal defects of the acd phenotype but has no effect on the perinatal lethality. The Trp63 gene encodes multiple isoforms, which play a role in proliferation, apoptosis, and stem/progenitor cell maintenance. Different p63 isoforms exhibit both proapoptotic (TAp63) and antiapoptotic (ΔNp63) functions. We hypothesized that deficiency of proapoptotic TAp63 isoforms might rescue the acd skeletal phenotype, similar to our previous observations with deficiency of p53. Mice heterozygous for a null allele of TAp63 were crossed to heterozygous acd mice to determine the effect of TAp63 deficiency on the acd mutant phenotype. In contrast to our results with the acd × p53 cross, skeletal anomalies were not rescued by deficiency of TAp63. In fact, the limb and vertebral anomalies observed in double-mutant embryos were more severe than those of embryos with the acd mutation alone, demonstrating a dose-dependent effect. These studies suggest that TAp63 isoforms do not facilitate p53-like apoptosis during development in response to acd-mediated telomere dysfunction and are consistent with the proposed roles of TAp63 in maintaining genomic stability.

The introduction of genetic counseling in Ethiopia: Results of a training workshop and lessons learned.

BACKGROUND: Over the past two decades non-communicable diseases (NCDs) have steadily increased as a cause of worldwide disability and mortality with a concomitant decrease in disease burden from communicable, maternal, neonatal and nutritional conditions. Congenital anomalies, the most common NCD affecting children, have recently become the fifth leading cause of under-five mortality worldwide, ahead of other conditions such as malaria, neonatal sepsis and malnutrition. Genetic counseling has been shown to be an effective method to decrease the impact of congenital anomalies and genetic conditions but is absent in almost all sub-Saharan Africa countries. To address this need for counseling services we designed and implemented the first broad-based genetic counseling curriculum in Ethiopia, launching it at St. Paul's Millennium Medical College (SPHMMC) in Addis Ababa, Ethiopia. METHODS: The curriculum, created by Michigan Medicine and SPHMMC specialists, consisted of medical knowledge and genetic counseling content and was delivered to two cohorts of nurses. Curriculum evaluation consisted of satisfaction surveys and pre- and post-assessments covering medical knowledge and genetic counseling content. Following Cohort 1 training, the curriculum was modified to increase the medical knowledge material and decrease Western genetic counseling principles material. RESULTS: Both cohorts reported high levels of satisfaction but felt the workshop was too short. No significant improvements in assessment scores were seen for Cohort 1 in terms of total scores and medical knowledge and genetic counseling-specific questions. Following curriculum modification, improvements were seen in Cohort 2 with an increase in total assessment scores from 63% to 73% (p = 0.043), with medical knowledge-specific questions increasing from 57% to 79% (p = 0.01) with no significant change in genetic counseling-specific scores. Multiple logistic, financial, cultural and systems-specific barriers were identified with recommendations for their consideration presented. CONCLUSION: Genetics medical knowledge of Ethiopian nurses increased significantly following curriculum delivery though difficulty was encountered with Western genetic counseling material.

Caudal regression in adrenocortical dysplasia (acd) mice is caused by telomere dysfunction with subsequent p53-dependent apoptosis.

Adrenocortical dysplasia (acd) is a spontaneous autosomal recessive mouse mutation that exhibits a pleiotropic phenotype with perinatal lethality. Mutant acd embryos have caudal truncation, vertebral segmentation defects, hydronephrosis, and limb hypoplasia, resembling humans with Caudal Regression syndrome. Acd encodes Tpp1, a component of the shelterin complex that maintains telomere integrity, and consequently acd mutant mice have telomere dysfunction and genomic instability. While the association between genomic instability and cancer is well documented, the association between genomic instability and birth defects is unexplored. To determine the relationship between telomere dysfunction and embryonic malformations, we investigated mechanisms leading to the caudal dysgenesis phenotype of acd mutant embryos. We report that the caudal truncation is caused primarily by apoptosis, not altered cell proliferation. We show that the apoptosis and consequent skeletal malformations in acd mutants are dependent upon the p53 pathway by genetic rescue of the limb hypoplasia and vertebral anomalies with p53 null mice. Furthermore, rescue of the acd phenotype by p53 deficiency is a dosage-sensitive process, as acd/acd, p53(-/-) double mutants exhibit preaxial polydactyly. These findings demonstrate that caudal dysgenesis in acd embryos is secondary to p53-dependent apoptosis. Importantly, this study reinforces a significant link between genomic instability and birth defects.

