ECEL1 related distal arthrogryposis 5D in an Indian cohort-Report of recognizable musculoskeletal phenotype and a possible founder variant.

Distal arthrogryposis type 5D (DA5D) is clinically characterized by knee extension contractures, distal joint contractures, clubfoot, micrognathia, ptosis, and scoliosis. We report nine affected individuals from eight unrelated Indian families with DA5D. Although the overall musculoskeletal phenotype is not very distinct from other distal arthrogryposis, the presence of fixed knee extension contractures with or without scoliosis could be an important early pointer to DA5D. We also report a possible founder variant in ECEL1 along with four novel variants and further expand the genotypic spectrum of DA5D.

The book is just being written: The enduring journey of parents of children with emerging- ultrarare disorders.

Ultra rare disorders are being diagnosed at an unprecedented rate, due to genomic sequencing. These diagnoses are often a new gene association, for which little is known, and few share the diagnosis. For these diagnoses, we use the term emerging-ultrarare disorder (E-URD), defined as <100 diagnosed individuals. We contacted 20 parents of children diagnosed with an E-URD through the Duke University Research Sequencing Clinic. Seventeen completed semi-structured interviews exploring parental perspectives (7/17 had children in publications describing the phenotype; 4/17 had children in the first publication establishing a new disorder). Data were analyzed using a directed content approach informed by an empowerment framework. Parents reported a range of responses, including benefits of a diagnosis and challenges of facing the unknown, some described feeling lost and confused, while others expressed empowerment. Empowerment characteristics were hope for the future, positive emotions, engagement, and confidence/self-efficacy to connect with similar others, partner with healthcare providers, and seek new knowledge. We identified a subset of parents who proactively engaged researchers, supported research and publications, and created patient advocacy and support organizations to connect with and bolster similarly diagnosed families. Other parents reported challenges of low social support, low tolerance for uncertainty, limited knowledge about their child's disorder, as well as difficulty partnering with HCPs and connecting to an E-URD community. An overarching classification was developed to describe parental actions taken after an E-URD diagnosis: adjusting, managing, and pioneering. These classifications may help genetic counselors identify and facilitate positive steps with parents of a child with an E-URD.

Quality Control of an Isogenic H/N/KRAS-Less Mouse Embryonic Fibroblast Cell Line Panel.

Isogenic H/N/KRAS-less mouse embryonic fibroblast (MEF) cell lines have been developed to assist in cell-based assays of RAS inhibitors. The quality control assessment of a panel of these isogenic MEFs is described here, with a focus on ensuring the proper insertion of the desired mutant RAS transgene, a determination of gene copy number, and an investigation of potential off-target mutations which could lead to phenotypes which are undesired in downstream experiments. Using this suite of quality control tools, a MEF cell line can be readily validated, and researchers can be assured of the rationale for an observed phenotype.

Novel compound heterozygous mutations in SCN4A as a potential genetic cause contributing to myopathic manifestations: A case report and literature review.

Background: SCN4A mutations account for a diverse array of clinical manifestations, encompassing periodic paralysis, myotonia, and newly recognized symptoms like classical congenital myopathy or congenital myasthenic syndromes. We describe the initial occurrence of myopathic features mimic with recessive classical CM in a Korean infant presenting with novel compound heterozygous SCN4A mutations. The infant exhibited profound hypotonia after birth, thereby expanding the spectrum of SCN4A-related channelopathy. Methods: The genetic analyses comprised targeted exome sequencing, employing a Celemics G-Mendeliome DES Panel, along with Sanger sequencing. Results: Considering the clinical manifestations observed in the proband, SCN4A variants emerged as the primary contenders for autosomal recessive (AR) congenital myopathy 22a, classic (#620351). Sanger sequencing validated the association of SCN4A variants with the phenotype, affirming the AR nature of the compound heterozygous variants in both the carrier mother (c.3533G > T/p.Gly1178Val) and the father (c.4216G > A/p.Ala1406Thr). Conclusion: Our report emphasizes the association of novel compound heterozygous mutations in SCN4A with myopathic features resembling CM, as supporting by muscle biopsy. It is essential to note that pathogenic SCN4A LoF mutations are exceedingly rare. This study contributes to our understanding of SCN4A mutations and their role in myopathic features mimic with classical CM.

A novel GLI3 frameshift mutation in a Chinese pedigree with polydactyly: A case report.

