Certain heterozygous variants in the kinase domain of the serine/threonine kinase NEK8 can cause an autosomal dominant form of polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) resulting from pathogenic variants in PKD1 and PKD2 is the most common form of PKD, but other genetic causes tied to primary cilia function have been identified. Biallelic pathogenic variants in the serine/threonine kinase NEK8 cause a syndromic ciliopathy with extra-kidney manifestations. Here we identify NEK8 as a disease gene for ADPKD in 12 families. Clinical evaluation was combined with functional studies using fibroblasts and tubuloids from affected individuals. Nek8 knockout mouse kidney epithelial (IMCD3) cells transfected with wild type or variant NEK8 were further used to study ciliogenesis, ciliary trafficking, kinase function, and DNA damage responses. Twenty-one affected monoallelic individuals uniformly exhibited cystic kidney disease (mostly neonatal) without consistent extra-kidney manifestations. Recurrent de novo mutations of the NEK8 missense variant p.Arg45Trp, including mosaicism, were seen in ten families. Missense variants elsewhere within the kinase domain (p.Ile150Met and p.Lys157Gln) were also identified. Functional studies demonstrated normal localization of the NEK8 protein to the proximal cilium and no consistent cilia formation defects in patient-derived cells. NEK8-wild type protein and all variant forms of the protein expressed in Nek8 knockout IMCD3 cells were localized to cilia and supported ciliogenesis. However, Nek8 knockout IMCD3 cells expressing NEK8-p.Arg45Trp and NEK8-p.Lys157Gln showed significantly decreased polycystin-2 but normal ANKS6 localization in cilia. Moreover, p.Arg45Trp NEK8 exhibited reduced kinase activity in vitro. In patient derived tubuloids and IMCD3 cells expressing NEK8-p.Arg45Trp, DNA damage signaling was increased compared to healthy passage-matched controls. Thus, we propose a dominant-negative effect for specific heterozygous missense variants in the NEK8 kinase domain as a new cause of PKD.

Genetics-first approach improves diagnostics of ESKD patients <50 years old.

BACKGROUND: Often only chronic kidney disease (CKD) patients with high likelihood of genetic disease are offered genetic testing. Early genetic testing could obviate the need for kidney biopsies, allowing for adequate prognostication and treatment. To test the viability of a 'genetics-first' approach for CKD, we performed genetic testing in a group of kidney transplant recipients aged <50 years, irrespective of cause of transplant. METHODS: From a cohort of 273 transplant patients, we selected 110 that were in care in the University Medical Center Utrecht, had DNA available and were without clear-cut non-genetic disease. Forty patients had been diagnosed with a genetic disease prior to enrollment; in 70 patients, we performed a whole-exome sequencing-based 379 gene panel analysis. RESULTS: Genetic analysis yielded a diagnosis in 51%. Extrapolated to the 273 patient cohort, who did not all fit the inclusion criteria, the diagnostic yield was still 21%. Retrospectively, in 43% of biopsied patients, the kidney biopsy would not have had added diagnostic value if genetic testing had been performed as a first-tier diagnostic. CONCLUSIONS: The burden of monogenic disease in transplant patients with end-stage kidney disease (ESKD) of any cause prior to the age of 50 years is between 21% and 51%. Early genetic testing can provide a non-invasive diagnostic, impacting prognostication and treatment, and obviating the need for an invasive biopsy. We conclude that in patients who expect to develop ESKD prior to the age of 50 years, genetic testing should be considered as first mode of diagnostics.

FOXL2 and TERT promoter mutation detection in circulating tumor DNA of adult granulosa cell tumors as biomarker for disease monitoring.

