Germline variants in tumor suppressor FBXW7 lead to impaired ubiquitination and a neurodevelopmental syndrome.

Neurodevelopmental disorders are highly heterogenous conditions resulting from abnormalities of brain architecture and/or function. FBXW7 (F-box and WD-repeat-domain-containing 7), a recognized developmental regulator and tumor suppressor, has been shown to regulate cell-cycle progression and cell growth and survival by targeting substrates including CYCLIN E1/2 and NOTCH for degradation via the ubiquitin proteasome system. We used a genotype-first approach and global data-sharing platforms to identify 35 individuals harboring de novo and inherited FBXW7 germline monoallelic chromosomal deletions and nonsense, frameshift, splice-site, and missense variants associated with a neurodevelopmental syndrome. The FBXW7 neurodevelopmental syndrome is distinguished by global developmental delay, borderline to severe intellectual disability, hypotonia, and gastrointestinal issues. Brain imaging detailed variable underlying structural abnormalities affecting the cerebellum, corpus collosum, and white matter. A crystal-structure model of FBXW7 predicted that missense variants were clustered at the substrate-binding surface of the WD40 domain and that these might reduce FBXW7 substrate binding affinity. Expression of recombinant FBXW7 missense variants in cultured cells demonstrated impaired CYCLIN E1 and CYCLIN E2 turnover. Pan-neuronal knockdown of the Drosophila ortholog, archipelago, impaired learning and neuronal function. Collectively, the data presented herein provide compelling evidence of an F-Box protein-related, phenotypically variable neurodevelopmental disorder associated with monoallelic variants in FBXW7.

Semiautomated approach focused on new genomic information results in time and effort-efficient reannotation of negative exome data.

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.

Complex balanced intrachromosomal rearrangement involving PITX2 identified as a cause of Axenfeld-Rieger Syndrome.

Axenfeld-Rieger Syndrome (ARS) type 1 is a rare autosomal dominant condition characterized by anterior chamber anomalies, umbilical defects, dental hypoplasia, and craniofacial anomalies, with Meckel's diverticulum in some individuals. Here, we describe a clinically ascertained female of childbearing age with ARS for whom clinical targeted sequencing and deletion/duplication analysis followed by clinical exome and genome sequencing resulted in no pathogenic variants or variants of unknown significance in PITX2 or FOXC1. Advanced bioinformatic analysis of the genome data identified a complex, balanced rearrangement disrupting PITX2. This case is the first reported intrachromosomal rearrangement leading to ARS, illustrating that for patients with compelling clinical phenotypes but negative genomic testing, additional bioinformatic analysis are essential to identify subtle genomic abnormalities in target genes.

Emergence of the natural history of Myhre syndrome: 47 patients evaluated in the Massachusetts General Hospital Myhre Syndrome Clinic (2016-2023).

Myhre syndrome is an increasingly diagnosed ultrarare condition caused by recurrent germline autosomal dominant de novo variants in SMAD4. Detailed multispecialty evaluations performed at the Massachusetts General Hospital (MGH) Myhre Syndrome Clinic (2016-2023) and by collaborating specialists have facilitated deep phenotyping, genotyping and natural history analysis. Of 47 patients (four previously reported), most (81%) patients returned to MGH at least once. For patients followed for at least 5 years, symptom progression was observed in all. 55% were female and 9% were older than 18 years at diagnosis. Pathogenic variants in SMAD4 involved protein residues p.Ile500Val (49%), p.Ile500Thr (11%), p.Ile500Leu (2%), and p.Arg496Cys (38%). Individuals with the SMAD4 variant p.Arg496Cys were less likely to have hearing loss, growth restriction, and aortic hypoplasia than the other variant groups. Those with the p.Ile500Thr variant had moderate/severe aortic hypoplasia in three patients (60%), however, the small number (n = 5) prevented statistical comparison with the other variants. Two deaths reported in this cohort involved complex cardiovascular disease and airway stenosis, respectively. We provide a foundation for ongoing natural history studies and emphasize the need for evidence-based guidelines in anticipation of disease-specific therapies.

The Importance of Mitochondrial Disease Testing in Young Adults With New Onset Sensorineural Hearing Loss.