Background: GLI3 gene mutations can result in various forms of polysyndactyly, such as Greig cephalopolysyndactyly syndrome (GCPS, MIM: #175700), Pallister-Hall syndrome (PHS, MIM: #146510), and isolated polydactyly (IPD, MIM: #174200, #174700). Reports on IPD-associated GLI3 mutations are rare. In this study, a novel GLI3 mutation was identified in a Chinese family with IPD. Results: We report a family with six members affected by IPD. The family members demonstrated several special phenotypes, including sex differences, abnormal finger joint development, and different polydactyly types. We identified a novel frameshift variant in the GLI3 gene (NM_000168.6: c.1820_1821del, NP_000159.3: p.Tyr607Cysfs*9) by whole-exome sequencing. Further analysis suggested that this mutation was the cause of polydactyly in this family. Conclusions: The discovery of this novel frameshift variant in our study further solidifies the relationship between IPD and GLI3 and expands the previously established spectrum of GLI3 mutations and associated phenotypes.

Novel KMT5B variant associated with neurodevelopmental disorder in a Chinese family: A case report.

Background: We report here the clinical and genetic features of KMT5B-related neurodevelopmental disorder caused by a novel heterozygous frameshift variant in KMT5B in a Chinese family. Case presentation: A 7-year-old Chinese boy with mild-to-moderate intellectual disability, significant language impairment, motor disability, and coordination difficulties presented to our hospital because he "could not speak and did not look at others." He was diagnosed with autism spectrum disorder previously owing to developmental delays in cognition, language expression, and understanding. The child also had variable nonspecific features including macrocephaly, wide button-hole space and nasal bridge, low ear, social behavior disorder, and foot deformities. Exome sequencing (ES) revealed that both the proband and his younger brother had inherited a novel heterozygous frameshift variant c.438_439ins[ASD; KT192064.1:1_310] of the KMT5B gene from their father. Bioinformatics analysis showed that the novel mutation affected the structure of the KMT5B pre-SET domain, mainly in the α-helix region. According to the American College of Medical Genetics and Genomics (ACMG) guidelines, this type of variant was eventually determined to be likely pathogenic (PVS1+PM2_P). Conclusions: Our investigation expands the mutation spectrum of KMT5B to help us to better understand KMT5B-related neurodevelopmental disorder.

Genotypic and phenotypic features of 39 Chinese patients with glycogen storage diseases type I, VI, and IX.

Glycogen storage diseases (GSDs) are abnormally inherited glycogen metabolism mainly affecting the liver, muscles, and heart. Deficiency of proteins involved in glycogen metabolism caused by genetic mutations are responsible for different subtype of GSDs. However, there are still some challenges in diagnosing GSD. This study includes 39 suspected GSDs patients from unrelated families in China. Next-generation sequencing (NGS) was used to investigate the reason for their diseases at the genetic level. Finally, all 39 patients were diagnosed with GSDs, including 20 GSD-Ia, 4 GSD-VI, and 15 GSD IX (12 GSD-IXa patients and 3 GSD-IXb patients). Thirty-two mutations in G6PC1, PYGL, PHKA2, and PHKB genes were identified, with 14 of them being novel variants. The pathogenicity of novel variants was classified according to ACMG guildlines and predicted by in slico algorithms. Mutations p.L216L and p.R83H in G6PC1 gene may be the hot spot mutation in Chinese. Hearing impairment is a rare clinical feature of GSD Ia, which has also been observed in our cohort. The severity of GSD VI and IX was indicated by our patients. Close follow-up should be applied to GSD VI and IX patients. Our findings provided evidence for building the phenotype-genotype of GSDs and expanded the mutation spectrum of related genes.

Extending the new era of genomic testing into pregnancy management: A proposed model for Australian prenatal services.