OBJECTIVE: Adult granulosa cell tumors (aGCTs) represent a rare, hormonally active subtype of ovarian cancer that has a tendency to relapse late and repeatedly. Current serum hormone markers are inaccurate in reflecting tumor burden in a subset of aGCT patients, indicating the need for a novel biomarker. We investigated the presence of circulating tumor DNA (ctDNA) harboring a FOXL2 or TERT promoter mutation in serial plasma samples of aGCT patients to determine its clinical value for monitoring disease. METHODS: In a national multicenter study, plasma samples (n = 110) were prospectively collected from 21 patients with primary (n = 3) or recurrent (n = 18) aGCT harboring a FOXL2 402C > G and/or TERT (C228T or C250T) promoter mutation. Circulating cell-free DNA was extracted and assessed for ctDNA containing one of either mutations using droplet digital PCR (ddPCR). Fractional abundance of FOXL2 mutant and TERT mutant ctDNA was correlated with clinical parameters. RESULTS: FOXL2 mutant ctDNA was found in plasma of 11 out of 14 patients (78.6%) with aGCT with a confirmed FOXL2 mutation. TERT C228T or TERT C250T mutant ctDNA was detected in plasma of 4 of 10 (40%) and 1 of 2 patients, respectively. Both FOXL2 mutant ctDNA and TERT promoter mutant ctDNA levels correlated with disease progression and treatment response in the majority of patients. CONCLUSIONS: FOXL2 mutant ctDNA was present in the majority of aGCT patients and TERT promoter mutant ctDNA has been identified in a smaller subset of patients. Both FOXL2 and TERT mutant ctDNA detection may have clinical value in disease monitoring.

NPHP1 (Nephrocystin-1) Gene Deletions Cause Adult-Onset ESRD.

Background Nephronophthisis (NPH) is the most prevalent genetic cause for ESRD in children. However, little is known about the prevalence of NPH in adult-onset ESRD. Homozygous full gene deletions of the NPHP1 gene encoding nephrocystin-1 are a prominent cause of NPH. We determined the prevalence of NPH in adults by assessing homozygous NPHP1 full gene deletions in adult-onset ESRD.Methods Adult renal transplant recipients from five cohorts of the International Genetics and Translational Research in Transplantation Network (iGeneTRAiN) underwent single-nucleotide polymorphism genotyping. After quality control, we determined autosomal copy number variants (such as deletions) on the basis of median log2 ratios and B-allele frequency patterns. The findings were independently validated in one cohort. Patients were included in the analysis if they had adult-onset ESRD, defined as start of RRT at ≥18 years old.Results We included 5606 patients with adult-onset ESRD; 26 (0.5%) showed homozygous NPHP1 deletions. No donor controls showed homozygosity for this deletion. Median age at ESRD onset was 30 (range, 18-61) years old for patients with NPH, with 54% of patients age ≥30 years old. Notably, only three (12%) patients were phenotypically classified as having NPH, whereas most patients were defined as having CKD with unknown etiology (n=11; 42%).Conclusions Considering that other mutation types in NPHP1 or mutations in other NPH-causing genes were not analyzed, NPH is a relatively frequent monogenic cause of adult-onset ESRD. Because 88% of patients had not been clinically diagnosed with NPH, wider application of genetic testing in adult-onset ESRD may be warranted.

Markers of hydration status in a 3-day trail running event.