OBJECTIVES: Sensorineural hearing loss (SNHL) occurs commonly as part of mitochondriopathies and varies in severity and onset. In this study, we characterized hearing with specific consideration for hearing loss as a potential early indicator of mitochondrial disease (MD). We hypothesize that genetic testing at the earliest detection of SNHL may lead to an earlier MD diagnosis. DESIGN: We reviewed the clinical and audiometric data of 49 patients undergoing genetic testing for MD. RESULTS: One-third of individuals with molecularly confirmed MD presented with SNHL. On average, patients had hearing loss at least 10 years before genetic testing. The collective audiometric profile includes mild to moderate SNHL at lower frequencies and moderate SNHL at 2 kHz and higher frequencies. CONCLUSIONS: This study suggests that screening for SNHL could be an early indicator of MD. We propose that the audiometric profile for those with a MD diagnosis may have clinical triage utility.

Implementation of genomic medicine for rare disease in a tertiary healthcare system: Mayo Clinic Program for Rare and Undiagnosed Diseases (PRaUD).

BACKGROUND: In the United States, rare disease (RD) is defined as a condition that affects fewer than 200,000 individuals. Collectively, RD affects an estimated 30 million Americans. A significant portion of RD has an underlying genetic cause; however, this may go undiagnosed. To better serve these patients, the Mayo Clinic Program for Rare and Undiagnosed Diseases (PRaUD) was created under the auspices of the Center for Individualized Medicine (CIM) aiming to integrate genomics into subspecialty practice including targeted genetic testing, research, and education. METHODS: Patients were identified by subspecialty healthcare providers from 11 clinical divisions/departments. Targeted multi-gene panels or custom exome/genome-based panels were utilized. To support the goals of PRaUD, a new clinical service model, the Genetic Testing and Counseling (GTAC) unit, was established to improve access and increase efficiency for genetic test facilitation. The GTAC unit includes genetic counselors, genetic counseling assistants, genetic nurses, and a medical geneticist. Patients receive abbreviated point-of-care genetic counseling and testing through a partnership with subspecialty providers. RESULTS: Implementation of PRaUD began in 2018 and GTAC unit launched in 2020 to support program expansion. Currently, 29 RD clinical indications are included in 11 specialty divisions/departments with over 142 referring providers. To date, 1152 patients have been evaluated with an overall solved or likely solved rate of 17.5% and as high as 66.7% depending on the phenotype. Noteworthy, 42.7% of the solved or likely solved patients underwent changes in medical management and outcome based on genetic test results. CONCLUSION: Implementation of PRaUD and GTAC have enabled subspecialty practices advance expertise in RD where genetic counselors have not historically been embedded in practice. Democratizing access to genetic testing and counseling can broaden the reach of patients with RD and increase the diagnostic yield of such indications leading to better medical management as well as expanding research opportunities.

New Tmc1 Deafness Mutations Impact Mechanotransduction in Auditory Hair Cells.

Transmembrane channel-like protein isoform 1 (TMC1) is a major component of the mechano-electrical transducer (MET) channel in cochlear hair cells and is subject to numerous mutations causing deafness. We report a new dominant human deafness mutation, TMC1 p.T422K, and have characterized the homologous mouse mutant, Tmc1 p.T416K, which caused deafness and outer hair cell (OHC) loss by the fourth postnatal week. MET channels showed decreased Ca2+ permeability and resting open probability, but no change in single-channel conductance or expression. Three adjacent deafness mutations are TMC1 p.L416R, p.G417R, and p.M418K, the last homologous to the mouse Beethoven that exhibits similar channel effects. All substitute a positive for a neutral residue, which could produce charge screening in the channel pore or influence binding of an accessory subunit. Channel properties were compared in mice of both sexes between dominant (Tmc1 p.T416K, Tmc1 p.D569N) and recessive (Tmc1 p.W554L, Tmc1 p.D528N) mutations of residues near the putative pore of the channel. Tmc1 p.W554L and p.D569N exhibit reduced maximum current with no effect on single-channel conductance, implying a smaller number of channels transported to the stereociliary tips; this may stem from impaired TMC1 binding to LHFPL5. Tmc1 p.D528N, located in the pore's narrowest region, uniquely caused large reductions in MET channel conductance and block by dihydrostreptomycin (DHS). For Tmc1 p.T416K and Tmc1 p.D528N, transduction loss occurred between P15 and P20. We propose two mechanisms linking channel mutations and deafness: decreased Ca2+ permeability, common to all mutants, and decreased resting open probability in low Ca2+, confined to dominant mutations.SIGNIFICANCE STATEMENT Transmembrane channel-like protein isoform 1 (TMC1) is thought to be a major component of the mechanotransducer channel in auditory hair cells, but the protein organization and channel structure are still uncertain. We made four mouse lines harboring Tmc1 point mutations that alter channel properties, causing hair cell degeneration and deafness. These include a mouse homolog of a new human deafness mutation pT416K that decreased channel Ca2+ permeability by introducing a positively-charged amino acid in the putative pore. All mutations are consistent with the channel structure predicted from modeling, but only one, p.D528N near the external face of the pore, substantially reduced channel conductance and Ca2+ permeability and virtually abolished block by dihydrostreptomycin (DHS), strongly endorsing its siting within the pore.

