A reverse genetics and genomics approach to gene paralog function and disease: Myokymia and the juxtaparanode.

The leucine-rich glioma-inactivated (LGI) family consists of four highly conserved paralogous genes, LGI1-4, that are highly expressed in mammalian central and/or peripheral nervous systems. LGI1 antibodies are detected in subjects with autoimmune limbic encephalitis and peripheral nerve hyperexcitability syndromes (PNHSs) such as Isaacs and Morvan syndromes. Pathogenic variations of LGI1 and LGI4 are associated with neurological disorders as disease traits including familial temporal lobe epilepsy and neurogenic arthrogryposis multiplex congenita 1 with myelin defects, respectively. No human disease has been reported associated with either LGI2 or LGI3. We implemented exome sequencing and family-based genomics to identify individuals with deleterious variants in LGI3 and utilized GeneMatcher to connect practitioners and researchers worldwide to investigate the clinical and electrophysiological phenotype in affected subjects. We also generated Lgi3-null mice and performed peripheral nerve dissection and immunohistochemistry to examine the juxtaparanode LGI3 microarchitecture. As a result, we identified 16 individuals from eight unrelated families with loss-of-function (LoF) bi-allelic variants in LGI3. Deep phenotypic characterization showed LGI3 LoF causes a potentially clinically recognizable PNHS trait characterized by global developmental delay, intellectual disability, distal deformities with diminished reflexes, visible facial myokymia, and distinctive electromyographic features suggestive of motor nerve instability. Lgi3-null mice showed reduced and mis-localized Kv1 channel complexes in myelinated peripheral axons. Our data demonstrate bi-allelic LoF variants in LGI3 cause a clinically distinguishable disease trait of PNHS, most likely caused by disturbed Kv1 channel distribution in the absence of LGI3.

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.

Belzutifan in adults with VHL-associated central nervous system hemangioblastoma: a single-center experience.

PURPOSE: Belzutifan is a selective inhibitor of hypoxia-inducible factor 2 alpha (HIF-2a) that has emerged as a targeted therapy option for Von Hippel-Lindau (VHL) syndrome-associated tumors with recent FDA approval. There is limited real-world evidence regarding safety and efficacy in CNS hemangioblastoma. Our objective was to report on our clinical experience with belzutifan in adult patients with VHL-associated CNS hemangioblastoma. METHODS: We retrospectively reviewed our institutional experience of belzutifan in adult patients (> 18 years of age at time of therapy) with VHL and craniospinal CNS hemangioblastomas not amenable to surgical resection. The period for study review was October 2021 to March 2023. RESULTS: 4 patients (all female) with a median age of 36 years at time of belzutifan initiation were included. Median duration of therapy at last follow-up was 11 months (6-17 months). All patients had radiographic response to therapy after a median of 3 months (2-5 months), with maximal response to therapy after a median of 8 months (3-17 months). Therapy was well tolerated, with the most common adverse effect being anemia. No patients had treatment pauses or dose adjustments due to belzutifan-related toxicity. No patients experienced hypoxia. CONCLUSION: We showed that belzutifan is safe and well-tolerated with strong disease response for CNS hemangioblastoma in adults with VHL, supporting continued use of belzutifan in this patient population. Future studies should assess duration of treatment, effects of cessation after long-term use, and markers of therapeutic response.

UBA2 variants underlie a recognizable syndrome with variable aplasia cutis congenita and ectrodactyly.

