Mechanisms of pathogenicity and the quest for genetic modifiers of kidney disease in branchiootorenal syndrome.

Backgound: Branchiootorenal (BOR) syndrome is an autosomal dominant disorder caused by pathogenic EYA1 variants and clinically characterized by auricular malformations with hearing loss, branchial arch anomalies, and congenital anomalies of the kidney and urinary tract. BOR phenotypes are highly variable and heterogenous. While random monoallelic expression is assumed to explain this phenotypic heterogeneity, the potential role of modifier genes has not yet been explored. Methods: Through thorough phenotyping and exome sequencing, we studied one family with disease presentation in at least four generations in both clinical and genetic terms. Functional investigation of the single associated EYA1 variant c.1698+1G>A included splice site analysis and assessment of EYA1 distribution in patient-derived fibroblasts. The candidate modifier gene CYP51A1 was evaluated by histopathological analysis of murine Cyp51+/- and Cyp51-/- kidneys. As the gene encodes the enzyme lanosterol 14α-demethylase, we assessed sterol intermediates in patient blood samples as well. Results: The EYA1 variant c.1698+1G>A resulted in functional deletion of the EYA domain by exon skipping. The EYA domain mediates protein-protein interactions between EYA1 and co-regulators of transcription. EYA1 abundance was reduced in the nuclear compartment of patient-derived fibroblasts, suggesting impaired nuclear translocation of these protein complexes. Within the affected family, renal phenotypes spanned from normal kidney function in adulthood to chronic kidney failure in infancy. By analyzing exome sequencing data for variants that potentially play roles as genetic modifiers, we identified a canonical splice site alteration in CYP51A1 as the strongest candidate variant. Conclusion: In this study, we demonstrate pathogenicity of EYA1 c.1698+1G>A, propose a mechanism for dysfunction of mutant EYA1, and conjecture CYP51A1 as a potential genetic modifier of renal involvement in BOR syndrome.

Clinical and Functional Assessment of Digenicity in Renal Phosphate Wasting.

Apart from increased fluid intake, patients with kidney stone disease (KSD) due to renal phosphate wasting require specific metaphylaxis. NaPi2a, NaPi2c, and NHERF1 regulate plasma phosphate concentration by reabsorbing phosphate in proximal kidney tubules and have been found altered in monogenic hypophosphatemia with a risk of KSD. In this study, we aimed at assessing the combined genetic alterations impacting NaPi2a, NaPi2c, and NHERF1. Therefore, we screened our hereditary KSD registry for cases of oligo- and digenicity, conducted reverse phenotyping, and undertook functional studies. As a result, we identified three patients from two families with digenic alterations in NaPi2a, NaPi2c, and NHERF1. In family 1, the index patient, who presented with severe renal calcifications and a bone mineralization disorder, carried digenic alterations affecting both NaPi transporter 2a and 2c. Functional analysis confirmed an additive genetic effect. In family 2, the index patient presented with kidney function decline, distinct musculature-related symptoms, and intracellular ATP depletion. Genetically, this individual was found to harbor variants in both NaPi2c and NHERF1 pointing towards genetic interaction. In summary, digenicity and gene dosage are likely to impact the severity of renal phosphate wasting and should be taken into account in terms of metaphylaxis through phosphate substitution.

Delta weight loss unlike genetic variation associates with hyperoxaluria after malabsorptive bariatric surgery.

The risk of enteric hyperoxaluria is significantly increased after malabsorptive bariatric surgery (MBS). However, its underlying determinants are only poorly characterized. In this case-control study, we aimed at identifying clinical and genetic factors to dissect their individual contributions to the development of post-surgical hyperoxaluria. We determined the prevalence of hyperoxaluria and nephrolithiasis after MBS by 24-h urine samples and clinical questionnaires at our obesity center. Both hyperoxaluric and non-hyperoxaluric patients were screened for sequence variations in known and candidate genes implicated in hyperoxaluria (AGXT, GRHPR, HOGA1, SLC26A1, SLC26A6, SLC26A7) by targeted next generation sequencing (tNGS). The cohort comprised 67 patients, 49 females (73%) and 18 males (27%). While hyperoxaluria was found in 29 patients (43%), only one patient reported postprocedural nephrolithiasis within 41 months of follow-up. Upon tNGS, we did not find a difference regarding the burden of (rare) variants between hyperoxaluric and non-hyperoxaluric patients. However, patients with hyperoxaluria showed significantly greater weight loss accompanied by markers of intestinal malabsorption compared to non-hyperoxaluric controls. While enteric hyperoxaluria is very common after MBS, genetic variation of known hyperoxaluria genes contributes little to its pathogenesis. In contrast, the degree of postsurgical weight loss and levels of malabsorption parameters may allow for predicting the risk of enteric hyperoxaluria and consecutive kidney stone formation.