BACKGROUND: Trio exome sequencing can be used to investigate congenital abnormalities identified on pregnancy ultrasound, but its use in an Australian context has not been assessed. AIMS: Assess clinical outcomes and changes in management after expedited genomic testing in the prenatal period to guide the development of a model for widespread implementation. MATERIALS AND METHODS: Forty-three prospective referrals for whole exome sequencing, including 40 trios (parents and pregnancy), two singletons and one duo were assessed in a tertiary hospital setting with access to a state-wide pathology laboratory. Diagnostic yield, turn-around time (TAT), gestational age at reporting, pregnancy outcome, change in management and future pregnancy status were assessed for each family. RESULTS: A clinically significant genomic diagnosis was made in 15/43 pregnancies (35%), with an average TAT of 12 days. Gestational age at time of report ranged from 16 + 5 to 31 + 6 weeks (median 21 + 3 weeks). Molecular diagnoses included neuromuscular and skeletal disorders, RASopathies and a range of other rare Mendelian disorders. The majority of families actively used the results in pregnancy decision making as well as in management of future pregnancies. CONCLUSIONS: Rapid second trimester prenatal genomic testing can be successfully delivered to investigate structural abnormalities in pregnancy, providing crucial guidance for current and future pregnancy management. The time-sensitive nature of this testing requires close laboratory and clinical collaboration to ensure appropriate referral and result communication. We found the establishment of a prenatal coordinator role and dedicated reporting team to be important facilitators. We propose this as a model for genomic testing in other prenatal services.

Von-Hipple Lindau syndrome with family history: a case report and seventeen years follow-up study.

Background: Von-Hipple Lindau syndrome is an uncommon autosomal dominant disorder. 17 years ago we diagnosed a young woman with VHL syndrome validated by Sanger sequencing, her family members were genetically tested as well, and 187 healthy people were randomly selected for VHL genetic testing as controls. We analyze the clinical and genetic characteristics of VHL syndrome in a Chinese lineage and with 17-year follow-up. Case presentation: A woman was finally diagnosed with VHL syndrome due to the detection of a missense mutation c.353T > C in exon 2 of the short arm of chromosome 3, which resulted in a leucine substitution at amino acid 118 of the encoded protein by a proline, which may be thought the main cause of the disease. The same mutation was observed in two other family members, their clinical symptoms are not entirely identical. However, this mutation was not found in other family members or 187 healthy controls. She clinically presented with central nervous system hemangioblastomas, clear renal cell carcinoma, and pancreatic neuroendocrine neoplasms, despite the multi-organ involvement and several relapses during the disease, the patients survive well for she was treated with aggressive surgery early in the course of the plaguing symptoms, whereas patients who are not aggressively treated have a poorer prognosis. Conclusion: The clinical presentation of VHL syndrome is atypical, and early identification and treatment of VHL syndrome is possible by genetic testing techniques. Multiple relapses occurred during the course of the disease, but early diagnosis and aggressive treatment allowed the patients to survive well.

Genetic Evaluation for Monogenic Disorders of Low Bone Mass and Increased Bone Fragility: What Clinicians Need to Know.

PURPOSE OF REVIEW: The purpose of this review is to outline the principles of clinical genetic testing and to provide practical guidance to clinicians in navigating genetic testing for patients with suspected monogenic forms of osteoporosis. RECENT FINDINGS: Heritability assessments and genome-wide association studies have clearly shown the significant contributions of genetic variations to the pathogenesis of osteoporosis. Currently, over 50 monogenic disorders that present primarily with low bone mass and increased risk of fractures have been described. The widespread availability of clinical genetic testing offers a valuable opportunity to correctly diagnose individuals with monogenic forms of osteoporosis, thus instituting appropriate surveillance and treatment. Clinical genetic testing may identify the appropriate diagnosis in a subset of patients with low bone mass, multiple or unusual fractures, and severe or early-onset osteoporosis, and thus clinicians should be aware of how to incorporate such testing into their clinical practices.

FBN2 pathogenic variants in congenital contractural arachnodactyly with severe cardiovascular manifestations.

PURPOSE: Congenital contractural arachnodactyly (CCA) is an extremely rare autosomal dominant connective tissue genetic disorder caused by pathogenic variants in FBN2. CCA is characterized by arachnodactyly, camptodactyly, contracture of major joints, scoliosis, pectus deformities, and crumpled ears, but rarely with lethal cardiovascular manifestations as in Marfan syndrome. It is imperative to conduct a comprehensive analysis and review of the pathogenesis of CCA resulting from pathogenic variants in FBN2 gene. MATERIALS AND METHODS: Using whole-exome sequencing and Sanger sequencing, we identified a novel pathogenic splice-altering variant (c.4472-3C>A) in intron 34 of FBN2 gene in a CCA pedigree. The transcriptional result of the splicing-altering variant was analyzed by RNA sequencing. We systematically analyzed the clinical manifestations of all reported cases of CCA caused by splicing-altering pathogenic variants and focused on all the pathogenic variants in FBN2 gene that are associated with severe cardiovascular manifestations. RESULTS: The splice-altering variant (c.4472-3C>A) in FBN2 was demonstrated to result in the exon 35 skipping and cause an in-frame deletion. Furthermore, we identified exons 31 to 35 may be a hotspot region in FBN2 gene associated with severe cardiovascular phenotype. CONCLUSIONS: This study enriched the pathogenic spectrum of CCA and identified a hotspot region in FBN2 gene associated with severe cardiovascular manifestations. We recommend that patients carrying pathogenic variants in exons 31 to 35 of FBN2 pay more attention to cardiac evaluation.