OBJECTIVE: To examine the relationships between changes in static prestage and poststage measures of commonly used hematological and urinary markers of hydration status and body mass (BM) in participants in a 3-day trail run. DESIGN: Descriptive field study. SETTING: Three Cranes Challenge trail run, South Africa. PARTICIPANTS: Twenty (6 men and 14 women) amateur runners. INTERVENTIONS: In stage 1 (S1), 29.3 km and 37.9 km in stage 2 (S2), and 27.8 km in stage 3 (S3). MAIN OUTCOME MEASURES: Prestage and poststage individual changes in serum osmolality (S osm), serum sodium (s[Na]), plasma volume (PV), urine osmolality (U osm), urine specific gravity (U sg), and BM. RESULTS: Consistently, mild environmental conditions were experienced on the 3 days of the race (ambient temperature range, 11.5-22.8°C). Mean S osm increased by 5 ± 6, 7 ± 9, and 3 ± 4 mOsm/kg during S1, S2, and S3, respectively, and returned to baseline pre-S2 and pre-S3. The correlation between individual prestage and poststage changes in S osm, Uosm, and U sg (n = 60) were nonsignificant (P > 0.05; r = 0.0047, r = 0.0074). There was a significant, but relatively low correlation between changes in S osm and percentage reduction in BM (r = 0.35; P < 0.01) and prechange and postchange in s[Na] (r = 0.45; P < 0.001). CONCLUSIONS: Serum osmality values confirm appropriate interstage rehydration. Changes in U osm, U sg, BM, s[Na+], and PV are not closely related to changes in S osm as markers of hydration assessment in multiday events in which single static measures of hydration status are required. These measures of hydration station status are therefore not recommended in this field setting.

Intestinal temperature, heart rate, and hydration status in multiday trail runners.

OBJECTIVE: To assess heart rate (HR), intestinal body temperature (T(intest)), and hydration status changes and relationships in 12 participants in a 3-day trail run. DESIGN: Descriptive field study. SETTING: : Three Cranes Challenge trail run, in Karkloof, KwaZulu-Natal, South Africa. PARTICIPANTS: Twelve (5 men and 7 women) amateur runners. INTERVENTIONS: Trail run of 95 km divided into 3 stages: elevation gains on the 3 days, 1020, 1226, and 680 m, respectively. MAIN OUTCOME MEASURES: Changes in HR, T(intest), serum osmolality, and body mass. RESULTS: Environmental conditions were consistently mild (ambient temperature range, 11.5-22.8°C; maximum relative humidity range, 95%-97%), average running speed varied from 9.00 to 5.14 minutes/km, and distance covered in the 3 stages ranged from 32 (stages 1 and 3) to 40 km (stage 2). Mean HR ranged from 134 to 171 beats per minute in the 12 athletes during the trail events and averaged at 150 beats per minute, whereas T(intest) ranged between 36.1 and 40.2°C. The correlation between maximum T(intest) and percent age-predicted maximum HR (n = 12) was significant (R = 0.58; P < 0.05), whereas the correlation between maximum T(intest) and serum osmolality or body mass did not reach significance (R = 0.16, 0.13; P > 0.05). CONCLUSIONS: This study provides evidence in support of the contention that maximum T(intest) is more closely related to metabolic rate during trail running than percent dehydration. The findings do not support an increase in core body temperature with a change in serum osmolality or body mass.

Upper respiratory tract infection symptoms in ultramarathon runners not related to immunoglobulin status.

OBJECTIVE: To determine the association between variation in exercise load, immunoglobulin (Ig) status, and self-reported symptoms of upper respiratory tract infection (URTI) in ultramarathon runners. DESIGN: Longitudinal observational field study. SETTING: Four weeks before and 2 weeks after an 86.5-km Comrades Marathon, South Africa. PARTICIPANTS: Fourteen randomly selected, amateur, male, ultramarathon runners. MAIN OUTCOME MEASURES: Daily record of training and URTI symptom incidence. Salivary IgA and IgM at 28 days, 14 days, and 1 day (01PRE) before the race, immediately post race (IPR) and 1 (01 PR), 3, and 14 days post race. Serum cortisol, IgG, IgM, and IgA concentrations at 01PRE, IPR, and 01PR. RESULTS: Mean weekly training distance varied from 89.4 kilometers per week (28.9 kilometers per week) to 4.2 kilometers per week (6.7 kilometers per week). Absolute and relative mucosal IgA and IgM concentrations were unaffected by pre-race taper in training volume (P > 0.05). IgA and IgM secretion rates decreased post race (P = 0.018; 0.008), returning to baseline by 01PR. Blood leukocyte, serum cortisol, and serum IgG concentrations increased at IPR (P < 0.001, <0001) and 01PR (P = 0.009), respectively. Upper respiratory tract infection symptom incidence was highest at 28PRE and 7 to 14 days post race but not related to salivary IgA and IgM secretion rates. Eight subjects (57%) who reported URTI symptoms pre race also reported these during days 7 to 14 post race. CONCLUSIONS: Upper respiratory tract infection symptom incidence was not associated with secretory Ig concentrations. Reactivation of pre-race viruses during the 2 weeks post race and exercise-induced inflammatory response are proposed as causes of the elevated URTI incidence at 28PRE and 7 to 14 days post race.