PURPOSE: The human chromosome 19q13.11 deletion syndrome is associated with a variable phenotype that includes aplasia cutis congenita (ACC) and ectrodactyly as specific features. UBA2 (ubiquitin-like modifier-activating enzyme 2) lies adjacent to the minimal deletion overlap region. We aimed to define the UBA2-related phenotypic spectrum in humans and zebrafish due to sequence variants and to establish the mechanism of disease. METHODS: Exome sequencing was used to detect UBA2 sequence variants in 16 subjects in 7 unrelated families. uba2 loss of function was modeled in zebrafish. Effects of human missense variants were assessed in zebrafish rescue experiments. RESULTS: Seven human UBA2 loss-of-function and missense sequence variants were detected. UBA2-phenotypes included ACC, ectrodactyly, neurodevelopmental abnormalities, ectodermal, skeletal, craniofacial, cardiac, renal, and genital anomalies. uba2 was expressed in zebrafish eye, brain, and pectoral fins; uba2-null fish showed deficient growth, microcephaly, microphthalmia, mandibular hypoplasia, and abnormal fins. uba2-mRNAs with human missense variants failed to rescue nullizygous zebrafish phenotypes. CONCLUSION: UBA2 variants cause a recognizable syndrome with a wide phenotypic spectrum. Our data suggest that loss of UBA2 function underlies the human UBA2 monogenic disorder and highlights the importance of SUMOylation in the development of affected tissues.

Impact of integrated translational research on clinical exome sequencing.

PURPOSE: Exome sequencing often identifies pathogenic genetic variants in patients with undiagnosed diseases. Nevertheless, frequent findings of variants of uncertain significance necessitate additional efforts to establish causality before reaching a conclusive diagnosis. To provide comprehensive genomic testing to patients with undiagnosed disease, we established an Individualized Medicine Clinic, which offered clinical exome testing and included a Translational Omics Program (TOP) that provided variant curation, research activities, or research exome sequencing. METHODS: From 2012 to 2018, 1101 unselected patients with undiagnosed diseases received exome testing. Outcomes were reviewed to assess impact of the TOP and patient characteristics on diagnostic rates through descriptive and multivariate analyses. RESULTS: The overall diagnostic yield was 24.9% (274 of 1101 patients), with 174 (15.8% of 1101) diagnosed on the basis of clinical exome sequencing alone. Four hundred twenty-three patients with nondiagnostic or without access to clinical exome sequencing were evaluated by the TOP, with 100 (9% of 1101) patients receiving a diagnosis, accounting for 36.5% of the diagnostic yield. The identification of a genetic diagnosis was influenced by the age at time of testing and the disease phenotype of the patient. CONCLUSION: Integration of translational research activities into clinical practice of a tertiary medical center can significantly increase the diagnostic yield of patients with undiagnosed disease.

Nail-patella-like renal disease masquerading as Fabry disease on kidney biopsy: a case report.

BACKGROUND: Genetic changes in the LIM homeobox transcription factor 1 beta (LMX1B) have been associated with focal segmental glomerulosclerosis (FSGS) without the extra-renal or ultrastructural manifestations of Nail-patella syndrome (NPS) known as Nail-patella-like renal disease (NPLRD). Fabry disease (FD) is an X-linked lysosomal disease caused by the deficiency of alpha-galactosidase A. The classic form of the disease is characterized by acroparesthesia, angiokeratomas, cornea verticillata, hypertrophic cardiomyopathy, strokes, and chronic kidney disease. Podocyte myelin bodies on ultrastructural examination of kidney tissue are very characteristic of FD; however some medications and other conditions may mimic this finding. CASE PRESENTATION: Here, we report on a female patient with chronic kidney disease (CKD), positive family history for kidney disease and kidney biopsy showing a FSGS lesion and presence of focal myelin figures within podocytes concerning for FD. However, genetic testing for FD was negative. After comprehensive clinical, biochemical, and genetic evaluation, including whole exome and RNA sequencing, she was ultimately diagnosed with NPLRD. CONCLUSIONS: This case illustrates the difficulties of diagnosing atypical forms of rare Mendelian kidney diseases and the role of a multidisciplinary team in an individualized medicine clinic setting in combination with state-of-the-art sequencing technologies to reach a definitive diagnosis.

De novo DDX3X missense variants in males appear viable and contribute to syndromic intellectual disability.

