The NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines) for Genetic/Familial High-Risk Assessment: Breast, Ovarian, Pancreatic, and Prostate are intended to serve as a resource for health care providers to identify individuals who may benefit from cancer risk assessment and genetic counseling and testing; help guide decisions related to genetic testing; and facilitate a multidisciplinary approach in the comprehensive care of individuals at increased risk for hereditary breast, ovarian, pancreatic, and prostate cancer. The current guidelines focus primarily on assessment of pathogenic and likely pathogenic (P/LP) variants associated with increased risk of breast, ovarian, pancreatic, and prostate cancer and recommended approaches to genetic counseling/testing and care strategies in individuals with these P/LP variants associated with increased risk of these cancers. These NCCN Guidelines Insights summarize the panel's most recent recommendations regarding screening for prostate cancer and pancreas cancer, as well as testing criteria for nonepithelial ovarian cancer.

The cohesin complex, composed of SMC1, SMC3, RAD21, and STAG1/STAG2, is essential for chromosome cohesion, DNA repair, and transcriptional regulation. Pathogenic variants in cohesin components cause cohesinopathies. The classical characteristics of cohesinopathies include developmental delay (DD), intellectual disability (ID), feeding difficulties, hypotonia, short stature, hearing loss, and dysmorphic features. Here, we present a 5-year-old boy with classical cohesinopathy features, including DD/ID and feeding difficulties, along with non-classical features such as hypoglycemia, recurrent fever, and immunodeficiency. Trio exome sequencing identified a novel de novo missense variant of uncertain significance (NM_005862.3:c.643G>A(p.Val215Ile)) in the STAG1 gene. The variant localizes to the RAD21 interaction interface, and molecular dynamics (MD) simulations revealed conformational changes comparable to other STAG1 variants reported as likely pathogenic in patients, supporting a deleterious effect which may disrupt the STAG1-RAD21 interaction interface. This case expands the phenotypic and molecular spectrum of STAG1-related cohesinopathy and advances our understanding of the disease mechanism.

Introduction The importance of prenatal determination of chorionicity for the management of twin pregnancies is well recognized. However, research on the contribution of prenatal evaluation of zygosity to the management of twins is limited. We assessed the utility of adding SNP-based cell-free DNA (cfDNA) zygosity testing to ultrasound chorionicity assessment for the clinical management of twin pregnancies.Methods Prospective observational study involving 13 United States practices with proficiency in prenatal ultrasound. Patients diagnosed by ultrasound with twins in the first trimester were assessed with cfDNA screening for zygosity. Ultrasound assessment of chorionicity was performed prior to cfDNA results. Placental pathology was used as the gold standard for chorionicity assessment. Gestational age at delivery and standardized birthweights were compared, based on chorionicity and zygosity.Results 110 twin pregnancies were included. Among 79 dichorionic (DC) cases confirmed by placental pathology, one (1.3%) was misclassified as monochorionic (MC) by ultrasound, but was dizygous (DZ) by cfDNA, consistent with DC. Of 31 monozygotic (MZ) twins by cfDNA, confirmed as MC by pathology, ultrasound misclassified one (3.6%) as DC. Median gestational age at delivery was earlier for MZ twin pregnancies (35.0 weeks) compared to DZ (36.9 weeks, p = 0.02). After adjusting for fetal sex and gestational age at birth, MZDC twins had significantly lower birthweights (p = 0.006) and birthweight percentiles (p = 0.004) than DZDC twins.Conclusions Based on postpartum placental pathology as the reference standard for determining MC versus DC, cfDNA zygosity testing appears to aid in the prenatal assignment of chorionicity. Larger studies are needed to confirm the value of zygosity testing in the management of twin pregnancies.

The alpha globin gene cluster on chromosome 16 encodes alpha-like globins whose expression changes during development. HBM located in this cluster was considered a pseudogene until its transcript was discovered in 2005, but without a detectable protein product. Here, we provide the first detailed description of HBM-encoded globin at the protein level (hemoglobin mu chain [HBM]) across different age groups using liquid chromatography coupled with tandem mass spectrometry. Using targeted analysis, we demonstrate that HBM protein is readily detectable in circulation with its levels falling with increasing gestational age. We also report HBM levels in relation to other hemoglobin chains in neonatal, pediatric, and adult blood. Furthermore, analysis of alpha thalassemia patient samples revealed that HBM levels were elevated 3- to 17-fold, indicating a compensatory role, which could be explored for its therapeutic potential. This study enhances our understanding of erythropoiesis and globin chain production through ontogeny.