The genetic landscape and clinical spectrum of nephronophthisis and related ciliopathies.

Nephronophthisis (NPH) is an autosomal-recessive ciliopathy representing one of the most frequent causes of kidney failure in childhood characterized by a broad clinical and genetic heterogeneity. Applied to one of the worldwide largest cohorts of patients with NPH, genetic analysis encompassing targeted and whole exome sequencing identified disease-causing variants in 600 patients from 496 families with a detection rate of 71%. Of 788 pathogenic variants, 40 known ciliopathy genes were identified. However, the majority of patients (53%) bore biallelic pathogenic variants in NPHP1. NPH-causing gene alterations affected all ciliary modules defined by structural and/or functional subdomains. Seventy six percent of these patients had progressed to kidney failure, of which 18% had an infantile form (under five years) and harbored variants affecting the Inversin compartment or intraflagellar transport complex A. Forty eight percent of patients showed a juvenile (5-15 years) and 34% a late-onset disease (over 15 years), the latter mostly carrying variants belonging to the Transition Zone module. Furthermore, while more than 85% of patients with an infantile form presented with extra-kidney manifestations, it only concerned half of juvenile and late onset cases. Eye involvement represented a predominant feature, followed by cerebellar hypoplasia and other brain abnormalities, liver and skeletal defects. The phenotypic variability was in a large part associated with mutation types, genes and corresponding ciliary modules with hypomorphic variants in ciliary genes playing a role in early steps of ciliogenesis associated with juvenile-to-late onset NPH forms. Thus, our data confirm a considerable proportion of late-onset NPH suggesting an underdiagnosis in adult chronic kidney disease.

Extended genomic HLA typing identifies previously unrecognized mismatches in living kidney transplantation.

Introduction: Antibody mediated rejection (ABMR) is the most common cause of long-term allograft loss in kidney transplantation (KT). Therefore, a low human leukocyte antigen (HLA) mismatch (MM) load is favorable for KT outcomes. Hitherto, serological or low-resolution molecular HLA typing have been adapted in parallel. Here, we aimed to identify previously missed HLA mismatches and corresponding antibodies by high resolution HLA genotyping in a living-donor KT cohort. Methods: 103 donor/recipient pairs transplanted at the University of Leipzig Medical Center between 1998 and 2018 were re-typed using next generation sequencing (NGS) of the HLA loci -A, -B, -C, -DRB1, -DRB345, -DQA1, -DQB1, -DPA1, and -DPB1. Based on these data, we compiled HLA MM counts for each pair and comparatively evaluated genomic HLA-typing with pre-transplant obtained serological/low-resolution HLA (=one-field) typing results. NGS HLA typing (=two-field) data was further used for reclassification of de novo HLA antibodies as "donor-specific". Results: By two-field HLA re-typing, we were able to identify additional MM in 64.1% (n=66) of cases for HLA loci -A, -B, -C, -DRB1 and -DQB1 that were not observed by one-field HLA typing. In patients with biopsy proven ABMR, two-field calculated MM count was significantly higher than by one-field HLA typing. For additional typed HLA loci -DRB345, -DQA1, -DPA1, and -DPB1 we observed 2, 26, 3, and 23 MM, respectively. In total, 37.3% (69/185) of de novo donor specific antibodies (DSA) formation was directed against these loci (DRB345 ➔ n=33, DQA1 ➔ n=33, DPA1 ➔ n=1, DPB1 ➔ n=10). Conclusion: Our results indicate that two-field HLA typing is feasible and provides significantly more sensitive HLA MM recognition in living-donor KT. Furthermore, accurate HLA typing plays an important role in graft management as it can improve discrimination between donor and non-donor HLA directed cellular and humoral alloreactivity in the long range. The inclusion of additional HLA loci against which antibodies can be readily detected, HLA-DRB345, -DQA1, -DQB1, -DPA1, and -DPB1, will allow a more precise virtual crossmatch and better prediction of potential DSA. Furthermore, in living KT, two-field HLA typing could contribute to the selection of the immunologically most suitable donors.