Phadiatop testing in assessing predisposition to respiratory tract symptoms of allergic origin in athletes.

OBJECTIVES: To validate the use of the Phadiatop test as a predictor of allergy-associated respiratory tract symptoms (RTS) in trail runners. METHODS: The incidence of self-reported RTS was documented in 16 runners for 31 days and related to the Phadiatop status and circulating markers of allergic responses (changes in concentrations of serum IgE (sIgE), differential leucocyte counts) at 8 time points before, during and after a 3-day 95 km trail run. RESULTS: Twelve (75%) athletes, of whom 7 (58%) were Phadiatoppositive, presented with post-race RTS. A peak sIgE concentration >100 IU/ml accompanied RTS in only 4 (57%) of the symptomatic Phadiatop-positive subjects. There was no significant difference between the eosinophil and basophil concentrations of the positive and negative groups (p>0.05). One Phadiatop-negative subject presented with RTS as well as a peak sIgE concentration >100 IU/ml. CONCLUSION: The Phadiatop assay does not accurately predict the development of post-exercise RTS of allergic origin in trail runners.

A mathematical model to describe fat oxidation kinetics during graded exercise.

PURPOSE: The purpose of this study was to develop a mathematical model (sine model, SIN) to describe fat oxidation kinetics as a function of the relative exercise intensity [% of maximal oxygen uptake (%VO2max)] during graded exercise and to determine the exercise intensity (Fatmax) that elicits maximal fat oxidation (MFO) and the intensity at which the fat oxidation becomes negligible (Fatmin). This model included three independent variables (dilatation, symmetry, and translation) that incorporated primary expected modulations of the curve because of training level or body composition. METHODS: Thirty-two healthy volunteers (17 women and 15 men) performed a graded exercise test on a cycle ergometer, with 3-min stages and 20-W increments. Substrate oxidation rates were determined using indirect calorimetry. SIN was compared with measured values (MV) and with other methods currently used [i.e., the RER method (MRER) and third polynomial curves (P3)]. RESULTS: There was no significant difference in the fitting accuracy between SIN and P3 (P = 0.157), whereas MRER was less precise than SIN (P < 0.001). Fatmax (44 +/- 10% VO2max) and MFO (0.37 +/- 0.16 g x min(-1)) determined using SIN were significantly correlated with MV, P3, and MRER (P < 0.001). The variable of dilatation was correlated with Fatmax, Fatmin, and MFO (r = 0.79, r = 0.67, and r = 0.60, respectively, P < 0.001). CONCLUSIONS: The SIN model presents the same precision as other methods currently used in the determination of Fatmax and MFO but in addition allows calculation of Fatmin. Moreover, the three independent variables are directly related to the main expected modulations of the fat oxidation curve. SIN, therefore, seems to be an appropriate tool in analyzing fat oxidation kinetics obtained during graded exercise.

A novel splicing mutation in KCNQ2 in a multigenerational family with BFNC followed for 25 years.