Novel Compound Heterozygous Mutations of LIG4 Gene in an Indian LIG4 Syndrome Patient with Severe Microcephaly: Case Report, In-silico Analysis and Systematic Review.

BACKGROUND: LIG4 syndrome, characterized by immunodeficiency, sensitivity to ionizing radiations, intrauterine growth retardation, postnatal growth retardation, and microcephaly, is a rare genetic disorder caused by pathogenic variants of the LIG4 gene. Few patients are presented with no immune dysregulation as well. CASE STUDY: We present here a male child of 2 years and 4 months of age with severe microcephaly and short stature. His birth weight was 1.9 Kg, and his current height, weight, and head circumference are 83.2 cm (z score = -2.37), 9.5 Kg (z score = -2.76), and 36 cm (z score = -9.24), respectively. Possible causative pathogenic compound heterozygous variants of the LIG4 gene, which were inherited from the parents, were identified by whole exome sequencing of the DNA of the patient and his parents. A systematic review of the literature is also performed to summarize the patients of LIG4 syndrome reported worldwide and summarize the associated genetic mutations of the LIG4 gene. Compound heterozygous variants (c.597_600delTCAG/ c.342del) of LIG4 gene were identified. The parents were found to be heterozygous carriers of one variant each. CONCLUSION: The in-silico analysis of identified variants explains their effect on the structure and function of the LIG4 protein hence explaining the genotype-phenotype correlation.

Reanalysis of Whole-Exome Sequencing Data of an Infant with Suspected Diagnosis of Jeune Syndrome Revealed a Likely Pathogenic Variant in GRK2: A Newly Associated Gene for Jeune Syndrome Phenotype.

Introduction: Ciliopathies with major skeletal involvement embrace a group of heterogeneous disorders caused by pathogenic variants in a group of diverse genes. A narrow thorax with shortening of long bones inspires a clinical entity underlined by dysfunction of primary cilia. Currently, more than 23 genes are listed in the OMIM database corresponding to this clinical entity: WDR19/34/35/60, IFT43/52/80/81/140/172, DYNC2LI1, TTC21B, DYNLT2B, EVC2, EVC, INTU, NEK1, CEP120, DYNC2H1, KIAA0586, SRTD1, KIAA0753, and SRTD12. Recently, individuals with biallelic loss-of-function variants in GRK2 are shown to demonstrate a phenotype compatible with Jeune syndrome. Experimental evidence has shown that impaired function of GRK2 compromises cilia-based signaling of Hedgehog pathway as well as Wnt signaling, while cilia morphology remains intact. Hence, GRK2 is now considered an essential protein in regulation of the skeletogenesis. Case Presentation: We presented a female infant born to a consanguineous marriage who was found to have a biallelic p.R474* alteration in GRK2 in reanalysis of the whole-exome sequencing (WES) data. The patient was exhibiting major clinical features of Jeune syndrome, such as shortened long bones, ribs, and narrow thorax. Discussion: Our reanalysis of WES data revealed a likely pathogenic biallelic variant in the GRK2 which is probably responsible for the Jeune syndrome phenotype in the patient. Hence, our report supports the recently discovered association of GRK2 loss-of-function variants with Jeune syndrome phenotype and emphasizes the significance of reanalysis of WES data, notably in patients with phenotypes suggestive of a such discernible Mendelian disorder.

A case of polyglucosan body myopathy caused by an RBCK1 gene variant and literature review.