The MAP3K7 gene: Further delineation of clinical characteristics and genotype/phenotype correlations.

Mitogen-activated protein 3 kinase 7 (MAP3K7) encodes the ubiquitously expressed transforming growth factor ß-activated kinase 1, which plays a crucial role in many cellular processes. Mutationsin the MAP3K7 gene have been linked to two distinct disorders: frontometaphyseal dysplasia type 2 (FMD2) and cardiospondylocarpofacial syndrome (CSCF). The fact that different mutations can induce two distinct phenotypes suggests a phenotype/genotype correlation, but no side-by-side comparison has been done thus far to confirm this. Here, we significantly expand the cohort and the description of clinical phenotypes for patients with CSCF and FMD2 who carry mutations in MAP3K7. Our findings support that in contrast to FMD2-causing mutations, CSCF-causing mutations in MAP3K7 have a loss-of-function effect. Additionally, patients with pathogenic mutations in MAP3K7 are at risk for (severe) cardiac disease, have symptoms associated with connective tissue disease, and we show overlap in clinical phenotypes of CSCF with Noonan syndrome (NS). Together, we confirm a molecular fingerprint of FMD2- versus CSCF-causing MAP3K7 mutations and conclude that mutations in MAP3K7 should be considered in the differential diagnosis of patients with syndromic congenital cardiac defects and/or cardiomyopathy, syndromic connective tissue disorders, and in the differential diagnosis of NS.

PUS7 deficiency in human patients causes profound neurodevelopmental phenotype by dysregulating protein translation.

Protein translation is a highly regulated process involving the interaction of numerous genes on every component of the protein translation machinery. Upregulated protein translation is a hallmark of cancer and is implicated in autism spectrum disorder, but the risks of developing each disease do not appear to be correlated with one another. In this study we identified two siblings from the NIH Undiagnosed Diseases Program with loss of function variants in PUS7, a gene previously implicated in the regulation of total protein translation. These patients exhibited a neurodevelopmental phenotype including autism spectrum disorder in the proband. Both patients also had features of Lesch-Nyhan syndrome, including hyperuricemia and self-injurious behavior, but without pathogenic variants in HPRT1. Patient fibroblasts demonstrated upregulation of protein synthesis, including elevated MYC protein, but did not exhibit increased rates of cell proliferation. Interestingly, the dysregulation of protein translation also resulted in mildly decreased levels of HPRT1 protein suggesting an association between dysregulated protein translation and the LNS-like phenotypic findings. These findings strengthen the correlation between neurodevelopmental disease, particularly autism spectrum disorders, and the rate of protein translation.

Expanding the phenotype of DNAJC30-associated Leigh syndrome.