DDX3X (Xp11.4) encodes a DEAD-box RNA helicase that escapes X chromosome inactivation. Pathogenic variants in DDX3X have been shown to cause X-linked intellectual disability (ID) (MRX102, MIM: 300958). The phenotypes associated with DDX3X variants are heterogeneous and include brain and behavioral abnormalities, microcephaly, hypotonia, and movement disorders and/or spasticity. The majority of DDX3X variants described are de novo mutations in females with ID. In contrast, most male DDX3X variants are inherited from an unaffected mother, with one documented exception being a recently identified de novo splice site variant. It has been suggested, therefore, that DDX3X exerts its effects through haploinsufficiency in females, and that affected males carry hypomorphic alleles that retain partial function. Given the lack of male de novo DDX3X variants, loss-of-function variants in this gene are suspected to be male lethal. Through whole-exome sequencing, we identified three unrelated males with hemizygous missense DDX3X variants and ID. All three variants were confirmed by Sanger sequencing, with two established as de novo. In silico analyses were supportive of pathogenicity. We report the male phenotypes and compare them to phenotypes observed in previously reported male and female patients. In conclusion, we propose that de novo DDX3X variants are not necessarily male lethal and should be considered as a cause of syndromic ID in both males and females.

Utility of DNA, RNA, Protein, and Functional Approaches to Solve Cryptic Immunodeficiencies.

PURPOSE: We report a female infant identified by newborn screening for severe combined immunodeficiencies (NBS SCID) with T cell lymphopenia (TCL). The patient had persistently elevated alpha-fetoprotein (AFP) with IgA deficiency, and elevated IgM. Gene sequencing for a SCID panel was uninformative. We sought to determine the cause of the immunodeficiency in this infant. METHODS: We performed whole-exome sequencing (WES) on the patient and parents to identify a genetic diagnosis. Based on the WES result, we developed a novel flow cytometric panel for rapid assessment of DNA repair defects using blood samples. We also performed whole transcriptome sequencing (WTS) on fibroblast RNA from the patient and father for abnormal transcript analysis. RESULTS: WES revealed a pathogenic paternally inherited indel in ATM. We used the flow panel to assess several proteins in the DNA repair pathway in lymphocyte subsets. The patient had absent phosphorylation of ATM, resulting in absent or aberrant phosphorylation of downstream proteins, including γH2AX. However, ataxia-telangiectasia (AT) is an autosomal recessive condition, and the abnormal functional data did not correspond with a single ATM variant. WTS revealed in-frame reciprocal fusion transcripts involving ATM and SLC35F2 indicating a chromosome 11 inversion within 11q22.3, of maternal origin. Inversion breakpoints were identified within ATM intron 16 and SLC35F2 intron 7. CONCLUSIONS: We identified a novel ATM-breaking chromosome 11 inversion in trans with a pathogenic indel (compound heterozygote) resulting in non-functional ATM protein, consistent with a diagnosis of AT. Utilization of several molecular and functional assays allowed successful resolution of this case.

Extension of the mutational and clinical spectrum of SOX2 related disorders: Description of six new cases and a novel association with suprasellar teratoma.

SOX2 is a transcription factor that is essential for maintenance of pluripotency and has several conserved roles in early embryonic development. Heterozygous loss-of-function variants in SOX2 are identified in approximately 40% of all cases of bilateral anophthalmia/micropthalmia (A/M). Increasingly SOX2 mutation-positive patients without major eye findings, but with a range of other developmental disorders including autism, mild to moderate intellectual disability with or without structural brain changes, esophageal atresia, urogenital anomalies, and endocrinopathy are being reported, suggesting that the clinical phenotype associated with SOX2 loss is much broader than previously appreciated. In this report we describe six new cases, four of which carry novel pathogenic SOX2 variants. Four cases presented with bilateral anophthalmia in addition to extraocular involvement. Another individual presented with only unilateral anophthalmia. One individual did not have any eye findings but presented with a suprasellar teratoma in infancy and was found to have the recurrent c.70del20 mutation in SOX2 (c.70_89del, p.Asn24Argfs*65). This is this first time this tumor type has been reported in the context of a de novo SOX2 mutation. Notably, individuals with hypothalamic hamartomas and slow-growing hypothalamo-pituitary tumors have been reported previously, but it is still unclear how SOX2 loss contributes to their formation.

Gain-of-function mutations in KCNK3 cause a developmental disorder with sleep apnea.