BACKGROUND:Li Fraumeni syndrome (LFS) is a hereditary multi-cancer syndrome caused by alterations in TP53 (MIM# 151623). Next generation sequencing (NGS) allows for the detection of TP53 variants at lower variant allele frequencies (VAFs). A TP53 variant with a VAF < 50% may represent mosaic LFS, aberrant clonal expansion (ACE), or possible circulating tumor DNA. Differentiation among these conditions is important for optimal patient management. METHODS:Genetic testing was performed using a custom gene panel, with an average read depth of 350× (range 166-553×). DNA extracted from four different tissues (blood, saliva, cultured skin fibroblasts, and colon) was sequenced. RESULTS:Here, we describe an adult female patient with a history of an adrenocortical neoplasm and osteosarcoma, diagnosed at 2 and 16 years of age, respectively. Testing of peripheral blood identified a pathogenic TP53 variant, c.733G>A, p.(Gly245Ser) (NM_000546.6); however, subsequent in vitro fertilization with preimplantation genetic testing for monogenic disorders (PGT-M) did not identify this TP53 variant in any of nine embryos tested. Testing for possible mosaicism in the proband was performed on four different specimens, revealing variable VAFs of the TP53 variant (saliva: 44%; blood: 31%; cultured skin fibroblasts: 18%; and colon tissue: 9%). These results suggest a post-zygotic event, consistent with mosaic LFS rather than ACE. CONCLUSION:This case highlights the complexity of interpreting mosaic variants in the TP53 gene, and we propose a testing algorithm to aid in the delineation of this phenomenon when relaying cancer and reproductive risk information in the context of mosaicism.

The NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines) for Genetic/Familial High-Risk Assessment: Breast, Ovarian, Pancreatic, and Prostate are intended to serve as a resource for health care providers to identify individuals who may benefit from cancer risk assessment and genetic counseling and testing; help guide decisions related to genetic testing; and facilitate a multidisciplinary approach in the comprehensive care of individuals at increased risk for hereditary breast, ovarian, pancreatic, and prostate cancer. The current guidelines focus primarily on assessment of pathogenic and likely pathogenic (P/LP) variants associated with increased risk of breast, ovarian, pancreatic, and prostate cancer and recommended approaches to genetic counseling/testing and care strategies in individuals with these P/LP variants associated with increased risk of these cancers. These NCCN Guidelines Insights summarize the panel’s most recent recommendations regarding screening for prostate cancer and pancreas cancer, as well as testing criteria for nonepithelial ovarian cancer.

Objective:We sought to establish a process for increasing somatic tumor testing for patients with germline BRCA negative advanced stage epithelial ovarian cancer (EOC) and to gain insight into patients' comprehension of their genetic testing. Methods:A multidisciplinary team utilized quality improvement framework to address clinical needs. After implementation of a new somatic testing process, we compared the rates of genetic testing referral, germline testing and somatic testing recommendations between a historic cohort (January 1, 2019-June 20, 2019) and implementation cohort (October 1, 2020 - March 31, 2021). Patients diagnosed with stage III-IV EOC who underwent surgery were included for analysis. To explore patients' comprehension of their genetic testing results, twenty-three patients in the historic cohort participated in semi-structured interviews. Results:Patients with advanced stage EOC without a germline BRCA mutation received recommendations for somatic testing 53.5 % (23/43) of the time in the historic cohort. An improvement in the rate of somatic testing recommendations was seen in patients without a germline mutation in the implementation cohort (84.6 % [22/26], P = 0.010). There was no decrease in germline testing after implementation (90 % [63/70] and 96.3 % [52/54,] P = 0.30). Most patients (21/23) in the historic cohort were not aware that both germline and somatic testing were completed for their oncology care. Conclusion:We successfully increased somatic testing recommendations in germline BRCA negative patients prior to completing upfront EOC treatment allowing for a timely, individualized discussion of maintenance PARP inhibitor use. Qualitative assessment of patients' comprehension of genetic testing for EOC shows a deficit in patient knowledge.