Novel somatic PBX1 mosaicism likely masking syndromic CAKUT in an adult with bilateral kidney hypoplasia.

Background: Congenital abnormalities of the kidney and urinary tract (CAKUT) are characterized by vast phenotypic heterogeneity and incomplete penetrance. Although CAKUT represent the main cause of pediatric chronic kidney disease, only ∼20% can be explained by single-gene disorders to date. While pathogenic alterations of PBX1 were recently associated with a severe form of syndromic CAKUT, most CAKUT patients survive childhood and adolescence to reach end-stage kidney disease later in life. Although somatic mosaicism is known to attenuate severity in other kidney diseases, it has rarely been described or systematically been assessed in CAKUT. Methods: We conducted an in-depth phenotypic characterization of the index patient and his family using targeted next-generation sequencing, segregation analysis and workup of mosaicism with DNA isolated from peripheral blood cells, oral mucosa and cultured urinary renal epithelial cells (URECs). Results: Somatic mosaicism was identified in a 20-year-old male with sporadic but mild syndromic renal hypoplasia. He was found to carry a novel de novo truncating variant in PBX1 [c.992C>A, p.(Ser331*)]. This variant was detected in 26% of sequencing reads from blood cells, 50% from oral mucosa and 20% from cultured URECs. Conclusions: PBX1-associated CAKUT is characterized by a wealth of de novo mutations. As in de novo cases, mutations can occur intra- or post-zygotically and genetic mosaicism might represent a more common phenomenon in PBX1 disease, accounting for variable expressivity on a general basis. Consequently we suggest ruling out somatic mosaicism in sporadic CAKUT, notably in attenuated and atypical clinical courses.

Matching clinical and genetic diagnoses in autosomal dominant polycystic kidney disease reveals novel phenocopies and potential candidate genes.

PURPOSE: Autosomal dominant polycystic kidney disease (ADPKD) represents the most common hereditary nephropathy. Despite growing evidence for genetic heterogeneity, ADPKD diagnosis is still primarily based upon clinical imaging criteria established before discovery of additional PKD genes. This study aimed at assessing the diagnostic value of genetic verification in clinical ADPKD. METHODS: In this prospective, diagnostic trial, 100 families with clinically diagnosed ADPKD were analyzed by PKD gene panel and multiplex ligation-dependent probe amplification (MLPA); exome sequencing (ES) was performed in panel/MLPA-negative families. RESULTS: Diagnostic PKD1/2 variants were identified in 81 families (81%), 70 of which in PKD1 and 11 in PKD2. PKD1 variants of unknown significance were detected in another 9 families (9%). Renal survival was significantly worse upon PKD1 truncation versus nontruncation and PKD2 alteration. Ten percent of the cohort were PKD1/2-negative, revealing alternative genetic diagnoses such as autosomal recessive PKD, Birt-Hogg-Dubé syndrome, and ALG9-associated PKD. In addition, among unsolved cases, ES yielded potential novel PKD candidates. CONCLUSION: By illustrating vast genetic heterogeneity, this study demonstrates the value of genetic testing in a real-world PKD cohort by diagnostic verification, falsification, and disease prediction. In the era of specific treatment for fast progressive ADPKD, genetic confirmation should form the basis of personalized patient care.

Retrospective genetic analysis illustrates the spectrum of autosomal Alport syndrome in a case of living-related donor kidney transplantation.

