Reverse phenotyping facilitates disease allele calling in exome sequencing of patients with CAKUT.

PURPOSE: Congenital anomalies of the kidneys and urinary tract (CAKUT) constitute the leading cause of chronic kidney disease in children. In total, 174 monogenic causes of isolated or syndromic CAKUT are known. However, syndromic features may be overlooked when the initial clinical diagnosis of CAKUT is made. We hypothesized that the yield of a molecular genetic diagnosis by exome sequencing (ES) can be increased by applying reverse phenotyping, by re-examining the case for signs/symptoms of the suspected clinical syndrome that results from the genetic variant detected by ES. METHODS: We conducted ES in an international cohort of 731 unrelated families with CAKUT. We evaluated ES data for variants in 174 genes, in which variants are known to cause isolated or syndromic CAKUT. In cases in which ES suggested a previously unreported syndromic phenotype, we conducted reverse phenotyping. RESULTS: In 83 of 731 (11.4%) families, we detected a likely CAKUT-causing genetic variant consistent with an isolated or syndromic CAKUT phenotype. In 19 of these 83 families (22.9%), reverse phenotyping yielded syndromic clinical findings, thereby strengthening the genotype-phenotype correlation. CONCLUSION: We conclude that employing reverse phenotyping in the evaluation of syndromic CAKUT genes by ES provides an important tool to facilitate molecular genetic diagnostics in CAKUT.

Beyond the tubule: pathological variants of LRP2, encoding the megalin receptor, result in glomerular loss and early progressive chronic kidney disease.

Pathogenic variants in the LRP2 gene, encoding the multiligand receptor megalin, cause a rare autosomal recessive syndrome: Donnai-Barrow/Facio-Oculo-Acoustico-Renal (DB/FOAR) syndrome. Because of the rarity of the syndrome, the long-term consequences of the tubulopathy on human renal health have been difficult to ascertain, and the human clinical condition has hitherto been characterized as a benign tubular condition with asymptomatic low-molecular-weight proteinuria. We investigated renal function and morphology in a murine model of DB/FOAR syndrome and in patients with DB/FOAR. We analyzed glomerular filtration rate in mice by FITC-inulin clearance and clinically characterized six families, including nine patients with DB/FOAR and nine family members. Urine samples from patients were analyzed by Western blot analysis and biopsy materials were analyzed by histology. In the mouse model, we used histological methods to assess nephrogenesis and postnatal renal structure and contrast-enhanced magnetic resonance imaging to assess glomerular number. In megalin-deficient mice, we found a lower glomerular filtration rate and an increase in the abundance of injury markers, such as kidney injury molecule-1 and N-acetyl-ß-d-glucosaminidase. Renal injury was validated in patients, who presented with increased urinary kidney injury molecule-1, classical markers of chronic kidney disease, and glomerular proteinuria early in life. Megalin-deficient mice had normal nephrogenesis, but they had 19% fewer nephrons in early adulthood and an increased fraction of nephrons with disconnected glomerulotubular junction. In conclusion, megalin dysfunction, as present in DB/FOAR syndrome, confers an increased risk of progression into chronic kidney disease.

Whole-Exome Sequencing Enables a Precision Medicine Approach for Kidney Transplant Recipients.

BACKGROUND: Whole-exome sequencing (WES) finds a CKD-related mutation in approximately 20% of patients presenting with CKD before 25 years of age. Although provision of a molecular diagnosis could have important implications for clinical management, evidence is lacking on the diagnostic yield and clinical utility of WES for pediatric renal transplant recipients. METHODS: To determine the diagnostic yield of WES in pediatric kidney transplant recipients, we recruited 104 patients who had received a transplant at Boston Children's Hospital from 2007 through 2017, performed WES, and analyzed results for likely deleterious variants in approximately 400 genes known to cause CKD. RESULTS: By WES, we identified a genetic cause of CKD in 34 out of 104 (32.7%) transplant recipients. The likelihood of detecting a molecular genetic diagnosis was highest for patients with urinary stone disease (three out of three individuals), followed by renal cystic ciliopathies (seven out of nine individuals), steroid-resistant nephrotic syndrome (nine out of 21 individuals), congenital anomalies of the kidney and urinary tract (ten out of 55 individuals), and chronic glomerulonephritis (one out of seven individuals). WES also yielded a molecular diagnosis for four out of nine individuals with ESRD of unknown etiology. The WES-related molecular genetic diagnosis had implications for clinical care for five patients. CONCLUSIONS: Nearly one third of pediatric renal transplant recipients had a genetic cause of their kidney disease identified by WES. Knowledge of this genetic information can help guide management of both transplant patients and potential living related donors.

Genetic variants in the LAMA5 gene in pediatric nephrotic syndrome.