PURPOSE: A large multigenerational family with benign familial neonatal convulsions (BFNC) was revisited to identify the disease-causing mutation and to assess long-term outcome. METHODS: We supplemented the original data with recent clinical and neurophysiologic data on patients and first-degree relatives, including information on seizure recurrence. We conducted linkage analysis at the EBN1 and EBN2 loci, followed by mutation analysis of KCNQ2. We evaluated the qualitative effect of the KCNQ2 mutation at the messenger RNA (mRNA) level by using reverse-transcribed total RNA isolated from leukocytes. RESULTS: Thirteen relatives had a history of neonatal convulsions, 11 of whom showed remission within 2 months. One patient showed an atypical course of neonatal convulsions, developing photosensitive myoclonic epilepsy at age 13 years. We found suggestive linkage of the BFNC phenotype to the 20q13-EBN1 locus (lod score, 2.03) and an intronic mutation IVS14-6 C>A in KCNQ2 segregating with the trait in all affected members, but absent in 100 unrelated control subjects. This mutation creates a new, preferentially used, splice site. Alternative splicing adds 4 nt containing a premature stop codon to the transcript, resulting in a truncated protein after position R588. CONCLUSIONS: We detected and characterized a novel splicing mutation in the brain-specific KCNQ2 gene by using easily accessible blood leukocytes. Aberrant splicing cosegregates with BFNC but not with photosensitivity.

Nutritional aspects in ultra-endurance exercise.

PURPOSE OF REVIEW: Despite much current debate regarding central and peripheral neural mechanisms which may be responsible for the onset of fatigue during prolonged exercise, maintenance of nutritional and hydration status remains critical for successful participation in ultra-endurance exercise. This review focuses on substrate and fluid homeostasis during ultra-endurance exercise and the use of nutritional supplementation both as ergogenic aid and to attenuate exercise-induced immunosuppression. RECENT FINDINGS: Current evidence continues to support mandatory high carbohydrate intakes (1). before the event to maximize muscle glycogen stores, (2). during the event to prevent hypoglycaemia and (3). after the event to optimize post-event repletion of endogenous carbohydrate stores. No consistent performance benefit has yet been shown following a high-fat diet. Greater utilization of intrafascicular triglyceride stores appears to account for additional fat utilization in females. Recent trends towards excessive fluid intake have resulted in frequent reports of hyponatraemic hyperhydration in ultra-distance athletes, with greater incidence in women than in men. Carbohydrate supplementation during the event attenuates immunosuppressive hormonal and cytokine responses to ultra-endurance exercise, but may impair vitamin C absorption, while the ergogenic value of caffeine supplementation in ultra-endurance performance is currently being questioned. SUMMARY: Meeting macronutrient and fluid intake demands remains an important priority for ultra-endurance athletes. Yet these athletes are reported to present with a high incidence of disordered eating patterns during periods of training, and excessive fluid replacement strategies have resulted in an increased incidence of water intoxication with resultant central nervous system dysfunction.

A physiological appraisal of aerobic riding in women.

The aim of this study was to compare selected acute cardiorespiratory and metabolic effects of exercise on a Fitness Flyer (FF) aerobic rider to those of treadmill (TM) running. Fourteen women, aged 23-35 years, performed incremental exercise tests to exhaustion on the TM and FF. Ratings of perceived exertion (RPE), heart rate (HR), minute ventilation (VE), VO2, and ventilatory equivalent (VEq) were compared in each subject during each phase of the exercise protocols, and blood lactate concentrations were measured before and 2-3 minutes after the exercise tests on the 2 modalities. Peak VO2 was higher (p < 0.05) on the TM than on the FF. Mean submaximal HR and VEq at a given VO2 was, however, higher on the FF than on the TM (p < 0.05). Maximum mean energy expenditure on the FF corresponded with mean energy expenditure on the TM at 8 km.h(-1) at an 18% gradient. Posttest blood lactate concentrations and RPE were higher on the FF than on the TM (p < 0.05). The results indicate that although exercising on an FF elicits less maximal cardiorespiratory response than does TM running, the FF may be better suited to developing local muscle endurance in the thigh muscles.