Purpose ATM germline pathogenic variants (GPVs) are associated with a moderately increased risk of female breast cancer, pancreatic cancer, and prostate cancer. Resources for managing ATM heterozygotes in clinical practice are limited. Methods An international workgroup developed a clinical practice resource to guide management of ATM heterozygotes using peer-reviewed publications and expert opinion. Results Although ATM is a moderate (intermediate) penetrance gene, cancer risks may be considered as a continuous variable, influenced by family history and other modifiers. ATM GPV heterozygotes should generally be offered enhanced breast surveillance according to their personalized risk estimate and country-specific guidelines and, generally, risk-reducing mastectomy is not recommended. Prostate cancer surveillance should be considered. Pancreatic cancer surveillance should be considered based on assessment of family history, ideally as part of a clinical trial, with existence of country-specific guidelines. For ATM GPV heterozygotes who develop cancer, radiation therapy decisions should not be influenced by the genetic result. Although poly-adenosine diphosphate ribose polymerase inhibitors are licensed for use in metastatic castration-resistant prostate cancer and ATM GPVs, the evidence-base is currently weak. Conclusion Systematic prospective data collection is needed to establish the spectrum of ATM-associated cancer and determine the outlines of surveillance, response to cancer treatment, and survival.

PURPOSE:RAD51C, RAD51D, and BRIP1 germline pathogenic variants (GPVs) are associated with increased lifetime risks of tubo-ovarian cancer. Resources for managing RAD51C, RAD51D, and BRIP1 heterozygotes in clinical practice are limited. METHODS:An international workgroup developed a Clinical Practice Resource to guide management of RAD51C, RAD51D, and BRIP1 heterozygotes using peer-reviewed publications and expert opinion. RESULTS:RAD51C, RAD51D, and BRIP1 are moderate (intermediate) penetrance ovarian cancer predisposition genes. Ovarian cancer risks for individuals with RAD51C, RAD51D, and BRIP1 GPVs may be influenced by family history and other modifiers. RAD51C and RAD51D GPVs are also associated with moderate risk of breast cancer, predominantly triple-negative subtype. RAD51C, RAD51D, and BRIP1 heterozygotes should be offered risk-reducing salpingo-oophorectomy close to the age of menopause based on age-specific risks and shared decision making. For RAD51C and RAD51D heterozygotes, enhanced breast surveillance may be indicated according to their personalized risk estimate and country-specific guidelines. Generally, risk-reducing mastectomy is not recommended. For RAD51C, RAD51D, and BRIP1 heterozygotes who develop cancer, there is insufficient evidence to guide any specific targeted treatment. CONCLUSION:Systematic prospective data collection is needed to establish the outcomes of RAD51C, RAD51D, and BRIP1 associated cancers and particularly response to cancer treatment and survival.

Li-Fraumeni syndrome (LFS) is an autosomal dominant cancer predisposition syndrome characterized by multiple primary tumors and early age of onset. While a diagnosis of LFS is confirmed by the presence of a heterozygous pathogenic variant in the TP53 gene, there are individuals with ‘phenotypic LFS’, where no genetic explanation was found although traditional diagnostic criteria are met. Although these individuals receive the same clinical management as classic-LFS patients, their care may be affected by late diagnosis, inability to confirm a diagnosis in other family members and/or pregnancy planning.

