Chronic kidney disease (CKD) represents a major health burden. Its central feature of renal fibrosis is not well understood. By exome sequencing, we identified mutations in FAN1 as a cause of ...karyomegalic interstitial nephritis (KIN), a disorder that serves as a model for renal fibrosis. Renal histology in KIN is indistinguishable from that of nephronophthisis, except for the presence of karyomegaly. The FAN1 protein has nuclease activity and acts in DNA interstrand cross-link (ICL) repair within the Fanconi anemia DNA damage response (DDR) pathway. We show that cells from individuals with FAN1 mutations have sensitivity to the ICL-inducing agent mitomycin C but do not exhibit chromosome breakage or cell cycle arrest after diepoxybutane treatment, unlike cells from individuals with Fanconi anemia. We complemented ICL sensitivity with wild-type FAN1 but not with cDNA having mutations found in individuals with KIN. Depletion of fan1 in zebrafish caused increased DDR, apoptosis and kidney cysts. Our findings implicate susceptibility to environmental genotoxins and inadequate DNA repair as novel mechanisms contributing to renal fibrosis and CKD.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Primary ciliary dyskinesia (PCD) is caused when defects of motile cilia lead to chronic airway infections, male infertility, and situs abnormalities. Multiple causative PCD mutations account for only ...65% of cases, suggesting that many genes essential for cilia function remain to be discovered. By using zebrafish morpholino knockdown of PCD candidate genes as an in vivo screening platform, we identified c21orf59, ccdc65, and c15orf26 as critical for cilia motility. c21orf59 and c15orf26 knockdown in zebrafish and planaria blocked outer dynein arm assembly, and ccdc65 knockdown altered cilia beat pattern. Biochemical analysis in Chlamydomonas revealed that the C21orf59 ortholog FBB18 is a flagellar matrix protein that accumulates specifically when cilia motility is impaired. The Chlamydomonas ida6 mutant identifies CCDC65/FAP250 as an essential component of the nexin-dynein regulatory complex. Analysis of 295 individuals with PCD identified recessive truncating mutations of C21orf59 in four families and CCDC65 in two families. Similar to findings in zebrafish and planaria, mutations in C21orf59 caused loss of both outer and inner dynein arm components. Our results characterize two genes associated with PCD-causing mutations and elucidate two distinct mechanisms critical for motile cilia function: dynein arm assembly for C21orf59 and assembly of the nexin-dynein regulatory complex for CCDC65.
Rare single-gene disorders cause chronic disease. However, half of the 6000 recessive single gene causes of disease are still unknown. Because recessive disease genes can illuminate, at least in ...part, disease pathomechanism, their identification offers direct opportunities for improved clinical management and potentially treatment. Rare diseases comprise the majority of chronic kidney disease (CKD) in children but are notoriously difficult to diagnose. Whole-exome resequencing facilitates identification of recessive disease genes. However, its utility is impeded by the large number of genetic variants detected. We here overcome this limitation by combining homozygosity mapping with whole-exome resequencing in 10 sib pairs with a nephronophthisis-related ciliopathy, which represents the most frequent genetic cause of CKD in the first three decades of life. In 7 of 10 sibships with a histologic or ultrasonographic diagnosis of nephronophthisis-related ciliopathy, we detect the causative gene. In six sibships, we identify mutations of known nephronophthisis-related ciliopathy genes, while in two additional sibships we found mutations in the known CKD-causing genes SLC4A1 and AGXT as phenocopies of nephronophthisis-related ciliopathy. Thus, whole-exome resequencing establishes an efficient, noninvasive approach towards early detection and causation-based diagnosis of rare kidney diseases. This approach can be extended to other rare recessive disorders, thereby providing accurate diagnosis and facilitating the study of disease mechanisms.
Despite the OlympiA trial demonstrating that early-stage, high-risk, HER2- germline BRCA1 and BRCA2 mutation (gBRCAm) positive breast cancer patients can benefit from PARPi in the adjuvant setting, ...the gBRCA testing rate in early-stage HR+/HER2− patients remains suboptimal compared to that in early-stage TNBC patients. To better understand the perceived barriers associated with gBRCA testing in HR+/HER2− disease, a quantitative survey was conducted across stakeholders (n = 430) including medical oncologists, surgeons, nurses, physician assistants, payers, and patients. This study revealed that while payers claim to cover gBRCA testing, poor clinician documentation and overutilization are key challenges. Therefore, payers place utilization management controls on gBRCA testing due to their impression that clinicians overtest. These controls have led to healthcare professionals experiencing payer pushback in the form of reimbursement limitations and denials. The perceived challenges to gBRCA testing stem from the lack of consensus dictating which patients are high risk and should be tested. While payers define high risk based on the CPS + EG score from the OlympiA trial, HCPs adopt a broader definition including genomic risk scores, lymph node involvement, and tumor grade and size. A dialogue to harmonize risk classification and testing eligibility across stakeholders is critical to address this disconnect and increase gBRCA testing in appropriate patients.