Leigh syndrome (LS) is a progressive neurodegenerative disease, characterized by extensive clinical, biochemical, and genetic heterogeneity. Recently, biallelic variants in DNAJC30 gene, encoding a protein crucial for the repair of mitochondrial complex I subunits, have been associated with Leber hereditary optic neuropathy and LS. It was suggested that clinical heterogeneity of DNAJC30-associated mitochondrial disease may be attributed to digenic inheritance. We describe three Polish patients, a 9-year-old boy, and female and male siblings, aged 17 and 11 years, with clinical and biochemical manifestations of LS. Exome sequencing (ES) identified a homozygous pathogenic variant in DNAJC30 c.152A>G, p.(Tyr51Cys) in the 9-year-old boy. In the siblings, ES identified two DNAJC30 variants: c.152A>G, p.(Tyr51Cys) and c.130_131del, p.(Ser44ValfsTer8) in a compound heterozygous state. In addition, both siblings carried a novel heterozygous c.484G>T, p.(Val162Leu) variant in NDUFS8 gene. This report provides further evidence for the association of DNAJC30 variants with LS. DNAJC30-associated LS is characterized by variable age at onset, movement disorder phenotype and normal or moderately elevated blood lactate level. Identification of a candidate heterozygous variant in NDUFS8 supports the hypothesis of digenic inheritance. Importantly, DNAJC30 pathogenic variants should be suspected in patients with LS irrespective of optic nerve involvement.

De novo PBX1 variant in a patient with glaucoma, kidney anomalies, and developmental delay: An expansion of the CAKUTHED phenotype.

An infant was referred for evaluation of congenital glaucoma and corneal clouding. In addition, he had a pelvic kidney, hypotonia, patent ductus arteriosus, abnormal pinnae, and developmental delay. Exome sequencing identified a previously unpublished de novo single nucleotide insertion in PBX1 c.400dupG (NM_002585.3), predicted to cause a frameshift resulting in a truncated protein with loss of function (p.Ala134Glyfs*65). Identification of this loss of function variant supports the diagnosis of congenital anomalies of the kidney and urinary tract syndrome with or without hearing loss, abnormal ears, or developmental delay (CAKUTHED). Here, we propose glaucoma as an extra-renal manifestation associated with PBX1-related disease due to the relationship of PBX1 with MEIS1, MEIS2, and FOXC1 transcription factors associated with eye development.

Disease-specific ACMG/AMP guidelines improve sequence variant interpretation for hearing loss.

PURPOSE: The ClinGen Variant Curation Expert Panels (VCEPs) provide disease-specific rules for accurate variant interpretation. Using the hearing loss-specific American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines, the Hearing Loss VCEP (HL VCEP) illustrates the utility of expert specifications in variant interpretation. METHODS: A total of 157 variants across nine HL genes, previously submitted to ClinVar, were curated by the HL VCEP. The curation process involved collecting published and unpublished data for each variant by biocurators, followed by bimonthly meetings of an expert curation subgroup that reviewed all evidence and applied the HL-specific ACMG/AMP guidelines to reach a final classification. RESULTS: Before expert curation, 75% (117/157) of variants had single or multiple variants of uncertain significance (VUS) submissions (17/157) or had conflicting interpretations in ClinVar (100/157). After applying the HL-specific ACMG/AMP guidelines, 24% (4/17) of VUS and 69% (69/100) of discordant variants were resolved into benign (B), likely benign (LB), likely pathogenic (LP), or pathogenic (P). Overall, 70% (109/157) variants had unambiguous classifications (B, LB, LP, P). We quantify the contribution of the HL-specified ACMG/AMP codes to variant classification. CONCLUSION: Expert specification and application of the HL-specific ACMG/AMP guidelines effectively resolved discordant interpretations in ClinVar. This study highlights the utility of ClinGen VCEPs in supporting more consistent clinical variant interpretation.

Growth hormone deficiency in a child with branchio-oto-renal spectrum disorder: Clinical evidence of EYA1 in pituitary development and a recommendation for pituitary function surveillance.

Branchio-oto-renal spectrum disorder (BORSD) is a rare autosomal dominant condition characterized by ear abnormalities with hard of hearing/deafness, second branchial arch malformations and renal anomalies. Pathogenic variations in EYA1 gene are found in the majority of clinically diagnosed individuals with BORSD. We describe an infant with BORSD related to a paternally inherited heterozygous pathogenic variation in EYA1 gene presenting with poor growth and hypoglycemia due to growth hormone deficiency. Magnetic resonance imaging revealed a diminutive pituitary gland and morphologically abnormal sella. Upon initiation of growth hormone therapy, the hypoglycemia resolved and catch up growth ensued. Pituitary abnormalities have not been reported previously in patients with BORSD. The zebrafish ortholog of eya1 is important for the development of adenohypophysis, suggesting that this patient's growth hormone deficiency and pituitary abnormality are part of BORSD. Inclusion of screening for pituitary hormone deficiency and pituitary imaging should be considered as a part of surveillance in patients with BORSD.