OBJECTIVE: To analyze the clinical and genetic characteristics of a patient with Polyglucosan body myopathy 1 (PGBM1) caused by a novel compound heterozygous variant in the RBCK1 gene. METHODS: The clinical data of the patient were collected, next-generation sequencing technology was used to determine the exome sequence of the patient, and the suspected pathogenic locus was verified by Sanger sequencing. RESULTS: Through whole-exome sequencing, we found that there were c.919G>T; p. (Glu307*) and c.723_730dup; p. (Glu244fs) variants of the RBCK1 gene in the patient, inherited from his parents, constituting a compound heterozygous variation. According to the guidelines of the American College of Medical Genetics and Genomics (ACMG), the two variants were rated as pathogenic, but there were no comparable cases. Previous literature reported 24 patients with RBCK1 gene variants, involving a total of 20 myocardial and 18 skeletal muscle cases. CONCLUSIONS: The patient was twice diagnosed with cardiac insufficiency, neglecting the usual manifestations of muscle weakness, resulting in misdiagnosis. Later, novel variants in the RBCK1 gene were discovered through whole-exome sequencing, and symptomatic treatment was given after diagnosis. The importance of whole-exome sequencing technology in disease diagnosis and genetic counseling was emphasized.

A cross-trait study of lung cancer and its related respiratory diseases based on large-scale exome sequencing population.

Background: Genome-wide association studies (GWASs) explain the genetic susceptibility between diseases and common variants. Nevertheless, with the appearance of large-scale sequencing profiles, we could explore the rare coding variants in disease pathogenesis. Methods: We estimated the genetic correlation of nine respiratory diseases and lung cancer in the UK Biobank (UKB) by linkage disequilibrium score regression (LDSC). Then, we performed exome-wide association studies at single-variant level and gene-level for lung cancer and lung cancer-related respiratory diseases using the whole-exome sequencing (WES) data of 427,934 European participants. Cross-trait meta-analysis was conducted by association analysis based on subsets (ASSET) to identify the pleiotropic variants, while in-silico functional analysis was performed to explore their function. Causal mediation analysis was used to explore whether these pleiotropic variants lead to lung cancer is mediated by affecting the chronic respiratory diseases. Results: Five respiratory diseases [emphysema, pneumonia, asthma, chronic obstructive pulmonary disease (COPD), and fibrosis] were genetically correlated with lung cancer. We identified 102 significant independent variants at single-variant levels for lung cancer and five lung cancer-related diseases. 15:78590583:G>A (missense variant in CHRNA5) was shared in lung cancer, emphysema, and COPD. Meanwhile, 14 significant genes and 87 suggestive genes were identified in gene-based association tests, including HSD3B7 (lung cancer), SRSF2 (pneumonia), TNXB (asthma), TERT (fibrosis), MOSPD3 (emphysema). Based on the cross-trait meta-analysis, we detected 145 independent pleiotropic variants. We further identified abundant pathways with significant enrichment effects, demonstrating that these pleiotropic genes were functional. Meanwhile, the proportion of mediation effects of these variants ranged from 6 to 23 (emphysema: 23%; COPD: 20%; pneumonia: 20%; fibrosis: 7%; asthma: 6%) through these five respiratory diseases to the incidence of lung cancer. Conclusions: The identified shared genetic variants, genes, biological pathways, and potential intermediate causal pathways provide a basis for further exploration of the relationship between lung cancer and respiratory diseases.

A combination of resistance genes confers high and durable resistance against stripe rust in wheat cultivar Lantian 26.

Lantian 26, a leading elite winter wheat cultivar in Gansu Province since its release in 2010, exhibits high resistance or immunization to stripe rust in adult-plant stage under a high disease pressure in Longnan (southeastern Gansu). Identifying the resistance genes in Lantian 26 could provide a basis for enhanced durability and high levels of resistance in wheat cultivars. Here, a segregating population was developed from a cross between a highly susceptible wheat cv. Mingxian 169 and the highly stripe rust-resistant cv. Lantian 26. The F2 and F2:3 progenies of the cross were inoculated with multiple prevalent virulent races of stripe rust for adult plant-stage resistance evaluation in two different environments. Exon sequence alignment analysis revealed that a stripe rust resistance gene on the 718.4-721.2 Mb region of chromosome 7BL, tentatively named as YrLT26, and a co-segregation STS marker GY17 was developed and validated using the F2:3 population and 103 wheat cultivars. The other two resistance genes, Yr9 and Yr30, were also identified in Lantian 26 using molecular markers. Therefore, the key to high and durable resistance to stripe rust at adult stage is the combination of Yr9, Yr30 and YrLT26 genes in Lantian 26. This could be a considerable strategy for improving the wheat cultivars with effective and durable resistance in the high-pressure region for stripe rust.