Stromal Antigen 1 (STAG1, OMIM 604358) is a core protein of the cohesin complex (Sumara, Toth, Carramolino) that regulates chromatid separation in mitosis, gene transcription, DNA repair and DNA replication (Peters). Pathogenic variants in components of the cohesin complex are rare and referred to as cohesinopathies. A published cohort of 17 patients with STAG1 pathogenic variants (7 deletions, 8 missense and 2 frameshift) notes a neurodevelopmental phenotype (Lehalle). De novo changes were noted in 76% of the cohort, while 12% were maternally inherited and 12% were unknown. All STAG1 patients had intellectual disability/developmental delay (100%). Additional findings included IUGR (18%), neonatal feeding difficulties (53%), hypotonia (29%), facial dysmorphisms (82%), epilepsy (41%), scoliosis (12%) and abnormal brain imaging (18%) (Lehalle). The authors conclude that the pathogenic STAG1 variant most likely resulted in haploinsufficiency of the gene product and in turn disrupted gene regulation resulting in the observed phenotypes. To add to the limited knowledge regarding STAG-1 cohesinopathies, we present a patient with a novel, missense, likely-pathogenic variant. In addition, we present mechanistic evidence supporting the deleterious nature of this variant. A male infant was born via uncomplicated vaginal delivery at 35-6/7 weeks to a 27yoG4P2; late preterm induction of labor was performed for debilitating maternal headaches. A course of betamethasone was administered prior to induction. The pregnancy was otherwise uncomplicated. Family history significant for bronchiectasis in an older sister. Physical exam and newborn screen evaluation were normal at birth, but poor suck was noted immediately. The infant was discharged on day 2 of life but readmitted day 11 due to dehydration, lack of weight gain and inability to coordinate feeds. At three months, persistent difficulty with oral skills and lack of weight gain were noted. Upper GI and swallowing studies were normal. A G-tube was placed. At 4 months, a history of recurrent ear and urinary infections were noted. At 7 months, the patient was diagnosed with laryngomalacia and developed unexplained right sided Horner's syndrome. MRI brain noted an incidental Chiari 1 malformation but no acute process. Genetics consult at 7 months noted anisocoria, mild bilateral conductive hearing loss and a presacral dimple. Metabolic testing was normal for lysosomal/peroxisomal storage disorders, aminoacidopathy, congenital disorders of glycosylation, carnitine, purine and pyrimidine disorders, and pyruvic defects. Chromosomal microarray was normal. Exome sequencing (ES) trio testing through GeneDx, a CAP/CLIA-certified lab, revealed a de novo, likely pathogenic, heterozygous variant in STAG1 c.643G>A, p.V215I consistent with STAG1-related disorders. Following initial WES, he was diagnosed with non-CF bronchiectasis (negative sweat test) and low IgM and IgA levels; he has had episodes of hypoglycemia, with negative metabolic evaluation. WES reanalysis with updated phenotype was non-diagnostic. At 3 years, height is 87.6cm (2%ile), weight 12.75 kg. He has diagnoses of developmental delay including expressive speech delay, receives IVIG infusions, chest therapy and remains G tube dependent with food aversion. He receives educational services and qualified for an IEP at age 3. Our patient's variant occurred at a highly conserved amino acid region in the cohesin complex interface between STAG1 and RAD21. Although reported as likely pathogenic, the deleteriousness of the variant was questioned because the physiochemical change from valine to isoleucine is small and isoleucine occurs in lower species (yeast and fruit fly). Molecular dynamic simulation on this variant and two published deleterious STAG1 variants showed similar altered protein conformation at the RAD21 interface. This report contributes to the expanding literature for STAG1 cohesinopathy phenotypes and mechanistic evidence of the pathogenicity of the genetic variant.

Prenatal diagnosis of chorionicity is critical for the management of twin pregnancies. Although ultrasound has been recommended for this, it has not been thoroughly validated. SNP-based cell-free DNA (cfDNA) can determine zygosity from 9 weeks’ gestation. Dizygotic cfDNA infers dichorionicity (DC). Monozygotic (MZ) cfDNA results may require managing the pregnancy as monochorionic (MC), regardless of chorionicity. The purpose of this study was to assess the accuracy of the diagnosis of chorionicity by ultrasound (C-US) prior to knowledge of cfDNA results. Prospective observational registry multi-institutional study (13 sites in the USA). Inclusion criteria were an ongoing twin pregnancy (< 20 weeks’ gestation) with C-US prior to cfDNA. Exclusion criteria were higher order multiples, contraindication for cfDNA, and twin to twin transfusion syndrome prior to enrollment. C-US, cfDNA results, perinatal outcome and placental pathology reports were collected. A total of 137 twin pregnancies were enrolled in the study. After exclusions for undetermined zygosity on cfDNA (1), missing placental pathology (15), incomplete follow-up (11), and unreported chorionicity on ultrasound (4), 106 pregnancies remained. Median gestation at C-US was 11 weeks. Two of 28 (7.1%) pregnancies reported to be MC on pathology were DC on C-US. All were monozygotic on cfDNA. One in 78 pregnancies (1.3%) reported to be DC on pathology was MC on C-US and was dizygotic on cfDNA. Overall, chorionicity was misdiagnosed by C-US in 2.8% (3/106) of twin pregnancies. Of the MZ-cfDNA twins (41.5%, 44/106), 36% were DC and 64% were MC on pathology. All DZ cfDNA twins (62/106, 58.5%) were DC on pathology. Surgical pathology confirmed the cfDNA zygosity diagnosis in all cases. Ultrasound assessment of chorionicity may be inaccurate in approximately 3% of twins. False negative MC diagnosis may preclude timely management of typical MC complications. False positive MC diagnosis may result in unnecessary surveillance or interventions. cfDNA screening for zygosity is essential in the management of twin pregnancies.