Sleep apnea is a common disorder that represents a global public health burden. KCNK3 encodes TASK-1, a K+ channel implicated in the control of breathing, but its link with sleep apnea remains poorly understood. Here we describe a new developmental disorder with associated sleep apnea (developmental delay with sleep apnea, or DDSA) caused by rare de novo gain-of-function mutations in KCNK3. The mutations cluster around the 'X-gate', a gating motif that controls channel opening, and produce overactive channels that no longer respond to inhibition by G-protein-coupled receptor pathways. However, despite their defective X-gating, these mutant channels can still be inhibited by a range of known TASK channel inhibitors. These results not only highlight an important new role for TASK-1 K+ channels and their link with sleep apnea but also identify possible therapeutic strategies.

Identification of Genetic Causes of Focal Segmental Glomerulosclerosis Increases With Proper Patient Selection.

OBJECTIVE: To increase the likelihood of finding a causative genetic variant in patients with a focal segmental glomerulosclerosis (FSGS) lesion, clinical and histologic characteristics were analyzed. PATIENTS AND METHODS: Individuals 18 years and older with an FSGS lesion on kidney biopsy evaluated at Mayo Clinic from November 1, 1999, through October 31, 2019, were divided into 4 groups based on clinical and histologic characteristics: primary FSGS, secondary FSGS with known cause, secondary FSGS without known cause, and undetermined FSGS. A targeted gene panel and a customized gene panel retrieved from exome sequencing were performed. RESULTS: The overall rate of detection of a monogenic cause was 42.9% (21/49). Individuals with undetermined FSGS had the highest rate of positivity (87.5%; 7/8) followed by secondary FSGS without an identifiable cause (61.5%; 8/13) and secondary FSGS with known cause (33.3%; 5/15). Four of 5 (80%) individuals in the latter group who had positive genetic testing results also had a family history of kidney disease. Univariate analysis showed that family history of kidney disease (odds ratio [OR], 13.8; 95% CI, 3.7 to 62.4; P<.001), absence of nephrotic syndrome (OR, 8.2; 95% CI, 1.9 to 58.1; P=.004), and female sex (OR, 5.1; 95% CI, 1.5 to 19.9; P=.01) were strong predictors of finding a causative genetic variant in the entire cohort. The most common variants were in the collagen genes (52.4%; 11/21), followed by the podocyte genes (38.1%; 8/21). CONCLUSION: In adults with FSGS lesions, proper selection of patients increases the rate of positive genetic testing significantly. The majority of individuals with undetermined FSGS in whom the clinical presentation and histologic parameters are discordant had a genetic diagnosis.

Genomics Integration Into Nephrology Practice.

RATIONALE & OBJECTIVE: The etiology of kidney disease remains unknown in many individuals with chronic kidney disease (CKD). We created the Mayo Clinic Nephrology Genomics Clinic to improve our ability to integrate genomic and clinical data to identify the etiology of unexplained CKD. STUDY DESIGN: Retrospective study. SETTING & PARTICIPANTS: An essential component of our program is the Nephrology Genomics Board which consists of nephrologists, geneticists, pathologists, translational omics scientists, and trainees who interpret the patient's clinical and genetic data. Since September 2016, the Board has reviewed 163 cases (15 cystic, 100 glomerular, 6 congenital anomalies of kidney and urinary tract (CAKUT), 20 stones, 15 tubulointerstitial, and 13 other). ANALYTICAL APPROACH: Testing was performed with targeted panels, single gene analysis, or analysis of kidney-related genes from exome sequencing. Variant classification was obtained based on the 2015 American College of Medical Genetics and Genomics and the Association for Molecular Pathology guidelines. RESULTS: A definitive genetic diagnosis was achieved for 50 families (30.7%). The highest diagnostic yield was obtained in individuals with tubulointerstitial diseases (53.3%), followed by congenital anomalies of the kidney and urological tract (33.3%), glomerular (31%), cysts (26.7%), stones (25%), and others (15.4%). A further 20 (12.3%) patients had variants of interest, and variant segregation, and research activities (exome, genome, or transcriptome sequencing) are ongoing for 44 (40%) unresolved families. LIMITATIONS: Possible overestimation of diagnostic rate due to inclusion of individuals with variants with evidence of pathogenicity but classified as of uncertain significance by the clinical laboratory. CONCLUSIONS: Integration of genomic and research testing and multidisciplinary evaluation in a nephrology cohort with CKD of unknown etiology or suspected monogenic disease provided a diagnosis in a third of families. These diagnoses had prognostic implications, and often changes in management were implemented.