1041 Background: Biomarkers play an essential role to inform treatment decisions in mBC. In hormone receptor positive/HER2 negative (HR+/HER2-) mBC, PIK3CA/AKT1/PTEN alterations are now actionable as ...pt selection biomarkers with the FDA’s Nov 2023 approval of the pan-AKT inhibitor capivasertib. Here we investigate the testing patterns and prevalence of PIK3CA/AKT1/PTEN alterations in mBC. Methods: This retrospective cohort study used two partially overlapping nationwide de-identified databases: the Flatiron Health electronic health record-derived database (FH DB) to assess next-generation sequencing (NGS) testing rates, and the Flatiron Health-Foundation Medicine clinico-genomic database (FH-FMI CGDB) to assess prevalence among tested. Pts were included if they were ≥18 years old and diagnosed with HR+/HER2- mBC from 1/1/17—6/30/22. Prevalence was assessed using any sample type or timing of testing post-mBC diagnosis and defined as pathogenic/likely pathogenic alterations as captured in FMI NGS testing. All analyses were summarized using descriptive statistics. Results: 8,049 (FH DB) and 2,912 (FH-FMI CGDB) pts were eligible. Overall, 37% of pts with mBC received NGS testing (3,002/8,049). Among pts in FH-FMI CGDB, 98% had PIK3CA tested (2,857/2,912) and 97% also had AKT1/PTEN tested (2,838/2,912) in the metastatic setting. The median time from mBC diagnosis to initial test was 148 days for PIK3CA and 188 days for AKT1/PTEN, and 92-93% of testing occurred after the start of first-line (1L) therapy (Table). 82% of PIK3CA testing was on tissue and 37% on liquid biopsy, with 19% of pts receiving both tissue and liquid biopsy. 77% of AKT1/PTEN testing was on tissue and 28% on liquid biopsy, with 5% of pts receiving both tissue and liquid biopsy. At least one PIK3CA/ AKT1/ PTEN alteration was detected in 55% of the pts (1,557/2857) tested for any of the markers, regardless of sample type. Conclusions: <40% of pts with mBC received NGS testing from 2017-2021, suggesting opportunities for improving pts’ access to testing. Coupled with the high rate of PIK3CA/AKT1/PTEN alterations, NGS testing should be incorporated in routine clinical practice to identify optimal treatments. Table: see text
e15033 Background: In the Phase 3 CAPItello-291 trial (NCT04305496), the addition of capivasertib (a potent, selective pan-AKT inhibitor) to fulvestrant significantly improved progression-free ...survival in patients with HR+/HER2− advanced breast cancer, especially in those with PIK3CA/AKT1/PTEN-altered tumors identified using tissue biopsy-based FoundationOneCDx. Given the growing utility of circulating tumor DNA (ctDNA) and interest in liquid biopsy-based tests to detect PIK3CA/AKT1/PTEN alterations, we performed a retrospective analytical comparison of FoundationOneCDx and blood-based FoundationOneLiquidCDx NGS data. Methods: We utilized the FoundationCORE database of breast cancer cases profiled during routine clinical care with FoundationOneCDx and/or FoundationOneLiquidCDx (covering the same genomic regions). Analytical comparison of all pathogenic PIK3CA/AKT1/PTEN alterations as well as alterations defined in the CAPItello-291 protocol (CAPItello-291 alterations) was performed in paired data from cases with both tissue and liquid biopsies, sampled within 90 days of each other. Subgroup analysis was conducted based on ctDNA tumor fraction (TF). Data from liquid biopsies were analyzed for co-occurring mutations. Results: CAPItello-291 alterations were detected in 44.9% of 35,730 tissue biopsies and 34.3% of 7056 liquid biopsies profiled from Jan 2017–Jun 2023. In paired NGS data (n=289), the overall positive percent agreement (PPA) for CAPItello-291 alterations across ctDNA TF subgroups was: TF ≥10% (n=99): 92.5%; TF 1–10% (n=66): 97.1%; TF <1% (n=124): 33.9%. The PPA for all PIK3CA/AKT1/PTEN short variants (base substitutions, indels) analyzed was: ctDNA TF ≥10%: PIK3CA, 93.9%; AKT1, 100%; PTEN, 100%, and ctDNA TF 1–10%: PIK3CA, 96.3%; AKT1, 100%; PTEN, 100%. For PTEN biallelic deletions, the PPA was 50% (2/4) in cases with ctDNA TF ≥10%. In liquid biopsies with CAPItello-291 alterations (n=2421), the most frequent co-occurring mutations were in TP53 (49.9%), ESR1 (31.2%), CDH1 (23.7%), NF1 (13.0%), RB1 (12.8%), CHEK2 (12.4%), ATM (11.9%), FGF3 (11.4%), FGF19 (11.0%), and FGF4 (10.5%). Mutations in BRCA1 (2.4%), BRCA2 (4.8%), and PALB2 (1.3%) were also observed. Conclusions: PPA between liquid and tissue biopsy NGS data for the detection of PIK3CA/AKT1/PTEN short variants was high in cases with ctDNA TF ≥1%. Liquid biopsies offer a minimally invasive approach for the detection of some CAPItello-291 alterations. However, limitations posed by ctDNA TF <1% and sample and variant types should be considered. Together, these data can help clinicians make informed decisions regarding suitable diagnostic tests to determine patient eligibility for breast cancer therapies.