To evaluate outcomes in twin pregnancies stratified by zygosity based on SNP-based cell-free DNA screening (cfDNA). Prospective study of twin pregnancies undergoing SNP-based cfDNA following prenatal ultrasound (< 20 weeks’ gestation) at 13 US sites (Oct 2021-March 2023). Pregnancies with twin-to-twin transfusion syndrome (TTTS) prior to enrollment or a contraindication to cfDNA screening were excluded. cfDNA results, ultrasound findings, placental pathology, and maternal and newborn outcomes were collected. Dizygotic twins (DZ) on cfDNA were classified as dichorionic (DC). Monozygotic (MZ) twins on cfDNA were classified as monochorionic (MC) or DC based on placental pathology. Odds ratios (95% confidence intervals) were calculated to compare the pregnancy outcomes for MZ MC and MZ DC twins with those of DZ twins. 137 patients enrolled in the study. After exclusions for undetermined cfDNA (1), incomplete follow-up (11), and MZ twins with no placental pathology (2), 123 patients remained in the cohort: 76 DZ, 31 MZ MC, and 16 MZ DC. Placental pathology was available for 63/76 (82.9%) DZ twins and was DC in all. Median gestation at cfDNA was 12+5 weeks. Pregnancy outcomes by zygosity and chorionicity are shown in Table 1. TTTS occurred in 3.2%, selective intrauterine growth restriction (SIUGR) in 22.6%, and twin-anemia-polycythemia sequence (TAPS) in 9.7% of MC twins. MZ MC twins had the highest incidence of pregnancy complications including preterm birth and NICU admission. The incidence of small-for-gestational age (SGA) (birth weight < 10th percentile) was higher in the MZ DC group compared to the DZ group (OR=2.51; 95% CI 1.08,5.87). SNP-based cfDNA in twin pregnancies can be used to determine the rate of complications by zygosity. This may help establish the actual rates of specific complications of MC twins, such as TTTS, SIUGR, and TAPS. The finding of a high incidence of SGA in MZ DC vs DZ DC twins may have important research and clinical implications, particularly on the frequency of growth surveillance.

Objective This study aimed to determine the prevalence and heteroplasmy level(s) of MT-RNR1 variants m.1555A > G and m.1494C > T, which are associated with aminoglycoside-induced hearing loss, in a general perinatal population. This study also aimed to characterize the association of these variants and their heteroplasmy levels with hearing loss outcomes with and without aminoglycoside exposure. Study Design Droplet digital polymerase chain reaction was performed on 479 maternal DNA samples from a general perinatal biobank at our institution to detect the presence and heteroplasmy levels of MT-RNR1 variants m.1555A > G and m.1494C > T. Testing of paired neonatal specimen(s) was planned for positive maternal tests. A retrospective chart review was performed to characterize the population, identify aminoglycoside exposures, and determine hearing outcomes. Results All maternal samples tested negative for MT-RNR1 variants m.1555A > G and m.1494C > T. Maternal and neonatal subjects had high rates of aminoglycoside exposure (15.9 and 13.9%, respectively). No subjects with sensorineural or mixed hearing loss had documented aminoglycoside exposure. Conclusion This study demonstrated that a larger sample size is needed to establish the prevalence of these variants as no subjects tested positive. Determination of variant prevalence in the neonatal population, association of variant heteroplasmy levels with hearing outcomes, and reliability of maternal testing as a surrogate for neonatal testing are important next steps toward universal prenatal or newborn screening. Key Points

Ring chromosomes are a relatively rare, usually de novo, genetic finding in which missing material from the ends of chromosomes join to form a ring. Mosaicism has also been reported. With the increased use of cell free fetal DNA screening, the potential for prenatal diagnosis has increased. However, in contrast to cases of complete trisomy, prenatal ultrasonographic findings and ultimately neonatal phenotypes of individuals with ring chromosomes may vary significantly.