Haploinsufficiency as a disease mechanism in GNB1-associated neurodevelopmental disorder.

BACKGROUND: GNB1 encodes a subunit of a heterotrimeric G-protein complex that transduces intracellular signaling cascades. Disruptions to the gene have previously been shown to be embryonic lethal in knockout mice and to cause complex neurodevelopmental disorders in humans. To date, the majority of variants associated with disease in humans have been missense variants in exons 5-7. METHODS: Genetic sequencing was performed on two patients presenting with complex neurological phenotypes including intellectual disability, hypotonia, and in one patient seizures. Reported variants were assessed using RNA sequencing and functional BRET/BiFC assays. RESULTS: A splice variant reported in patient 1 was confirmed to cause usage of a cryptic splice site leading to a truncated protein product. Patient 2 was reported to have a truncating variant. BRET and BiFC assays of both patient variants confirmed both were deficient in inducing GPCR-induced G protein activation due to lack of dimer formation with the Gγ subunit. CONCLUSION: Here, we report two patients with functionally confirmed loss of function variants in GNB1 and neurodevelopmental phenotypes including intellectual disability, hypotonia, and seizures in one patient. These results suggest haploinsufficiency of GNB1 is a mechanism for neurodevelopmental disorders in humans.

A second cohort of CHD3 patients expands the molecular mechanisms known to cause Snijders Blok-Campeau syndrome.

There has been one previous report of a cohort of patients with variants in Chromodomain Helicase DNA-binding 3 (CHD3), now recognized as Snijders Blok-Campeau syndrome. However, with only three previously-reported patients with variants outside the ATPase/helicase domain, it was unclear if variants outside of this domain caused a clinically similar phenotype. We have analyzed 24 new patients with CHD3 variants, including nine outside the ATPase/helicase domain. All patients were detected with unbiased molecular genetic methods. There is not a significant difference in the clinical or facial features of patients with variants in or outside this domain. These additional patients further expand the clinical and molecular data associated with CHD3 variants. Importantly we conclude that there is not a significant difference in the phenotypic features of patients with various molecular disruptions, including whole gene deletions and duplications, and missense variants outside the ATPase/helicase domain. This data will aid both clinical geneticists and molecular geneticists in the diagnosis of this emerging syndrome.

Cushing syndrome: uncovering Carney complex due to novel PRKAR1A mutation.

Carney complex (CNC) is a rare multiple neoplasia syndrome characterized by spotty pigmentation of the skin and mucosa in association with various non-endocrine and endocrine tumors, including primary pigmented nodular adrenocortical disease (PPNAD). A 20-year-old woman was referred for suspected Cushing syndrome. She had signs of cortisol excess as well as skin lentigines on physical examination. Biochemical investigation was suggestive of corticotropin (ACTH)-independent Cushing syndrome. Unenhanced computed tomography scan of the abdomen did not reveal an obvious adrenal mass. She subsequently underwent bilateral laparoscopic adrenalectomy, and histopathology was consistent with PPNAD. Genetic testing revealed a novel frameshift pathogenic variant c.488delC/p.Thr163MetfsX2 (ClinVar Variation ID: 424516) in the PRKAR1A gene, consistent with clinical suspicion for CNC. Evaluation for other clinical features of the complex was unrevealing. We present a case of PPNAD-associated Cushing syndrome leading to the diagnosis of CNC due to a novel PRKAR1A pathogenic variant. Learning points: PPNAD should be considered in the differential for ACTH-independent Cushing syndrome, especially when adrenal imaging appears normal. The diagnosis of PPNAD should prompt screening for CNC. CNC is a rare multiple neoplasia syndrome caused by inactivating pathogenic variants in the PRKAR1A gene. Timely diagnosis of CNC and careful surveillance can help prevent potentially fatal complications of the disease.