BACKGROUND: Nephrotic syndrome (NS), a chronic kidney disease, is characterized by significant loss of protein in the urine causing hypoalbuminemia and edema. In general, ∼15% of childhood-onset cases do not respond to steroid therapy and are classified as steroid-resistant NS (SRNS). In ∼30% of cases with SRNS, a causative mutation can be detected in one of 44 monogenic SRNS genes. The gene LAMA5 encodes laminin-α5, an essential component of the glomerular basement membrane. Mice with a hypomorphic mutation in the orthologous gene Lama5 develop proteinuria and hematuria. METHODS: To identify additional monogenic causes of NS, we performed whole exome sequencing in 300 families with pediatric NS. In consanguineous families we applied homozygosity mapping to identify genomic candidate loci for the underlying recessive mutation. RESULTS: In three families, in whom mutations in known NS genes were excluded, but in whom a recessive, monogenic cause of NS was strongly suspected based on pedigree information, we identified homozygous variants of unknown significance (VUS) in the gene LAMA5. While all affected individuals had nonsyndromic NS with an early onset of disease, their clinical outcome and response to immunosuppressive therapy differed notably. CONCLUSION: We here identify recessive VUS in the gene LAMA5 in patients with partially treatment-responsive NS. More data will be needed to determine the impact of these VUS in disease management. However, familial occurrence of disease, data from genetic mapping and a mouse model that recapitulates the NS phenotypes suggest that these genetic variants may be inherited factors that contribute to the development of NS in pediatric patients.

Whole-Exome Sequencing Identifies Causative Mutations in Families with Congenital Anomalies of the Kidney and Urinary Tract.

BACKGROUND: Congenital anomalies of the kidney and urinary tract (CAKUT) are the most prevalent cause of kidney disease in the first three decades of life. Previous gene panel studies showed monogenic causation in up to 12% of patients with CAKUT. METHODS: We applied whole-exome sequencing to analyze the genotypes of individuals from 232 families with CAKUT, evaluating for mutations in single genes known to cause human CAKUT and genes known to cause CAKUT in mice. In consanguineous or multiplex families, we additionally performed a search for novel monogenic causes of CAKUT. RESULTS: In 29 families (13%), we detected a causative mutation in a known gene for isolated or syndromic CAKUT that sufficiently explained the patient's CAKUT phenotype. In three families (1%), we detected a mutation in a gene reported to cause a phenocopy of CAKUT. In 15 of 155 families with isolated CAKUT, we detected deleterious mutations in syndromic CAKUT genes. Our additional search for novel monogenic causes of CAKUT in consanguineous and multiplex families revealed a potential single, novel monogenic CAKUT gene in 19 of 232 families (8%). CONCLUSIONS: We identified monogenic mutations in a known human CAKUT gene or CAKUT phenocopy gene as the cause of disease in 14% of the CAKUT families in this study. Whole-exome sequencing provides an etiologic diagnosis in a high fraction of patients with CAKUT and will provide a new basis for the mechanistic understanding of CAKUT.

A Multi-layered Quantitative In Vivo Expression Atlas of the Podocyte Unravels Kidney Disease Candidate Genes.

Damage to and loss of glomerular podocytes has been identified as the culprit lesion in progressive kidney diseases. Here, we combine mass spectrometry-based proteomics with mRNA sequencing, bioinformatics, and hypothesis-driven studies to provide a comprehensive and quantitative map of mammalian podocytes that identifies unanticipated signaling pathways. Comparison of the in vivo datasets with proteomics data from podocyte cell cultures showed a limited value of available cell culture models. Moreover, in vivo stable isotope labeling by amino acids uncovered surprisingly rapid synthesis of mitochondrial proteins under steady-state conditions that was perturbed under autophagy-deficient, disease-susceptible conditions. Integration of acquired omics dimensions suggested FARP1 as a candidate essential for podocyte function, which could be substantiated by genetic analysis in humans and knockdown experiments in zebrafish. This work exemplifies how the integration of multi-omics datasets can identify a framework of cell-type-specific features relevant for organ health and disease.

Whole Exome Sequencing Reveals a Monogenic Cause of Disease in ≈43% of 35 Families With Midaortic Syndrome.

Midaortic syndrome (MAS) is a rare cause of severe childhood hypertension characterized by narrowing of the abdominal aorta in children and is associated with extensive vascular disease. It may occur as part of a genetic syndrome, such as neurofibromatosis, or as consequence of a pathological inflammatory disease. However, most cases are considered idiopathic. We hypothesized that in a high percentage of these patients, a monogenic cause of disease may be detected by evaluating whole exome sequencing data for mutations in 1 of 38 candidate genes previously described to cause vasculopathy. We studied a cohort of 36 individuals from 35 different families with MAS by exome sequencing. In 15 of 35 families (42.9%), we detected likely causal dominant mutations. In 15 of 35 (42.9%) families with MAS, whole exome sequencing revealed a mutation in one of the genes previously associated with vascular disease (NF1, JAG1, ELN, GATA6, and RNF213). Ten of the 15 mutations have not previously been reported. This is the first report of ELN, RNF213, or GATA6 mutations in individuals with MAS. Mutations were detected in NF1 (6/15 families), JAG1 (4/15 families), ELN (3/15 families), and one family each for GATA6 and RNF213 Eight individuals had syndromic disease and 7 individuals had isolated MAS. Whole exome sequencing can provide conclusive molecular genetic diagnosis in a high fraction of individuals with syndromic or isolated MAS. Establishing an etiologic diagnosis may reveal genotype/phenotype correlations for MAS in the future and should, therefore, be performed routinely in MAS.