The LMNA gene encodes lamin A and lamin C, which play important roles in nuclear organization. Pathogenic variants in LMNA cause laminopathies, a group of disorders with diverse phenotypes. There are two main groups of disease-causing variants: missense variants affecting dimerization and intermolecular interactions, and heterozygous substitutions activating cryptic splice sites. These variants lead to different disorders, such as dilated cardiomyopathy and Hutchinson–Gilford progeria (HGP). Among these, the phenotypic terms for LMNA-associated cardiocutaneous progeria syndrome (LCPS), which does not alter lamin A processing and has an older age of onset, have been described. Here, we present the workup of an LMNA variant of uncertain significance, NM_170707.2 c. 4G>A, p.(Glu2Lys), in a 36-year-old female with severe calcific aortic stenosis, a calcified mitral valve, premature aging, and a family history of similar symptoms. Due to the uncertainty of in silico predictions for this variant, an assessment of nuclear morphology was performed using the immunocytochemistry of stable cell lines to indicate whether the p.(Glu2Lys) had a similar pathogenic mechanism as a previously described pathogenic variant associated with LCPS, p.Asp300Gly. Indirect immunofluorescence analysis of nuclei from stable cell lines showed abnormal morphology, including lobulation and occasional ringed nuclei. Relative to the controls, p.Glu2Lys and p.Asp300Gly nuclei had significantly (p < 0.001) smaller average nuclear areas than controls (mean = 0.10 units, SD = 0.06 for p.Glu2Lys; and mean = 0.09 units, SD = 0.05 for p.Asp300Gly versus mean = 0.12, SD = 0.05 for WT). After functional studies and segregation studies, this variant was upgraded to likely pathogenic. In summary, our findings suggest that p.Glu2Lys impacts nuclear morphology in a manner comparable to what was observed in p.Asp300Gly cells, indicating that the variant is the likely cause of the LCPS segregating within this family.

There is a broad phenotypic spectrum of monogenic polycystic kidney diseases (PKDs). These disorders often involve cilia-related genes and lead to the development of fluid-filled cysts and eventual kidney function decline and failure. Preimplantation genetic testing for monogenic (PGT-M) disorders has moved into the clinical realm. It allows prospective parents to avoid passing on heritable diseases to their children, including monogenic PKD. The PGT-M process involves embryo generation through in vitro fertilization, with subsequent testing of embryos and selective transfer of those that do not harbor the specific disease-causing variant(s). There is a growing body of literature supporting the success of PGT-M for autosomal-dominant and autosomal-recessive PKD, although with important technical limitations in some cases. This technology can be applied to many other types of monogenic PKD and ciliopathies despite the lack of existing reports in the literature. PGT-M for monogenic PKD, like other forms of assisted reproductive technology, raises important ethical questions. When considering PGT-M for kidney diseases, as well as the potential to avoid disease in future generations, there are regulatory and ethical considerations. These include limited government regulation and unstandardized consent processes, potential technical errors, high cost and equity concerns, risks associated with pregnancy for mothers with kidney disease, and the impact on all involved in the process, including the children who were made possible with this technology.

Most rare disease patients (75–50%) undergoing genomic sequencing remain unsolved, often due to lack of information about variants identified. Data review over time can leverage novel information regarding disease-causing variants and genes, increasing this diagnostic yield. However, time and resource constraints have limited reanalysis of genetic data in clinical laboratories setting. We developed RENEW, (REannotation of NEgative WES/WGS) an automated reannotation procedure that uses relevant new information in on-line genomic databases to enable rapid review of genomic findings. We tested RENEW in an unselected cohort of 1066 undiagnosed cases with a broad spectrum of phenotypes from the Mayo Clinic Center for Individualized Medicine using new information in ClinVar, HGMD and OMIM between the date of previous analysis/testing and April of 2022. 5741 variants prioritized by RENEW were rapidly reviewed by variant interpretation specialists. Mean analysis time was approximately 20 s per variant (32 h total time). Reviewed cases were classified as: 879 (93.0%) undiagnosed, 63 (6.6%) putatively diagnosed, and 4 (0.4%) definitively diagnosed. New strategies are needed to enable efficient review of genomic findings in unsolved cases. We report on a fast and practical approach to address this need and improve overall diagnostic success in patient testing through a recurrent reannotation process.