Genetic underpinnings explored: OPA1 deletion and complex phenotypes on chromosome 3q29.

BACKGROUND: Copy number variations (CNVs) have emerged as significant contributors to the elusive genetic causality of inherited eye diseases. In this study, we describe a case with optic atrophy and a brain aneurysm, in which a de novo CNV 3q29 deletion was identified. CASE PRESENTATION: A 40-year-old female patient was referred to our department after undergoing aneurysm transcatheter arterial embolization for a brain aneurysm. She had no history of systemic diseases, except for unsatisfactory best-corrected visual acuity (BCVA) since elementary school. Electrophysiological tests confirmed the findings in retinal images, indicating optic nerve atrophy. Chromosomal microarray analysis revealed a de novo deletion spanning 960 kb on chromosome 3q29, encompassing OPA1 and six neighboring genes. Unlike previously reported deletions in this region associated with optic atrophy, neuropsychiatric disorders, and obesity, this patient displayed a unique combination of optic atrophy and a brain aneurysm. However, there is no causal relationship between the brain aneurysm and the CNV. CONCLUSION: In conclusion, the optic atrophy is conclusively attributed to the OPA1 deletion, and the aneurysm could be a coincidental association. The report emphasizes the likelihood of underestimating OPA1 deletions due to sequencing technology limitations. Recognizing these constraints, healthcare professionals must acknowledge these limitations and consistently search for OPA1 variants/deletions in Autosomal Dominant Optic Atrophy (ADOA) patients with negative sequencing results. This strategic approach ensures a more comprehensive exploration of copy-number variations, ultimately enhancing diagnostic precision in the field of genetic disorders.

Identification of a Novel Homozygous GLS Gene Variant Associated with Developmental and Epileptic Encephalopathy (DEE) Type 71.

Developmental and epileptic encephalopathy (DEEs) (OMIM#618,328) is characterized by seizures, hypotonia, and brain abnormalities, often arising from mutations in genes crucial for brain function. Among these genes, GLS stands out due to its vital role in the central nervous system (CNS), with homozygous variants potentially causing DEE type 71. Using Whole Exome Sequencing (WES) on a patient exhibiting symptoms of epileptic encephalopathy, we identified a novel homozygous variant, NM_014905.5:c.1849G > T; p.(Asp617Tyr), in the GLS gene. The 5-year-old patient, born to consanguineous parents, presented with developmental delay, encephalopathy, frequent seizures, and hypotonia. Sanger sequencing further validated the GLS gene variant in both the patient and his family. Furthermore, our bioinformatics analysis indicated that this missense variant could lead to alteration of splicing, resulting in the activation of a cryptic donor site and potentially causing loss of protein function. Our finding highlights the pathogenic significance of the GLS gene, particularly in the context of brain disorders, specifically DEE71.

Exome copy number variant detection, analysis, and classification in a large cohort of families with undiagnosed rare genetic disease.

Copy number variants (CNVs) are significant contributors to the pathogenicity of rare genetic diseases and, with new innovative methods, can now reliably be identified from exome sequencing. Challenges still remain in accurate classification of CNV pathogenicity. CNV calling using GATK-gCNV was performed on exomes from a cohort of 6,633 families (15,759 individuals) with heterogeneous phenotypes and variable prior genetic testing collected at the Broad Institute Center for Mendelian Genomics of the Genomics Research to Elucidate the Genetics of Rare Diseases consortium and analyzed using the seqr platform. The addition of CNV detection to exome analysis identified causal CNVs for 171 families (2.6%). The estimated sizes of CNVs ranged from 293 bp to 80 Mb. The causal CNVs consisted of 140 deletions, 15 duplications, 3 suspected complex structural variants (SVs), 3 insertions, and 10 complex SVs, the latter two groups being identified by orthogonal confirmation methods. To classify CNV variant pathogenicity, we used the 2020 American College of Medical Genetics and Genomics/ClinGen CNV interpretation standards and developed additional criteria to evaluate allelic and functional data as well as variants on the X chromosome to further advance the framework. We interpreted 151 CNVs as likely pathogenic/pathogenic and 20 CNVs as high-interest variants of uncertain significance. Calling CNVs from existing exome data increases the diagnostic yield for individuals undiagnosed after standard testing approaches, providing a higher-resolution alternative to arrays at a fraction of the cost of genome sequencing. Our improvements to the classification approach advances the systematic framework to assess the pathogenicity of CNVs.