Congenital ichthyosis in Prader-Willi syndrome associated with maternal chromosome 15 uniparental disomy: Case report and review of autosomal recessive conditions unmasked by UPD.

Prader-Willi syndrome (PWS) is a prototypic genetic condition related to imprinting. Causative mechanisms include paternal 15q11-q13 deletion, maternal chromosome 15 uniparental disomy (UPD15), Prader-Willi Syndrome/Angelman Syndrome (PWS/AS) critical region imprinting defects, and complex chromosomal rearrangements. Maternal UPD15-related PWS poses risks of concomitant autosomal recessive (AR) disorders when the mother carries a pathogenic variant in one of the genes on chromosome 15 associated with autosomal recessive inherited disease. Co-occurrence of autosomal recessive conditions in the setting of UPD leads to increased complexity of the clinical phenotype, and may delay the diagnosis of PWS. We report a patient with PWS and associated congenital ichthyosis due to maternal UPD15, and a homozygous novel pathogenic variant in ceramide synthase 3 (CERS3). We also review the literature of associated disorders reported in the setting of maternal UPD15-related PWS and provide a summary of the previously described CERS3 variants. This represents the second case of autosomal recessive congenital ichthyosis (ARCI) in the setting of PWS and UPD15. There needs to be a high index of suspicion of this genetic mechanism when there is unexpected phenotype or evolution of the clinical course in a patient with PWS.

ClinGen expert clinical validity curation of 164 hearing loss gene-disease pairs.

PURPOSE: Proper interpretation of genomic variants is critical to successful medical decision making based on genetic testing results. A fundamental prerequisite to accurate variant interpretation is the clear understanding of the clinical validity of gene-disease relationships. The Clinical Genome Resource (ClinGen) has developed a semiquantitative framework to assign clinical validity to gene-disease relationships. METHODS: The ClinGen Hearing Loss Gene Curation Expert Panel (HL GCEP) uses this framework to perform evidence-based curations of genes present on testing panels from 17 clinical laboratories in the Genetic Testing Registry. The HL GCEP curated and reviewed 142 genes and 164 gene-disease pairs, including 105 nonsyndromic and 59 syndromic forms of hearing loss. RESULTS: The final outcome included 82 Definitive (50%), 12 Strong (7%), 25 Moderate (15%), 32 Limited (20%), 10 Disputed (6%), and 3 Refuted (2%) classifications. The summary of each curation is date stamped with the HL GCEP approval, is live, and will be kept up-to-date on the ClinGen website ( https://search.clinicalgenome.org/kb/gene-validity ). CONCLUSION: This gene curation approach serves to optimize the clinical sensitivity of genetic testing while reducing the rate of uncertain or ambiguous test results caused by the interrogation of genes with insufficient evidence of a disease link.

Correction: ClinGen expert clinical validity curation of 164 hearing loss gene-disease pairs.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Expert specification of the ACMG/AMP variant interpretation guidelines for genetic hearing loss.

Due to the high genetic heterogeneity of hearing loss (HL), current clinical testing includes sequencing large numbers of genes, which often yields a significant number of novel variants. Therefore, the standardization of variant interpretation is crucial to provide consistent and accurate diagnoses. The Hearing Loss Variant Curation Expert Panel was created within the Clinical Genome Resource to provide expert guidance for standardized genomic interpretation in the context of HL. As one of its major tasks, our Expert Panel has adapted the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines for the interpretation of sequence variants in HL genes. Here, we provide a comprehensive illustration of the newly specified ACMG/AMP HL rules. Three rules remained unchanged, four rules were removed, and the remaining 21 rules were specified. These rules were further validated and refined using a pilot set of 51 variants assessed by curators and disease experts. Of the 51 variants evaluated in the pilot, 37% (19/51) changed category based upon application of the expert panel specified rules and/or aggregation of evidence across laboratories. These HL-specific ACMG/AMP rules will help standardize variant interpretation, ultimately leading to better care for individuals with HL.