Pathogenetic basis of Takenouchi-Kosaki syndrome: Electron microscopy study using platelets in patients and functional studies in a Caenorhabditis elegans model.

The combined phenotype of thrombocytopenia accompanied by intellectual disability in patients with a de novo heterozygous mutation, i.e., p.Tyr64Cys in CDC42, signifies a clinically recognizable novel syndrome that has been eponymized as "Takenouchi-Kosaki syndrome" (OMIM #616737). In the present study, a detailed phenotypic analysis performed for a total of five patients with Takenouchi-Kosaki syndrome revealed that intellectual disability, macrothrombocytopenia, camptodactyly, structural brain abnormalities with sensorineural deafness, hypothyroidism, and frequent infections comprise the cardinal features of this condition. A morphologic analysis of platelets derived from three affected individuals was performed using electron microscopy. The platelets of the three patients were large and spherical in shape. Furthermore, platelet α-granules were decreased, while vacuoles were increased. We further performed a functional analysis of p.Tyr64Cys in CDC42 through CRISPR/Cas9-mediated gene editing in a Caenorhabditis elegans model. This functional analysis suggested that the mutant allele has hypomorphic effects. Takenouchi-Kosaki syndrome is clinically recognizable by the combined phenotype of intellectual disability, macrothrombocytopenia, camptodactyly, structural brain abnormalities with sensorineural deafness, hypothyroidism, and frequent infections as well as the identification of a heterozygous de novo mutation in CDC42, i.e., p.Tyr64Cys.

High-throughput gene expression analysis identifies p53-dependent and -independent pathways contributing to the adrenocortical dysplasia (acd) phenotype.

In mammalian cells TPP1, encoded by the Acd gene, is a key component of the shelterin complex, which is required for telomere length maintenance and telomere protection. In mice, a hypomorphic mutation in Acd causes the adrenocortical dysplasia (acd) phenotype, which includes limb and body axis anomalies, and perinatal lethality. p53 deficiency partially rescues limb and body axis anomalies in acd mutant embryos, but not perinatal lethality, implicating p53-independent mechanisms in the acd phenotype. Loss of function of most shelterin proteins results in early embryonic lethality. Thus, study of the hypomorphic acd allele provides a unique opportunity to understand telomere dysfunction at an organismal level. The aim of this study was to identify transcriptome alterations in acd mutant and acd, p53 double mutant embryos to understand the p53-dependent and -independent factors that contribute to the mutant phenotypes in the context of the whole organism. Genes involved in developmental processes, cell cycle, metabolic pathways, tight junctions, axon guidance and signaling pathways were regulated by p53-driven mechanisms in acd mutant embryos, while genes functioning in immune response, and RNA processing were altered independently of p53 in acd, p53 double mutant embryos. To our best of knowledge, this is the first study revealing detailed transcriptomic alterations, reflecting novel p53-dependent and -independent pathways contributing to the acd phenotype. Our data confirm the importance of cell cycle and DNA repair pathways, and suggest novel links between telomere dysfunction and immune system regulation and the splicing machinery. Given the broad applicability of telomere maintenance in growth, development, and genome stability, our data will also provide a rich resource for others studying telomere maintenance and DNA damage responses in mammalian model systems.

PIK3CA-associated developmental disorders exhibit distinct classes of mutations with variable expression and tissue distribution.

Mosaicism is increasingly recognized as a cause of developmental disorders with the advent of next-generation sequencing (NGS). Mosaic mutations of PIK3CA have been associated with the widest spectrum of phenotypes associated with overgrowth and vascular malformations. We performed targeted NGS using 2 independent deep-coverage methods that utilize molecular inversion probes and amplicon sequencing in a cohort of 241 samples from 181 individuals with brain and/or body overgrowth. We identified PIK3CA mutations in 60 individuals. Several other individuals (n = 12) were identified separately to have mutations in PIK3CA by clinical targeted-panel testing (n = 6), whole-exome sequencing (n = 5), or Sanger sequencing (n = 1). Based on the clinical and molecular features, this cohort segregated into three distinct groups: (a) severe focal overgrowth due to low-level but highly activating (hotspot) mutations, (b) predominantly brain overgrowth and less severe somatic overgrowth due to less-activating mutations, and (c) intermediate phenotypes (capillary malformations with overgrowth) with intermediately activating mutations. Sixteen of 29 PIK3CA mutations were novel. We also identified constitutional PIK3CA mutations in 10 patients. Our molecular data, combined with review of the literature, show that PIK3CA-related overgrowth disorders comprise a discontinuous spectrum of disorders that correlate with the severity and distribution of mutations.