Klee, Eric W.

Abstract 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, 1,152 patients have been evaluated with an overall solve rate of 14.1% 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.

Heterozygous loss-of-function (LOF) mutations in PIK3R1 (encoding phosphatidylinositol 3-kinase [PI3K] regulatory subunits) cause activated PI3Kδ syndrome 2 (APDS2), which has a similar clinical profile to APDS1, caused by heterozygous gain-of-function (GOF) mutations in PIK3CD (encoding the PI3K p110δ catalytic subunit). While several studies have established how PIK3CD GOF leads to immune dysregulation, less is known about how PIK3R1 LOF mutations alter cellular function. By studying a novel CRISPR/Cas9 mouse model and patients’ immune cells, we determined how PIK3R1 LOF alters cellular function. We observed some overlap in cellular defects in APDS1 and APDS2, including decreased intrinsic B cell class switching and defective Tfh cell function. However, we also identified unique APDS2 phenotypes including defective expansion and affinity maturation of Pik3r1 LOF B cells following immunization, and decreased survival of Pik3r1 LOF pups. Further, we observed clear differences in the way Pik3r1 LOF and Pik3cd GOF altered signaling. Together these results demonstrate crucial differences between these two genetic etiologies.

Abstract 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.

The chromokinesin KIF22 generates forces that contribute to mitotic chromosome congression and alignment. Mutations in the α2 helix of the motor domain of KIF22 have been identified in patients with abnormal skeletal development, and we report the identification of a patient with a novel mutation in the KIF22 tail. We demonstrate that pathogenic mutations do not result in a loss of KIF22’s functions in early mitosis. Instead, mutations disrupt chromosome segregation in anaphase, resulting in reduced proliferation, abnormal daughter cell nuclear morphology, and, in a subset of cells, cytokinesis failure. This phenotype could be explained by a failure of KIF22 to inactivate in anaphase. Consistent with this model, constitutive activation of the motor via a known site of phosphoregulation in the tail phenocopied the effects of pathogenic mutations. These results suggest that the motor domain α2 helix may be an important site for regulation of KIF22 activity at the metaphase to anaphase transition. In support of this conclusion, mimicking phosphorylation of α2 helix residue T158 also prevents inactivation of KIF22 in anaphase. These findings demonstrate the importance of both the head and tail of the motor in regulating the activity of KIF22 and offer insight into the cellular consequences of preventing KIF22 inactivation and disrupting force balance in anaphase.

Abstract Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype with low overall survival rates and high molecular heterogeneity; therefore, few targeted therapies are available. The luminal androgen receptor (LAR) is the most consistently identified TNBC subtype, but the clinical utility has yet to be established. Here, we constructed a novel genomic classifier, LAR-Sig, that distinguishes the LAR subtype from other TNBC subtypes and provide evidence that it is a clinically distinct disease. A meta-analysis of seven TNBC datasets (n = 1086 samples) from neoadjuvant clinical trials demonstrated that LAR patients have significantly reduced response (pCR) rates than non-LAR TNBC patients (odds ratio = 2.11, 95% CI: 1.33, 2.89). Moreover, deconvolution of the tumor microenvironment confirmed an enrichment of luminal epithelium corresponding with a decrease in basal and myoepithelium in LAR TNBC tumors. Increased immunosuppression in LAR patients may lead to a decreased presence of cycling T-cells and plasma cells. While, an increased presence of myofibroblast-like cancer-associated cells may impede drug delivery and treatment. In summary, the lower levels of tumor infiltrating lymphocytes (TILs), reduced immune activity in the micro-environment, and lower pCR rates after NAC, suggest that new therapeutic strategies for the LAR TNBC subtype need to be developed.

AbstractBackgroundAchalasia‐addisonianism‐alacrima syndrome, frequently referred to as Allgrove syndrome or Triple A syndrome, is a multisystem disorder resulting from homozygous or compound heterozygous pathogenic variants in the gene encoding aladin (AAAS). Aladin is a member of the WD‐repeat family of proteins and is a component of the nuclear pore complex. It is thought to be involved in nuclear import and export of molecules. Here, we describe an individual with a paternally inherited truncating variant and a maternally inherited, novel missense variant in AAAS presenting with alacrima, achalasia, anejaculation, optic atrophy, muscle weakness, dysarthria, and autonomic dysfunction.MethodsWhole‐exome sequencing was performed in the proband, sister, and parents. Variants were confirmed by Sanger sequencing. The localization of aladin to the nuclear pore was assessed in cells expressing the patient variant.ResultsFunctional testing of the maternally inherited variant, p.(Arg270Pro), demonstrated decreased localization of aladin to the nuclear pore in cells expressing the variant, indicating a deleterious effect. Follow‐up testing in the proband's affected sister revealed that she also inherited the biallelic AAAS variants.ConclusionsReview of the patient's clinical, pathological, and genetic findings resulted in a diagnosis of Triple A syndrome.

Abstract The pre‐mRNA‐processing factor 8, encoded by PRPF8, is a scaffolding component of a spliceosome complex involved in the removal of introns from mRNA precursors. Previously, heterozygous pathogenic variants in PRPF8 have been associated with autosomal dominant retinitis pigmentosa. More recently, PRPF8 was suggested as a candidate gene for autism spectrum disorder due to the enrichment of sequence variants in this gene in individuals with neurodevelopmental disorders. We report 14 individuals with various forms of neurodevelopmental conditions, found to have heterozygous, predominantly de novo, missense, and loss‐of‐function variants in PRPF8 . These individuals have clinical features that may represent a new neurodevelopmental syndrome.

Background High-impact pathogenic variants in more than a thousand genes are involved in Mendelian forms of neurodevelopmental disorders (NDD). Methods This study describes the molecular and clinical characterisation of 28 probands with NDD harbouring heterozygous AGO1 coding variants, occurring de novo for all those whose transmission could have been verified (26/28). Results A total of 15 unique variants leading to amino acid changes or deletions were identified: 12 missense variants, two in-frame deletions of one codon, and one canonical splice variant leading to a deletion of two amino acid residues. Recurrently identified variants were present in several unrelated individuals: p.(Phe180del), p.(Leu190Pro), p.(Leu190Arg), p.(Gly199Ser), p.(Val254Ile) and p.(Glu376del). AGO1 encodes the Argonaute 1 protein, which functions in gene-silencing pathways mediated by small non-coding RNAs. Three-dimensional protein structure predictions suggest that these variants might alter the flexibility of the AGO1 linker domains, which likely would impair its function in mRNA processing. Affected individuals present with intellectual disability of varying severity, as well as speech and motor delay, autistic behaviour and additional behavioural manifestations. Conclusion Our study establishes that de novo coding variants in AGO1 are involved in a novel monogenic form of NDD, highly similar to the recently reported AGO2 -related NDD.