BACKGROUND: Kidney transplantation is the treatment of choice in end-stage renal disease due to Alport syndrome (AS). However, the chances of finding an adequate living-related donor in AS are much worse compared to non-heritable conditions. Successful cases of related living-donor transplantation mostly refer to X-linked AS but are rarely reported in genetically confirmed autosomal AS. CASE PRESENTATION: We describe the outcome of an exceptional AB0-incompatible kidney donation from father to son in a family with altered COL4A3. While decision-making was based on extensive clinical donor evaluation prior to transplantation, we analyzed the underlying genetic background in retrospect and associated these findings with the phenotype in all available family members. While biallelic COL4A3 variants caused autosomal recessive AS (ARAS) in the son (recipient), heterozygous family members, including the father (donor), showed minimal renal involvement and high-frequency sensorineural hearing impairment later in life indicating mild autosomal dominant Alport syndrome (ADAS). The recipient's successful participation in the European and World Transplant Games is a testament to the positive outcome of transplantation. CONCLUSIONS: In summary, living-related donor transplantation may be successful in autosomal AS, provided that thorough clinical and genetic evaluation of potential donors is performed. However, unrelated kidney transplantation should be given priority upon unpredictable genetic risk. Individual genetic variant interpretation is an important component of personalized donor assessment and will help to better predict genetic risk in the future.

Evaluating pathogenicity of SLC34A3-Ser192Leu, a frequent European missense variant in disorders of renal phosphate wasting.

Loss-of-function mutations of SLC34A3 represent an established cause of a distinct renal phosphate wasting disorder termed hereditary hypophosphatemic rickets with hypercalciuria (HHRH). SLC34A3 encodes the renal phosphate transporter NaPi2c expressed at the apical brush border of proximal renal tubules. Substitution of p.Ser192Leu is one of the most frequent genetic changes among HHRH patients in Europe, but has never been systematically evaluated, clinically or on a cellular level. Identification of a 32-year-old female with a homozgyous c.575C>T, p.Ser192Leu substitution enabled a more comprehensive assessment of the impact of this missense variant. Clinically, the patient showed renal phosphate wasting and nephrocalcinosis without any bone abnormalities. Heterozygous carriers of deleterious SLC34A3 variants were previously described to harbor an increased risk of kidney stone formation and renal calcification. We hence examined the frequency of p.Ser192Leu variants in our adult kidney stone cohort and compared the results to clinical findings of previously published cases of both mono- and biallelic p.Ser192Leu changes. On a cellular level, p.Ser192Leu-mutated transporters localize to the plasma membrane in different cellular systems, but lead to significantly reduced transport activity of inorganic phosphate upon overexpression in Xenopus oocytes. Despite the reduced function in ectopic cellular systems, the clinical consequences of p.Ser192Leu may appear relatively mild, at least in our index patient, and can potentially be missed in clinical practice.

Diffusion imaging-based subdivision of the human hypothalamus: a magnetic resonance study with clinical implications.

The hypothalamus and its subdivisions are involved in many neuropsychiatric conditions such as affective disorders, schizophrenia, or narcolepsy, but parcellations of hypothalamic subnuclei have hitherto been feasible only with histological techniques in postmortem brains. In an attempt to map subdivisions of the hypothalamus in vivo, we analyzed the directionality information from high-resolution diffusion-weighted magnetic resonance images of healthy volunteers. We acquired T1-weighted and diffusion-weighted scans in ten healthy subjects at 3 T. In the T1-weighted images, we manually delineated an individual mask of the hypothalamus in each subject and computed in the co-registered diffusion-weighted images the similarity of the principal diffusion direction for each pair of mask voxels. By clustering the similarity matrix into three regions with a k-means algorithm, we obtained an anatomically coherent arrangement of subdivisions across hemispheres and subjects. In each hypothalamus mask, we found an anterior region with dorsoventral principal diffusion direction, a posteromedial region with rostro-caudal direction, and a lateral region with mediolateral direction. A comparative analysis with microstructural hypothalamus parcellations from the literature reveals that each of these regions corresponds to a specific group of hypothalamic subnuclei as defined in postmortem brains. This is to our best knowledge the first in vivo study that attempts a delineation of hypothalamic subdivisions by clustering diffusion-weighted magnetic resonance imaging data. When applied in a larger sample of neuropsychiatric patients, a structural analysis of hypothalamic subnuclei should contribute to a better understanding of the pathogenesis of neuropsychiatric conditions such as affective disorders.