Adrenal Cortical Carcinoma Associated With Lynch Syndrome: A Case Report and Review of Literature.

OBJECTIVE: Adrenocortical carcinoma (ACC) is a rare malignancy with poor prognosis. ACC was reported in 3.2% patients with Lynch syndrome (LS), however no particular case-detection strategies have been recommended. PARTICIPANTS: We report a case of a 65-year-old woman who was incidentally discovered with a large adrenal mass during work-up of postmenopausal uterine bleeding. She was recently diagnosed with MSH6 germline mutation after her sister presented with uterine carcinoma in the setting of LS. RESULTS: Whereas the patient was asymptomatic for overt hormonal excess, biochemical work-up confirmed glucocorticoid autonomy and androgen and estrogen excess. Urine steroid profiling was suggestive of ACC. Adrenalectomy confirmed an oncocytic ACC with focal extracapsular extension into the periadrenal adipose tissue with a Ki-67 of 15% and a peak mitotic count of 40/50 high-power fields. CONCLUSION: ACC can be the only manifestation of LS. A best case-detection approach for ACC in the asymptomatic patient with LS is unclear, however urine steroid profiling could be considered.

Shared and unique susceptibility genes in a mouse model of Graves' disease determined in BXH and CXB recombinant inbred mice.

Susceptibility genes for TSH receptor (TSHR) antibodies and hyperthyroidism can be probed in recombinant inbred (RI) mice immunized with adenovirus expressing the TSHR A-subunit. The RI set of CXB strains, derived from susceptible BALB/c and resistant C57BL/6 (B6) mice, were studied previously. High-resolution genetic maps are also available for RI BXH strains, derived from B6 and C3H/He parents. We found that C3H/He mice develop TSHR antibodies, and some animals become hyperthyroid after A-subunit immunization. In contrast, the responses of the F1 progeny of C3H/He x B6 mice, as well as most BXH RI strains, are dominated by the B6 resistance to hyperthyroidism. As in the CXB set, linkage analysis of BXH strains implicates different chromosomes (Chr) or loci in the susceptibility to induced TSHR antibodies vs. hyperthyroidism. Importantly, BXH and CXB mice share genetic loci controlling the generation of TSHR antibodies (Chr 17, major histocompatibility complex region, and Chr X) and development of hyperthyroidism (Chr 1 and 3). Moreover, some chromosomal linkages are unique to either BXH or CXB strains. An interesting candidate gene linked to thyroid-stimulating antibody generation in BXH mice is the Ig heavy chain locus, suggesting a role for particular germline region genes as precursors for these antibodies. In conclusion, our findings reinforce the importance of major histocompatibility complex region genes in controlling the generation of TSHR antibodies measured by TSH binding inhibition. Moreover, these data emphasize the value of RI strains to dissect the genetic basis for induced TSHR antibodies vs. their effects on thyroid function in Graves' disease.

Phenotypic Variability of c.436delC DCAF17 Gene Mutation in Woodhouse-Sakati Syndrome.

BACKGROUND Woodhouse-Sakati syndrome (WSS) is a rare autosomal recessive genetic condition that was first described in 1983. Since its original description, approximately 50 cases have been reported with various clinical signs and symptoms. Characteristics include progressive neurologic deterioration with extrapyramidal involvement and polyendocrinopathy most notable for hypogonadism starting in early adolescence. Clinical presentation is variable, and a subset of patients may have additional features, such as premature aging, alopecia, distinctive facial features, cognitive impairment, or deafness. CASE REPORT We illustrate the phenotypic variability of 5 patients with WSS due to the previously reported homozygous single nucleotide deletion c.436delC in the DCAF17 gene, identified in 2008. Despite identical genetic alteration, our 5 patients had various clinical features among them and compared with previously reported cases with the same pathogenic mutation. CONCLUSIONS The phenotypic variability of WSS due to c.436delC founder mutation may have a wider range than previously recognized.