Tyrosinase (TYR) is a multifunctional copper-containing glycoenzyme (∼80
kDa), which plays a key role in the rate-limiting steps of the melanin biosynthetic pathway. This membrane-bound protein, ...possibly evolved by the fusion of two different copper-binding proteins, is mainly expressed in epidermal, ocular and follicular melanocytes. In the melanocytes, TYR functions as an integrated unit with other TYR-related proteins (TYRP1, TYRP2), lysosome-associated membrane protein 1 (LAMP1) and melanocyte-stimulating hormone receptors; thus forming a melanogenic complex. Mutations in the TYR gene (
TYR, 11q14-21, MIM 606933) cause oculocutaneous albinism type 1 (OCA1, MIM 203100), a developmental disorder having an autosomal recessive mode of inheritance. In addition,
TYR can act as a modifier locus for primary congenital glaucoma (PCG) and it also contributes significantly in the eye developmental process. Expression of
TYR during neuroblast division helps in later pathfinding by retinal ganglion cells from retina to the dorsal lateral geniculate nucleus. However, mutation screening of
TYR is complicated by the presence of a pseudogene–TYR like segment (
TYRL, 11p11.2, MIM 191270), sharing ∼98% sequence identity with the 3′ region of
TYR. Thus, in absence of a full-proof strategy, any nucleotide variants identified in the 3′ region of
TYR could actually be present in
TYRL. Interestingly, despite extensive search, the second
TYR mutation in 15% of the OCA1 cases remains unidentified. Several possible locations of these “uncharacterized mutations” (UCMs) have been speculated so far. Based on the structure of
TYR gene, its sequence context and some experimental evidences, we propose two additional possibilities, which on further investigations might shed light on the molecular basis of UCMs in
TYR of OCA1 patients; (i) partial deletion of the exons 4 and 5 region of
TYR that is homologous with
TYRL and (ii) variations in the polymorphic GA complex repeat located between distal and proximal elements of the human
TYR promoter that can modulate the expression of the gene leading to disease pathogenesis.
Nephronophthisis-related ciliopathies (NPHP-RC) are autosomal-recessive cystic kidney diseases. More than 13 genes are implicated in its pathogenesis to date, accounting for only 40 % of all cases. ...High-throughput mutation screenings of large patient cohorts represent a powerful tool for diagnostics and identification of novel
NPHP
genes. We here performed a new high-throughput mutation analysis method to study 13 established
NPHP
genes (
NPHP1
–
NPHP13
) in a worldwide cohort of 1,056 patients diagnosed with NPHP-RC. We first applied multiplexed PCR-based amplification using Fluidigm Access-Array™ technology followed by barcoding and next-generation resequencing on an Illumina platform. As a result, we established the molecular diagnosis in 127/1,056 independent individuals (12.0 %) and identified a single heterozygous truncating mutation in an additional 31 individuals (2.9 %). Altogether, we detected 159 different mutations in 11 out of 13 different
NPHP
genes, 99 of which were novel. Phenotypically most remarkable were two patients with truncating mutations in
INVS/NPHP2
who did not present as infants and did not exhibit extrarenal manifestations. In addition, we present the first case of Caroli disease due to mutations in
WDR19/NPHP13
and the second case ever with a recessive mutation in
GLIS2/NPHP7
. This study represents the most comprehensive mutation analysis in NPHP-RC patients, identifying the largest number of novel mutations in a single study worldwide.
Enlargement of kidney tubules is a common feature of multiple cystic kidney diseases in humans and mice. However, while some of these pathologies are characterized by cyst expansion and organ ...enlargement, in others, progressive interstitial fibrosis and kidney atrophy prevail. The Kif3a knockout mouse is an established non-orthologous mouse model of cystic kidney disease. Conditional inactivation of Kif3a in kidney tubular cells results in loss of primary cilia and rapid cyst growth. Conversely, loss of function of the gene GLIS2/NPHP7 causes progressive kidney atrophy, interstitial inflammatory infiltration, and fibrosis. Kif3a null tubular cells have unrestrained proliferation and reduced stabilization of p53 resulting in a loss of cell cycle arrest in the presence of DNA damage. In contrast, loss of Glis2 is associated with activation of checkpoint kinase 1, stabilization of p53, and induction of cell senescence. Interestingly, the cystic phenotype of Kif3a knockout mice is partially rescued by genetic ablation of Glis2 and pharmacological stabilization of p53. Thus, Kif3a is required for cell cycle regulation and the DNA damage response, whereas cell senescence is significantly enhanced in Glis2 null cells. Hence, cell senescence is a central feature in nephronophthisis type 7 and Kif3a is unexpectedly required for efficient DNA damage response and cell cycle arrest.
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