Analysis of 24 genes reveals a monogenic cause in 11.1% of cases with steroid-resistant nephrotic syndrome at a single center.

BACKGROUND: Steroid-resistant nephrotic syndrome (SRNS) is the second most frequent cause of end-stage renal disease (ESRD) among patients manifesting at under 25 years of age. We performed mutation analysis using a high-throughput PCR-based microfluidic technology in 24 single-gene causes of SRNS in a cohort of 72 families, who presented with SRNS before the age of 25 years. METHODS: Within an 18-month interval, we obtained DNA samples, pedigree information, and clinical information from 77 consecutive children with SRNS from 72 different families seen at Boston Children's Hospital (BCH). Mutation analysis was completed by combining high-throughput multiplex PCR with next-generation sequencing. We analyzed the sequences of 18 recessive and 6 dominant genes of SRNS in all 72 families for disease-causing variants. RESULTS: We identified the disease-causing mutation in 8 out of 72 (11.1%) families. Mutations were detected in the six genes: NPHS1 (2 out of 72), WT1 (2 out of 72), NPHS2, MYO1E, TRPC6, and INF2. Median age at onset was 4.1 years in patients without a mutation (range 0.5-18.8), and 3.2 years in those in whom the causative mutation was detected (range 0.1-14.3). Mutations in dominant genes presented with a median onset of 4.5 years (range 3.2-14.3). Mutations in recessive genes presented with a median onset of 0.5 years (range 0.1-3.2). CONCLUSION: Our molecular genetic diagnostic study identified underlying monogenic causes of steroid-resistant nephrotic syndrome in ~11% of patients with SRNS using a cost-effective technique. We delineated some of the therapeutic, diagnostic, and prognostic implications. Our study confirms that genetic testing is indicated in pediatric patients with SRNS.

Whole exome sequencing frequently detects a monogenic cause in early onset nephrolithiasis and nephrocalcinosis.

The incidence of nephrolithiasis continues to rise. Previously, we showed that a monogenic cause could be detected in 11.4% of individuals with adult-onset nephrolithiasis or nephrocalcinosis and in 16.7-20.8% of individuals with onset before 18 years of age, using gene panel sequencing of 30 genes known to cause nephrolithiasis/nephrocalcinosis. To overcome the limitations of panel sequencing, we utilized whole exome sequencing in 51 families, who presented before age 25 years with at least one renal stone or with a renal ultrasound finding of nephrocalcinosis to identify the underlying molecular genetic cause of disease. In 15 of 51 families, we detected a monogenic causative mutation by whole exome sequencing. A mutation in seven recessive genes (AGXT, ATP6V1B1, CLDN16, CLDN19, GRHPR, SLC3A1, SLC12A1), in one dominant gene (SLC9A3R1), and in one gene (SLC34A1) with both recessive and dominant inheritance was detected. Seven of the 19 different mutations were not previously described as disease-causing. In one family, a causative mutation in one of 117 genes that may represent phenocopies of nephrolithiasis-causing genes was detected. In nine of 15 families, the genetic diagnosis may have specific implications for stone management and prevention. Several factors that correlated with the higher detection rate in our cohort were younger age at onset of nephrolithiasis/nephrocalcinosis, presence of multiple affected members in a family, and presence of consanguinity. Thus, we established whole exome sequencing as an efficient approach toward a molecular genetic diagnosis in individuals with nephrolithiasis/nephrocalcinosis who manifest before age 25 years.

Whole Exome Sequencing of Patients with Steroid-Resistant Nephrotic Syndrome.

BACKGROUND AND OBJECTIVES: Steroid-resistant nephrotic syndrome overwhelmingly progresses to ESRD. More than 30 monogenic genes have been identified to cause steroid-resistant nephrotic syndrome. We previously detected causative mutations using targeted panel sequencing in 30% of patients with steroid-resistant nephrotic syndrome. Panel sequencing has a number of limitations when compared with whole exome sequencing. We employed whole exome sequencing to detect monogenic causes of steroid-resistant nephrotic syndrome in an international cohort of 300 families. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Three hundred thirty-five individuals with steroid-resistant nephrotic syndrome from 300 families were recruited from April of 1998 to June of 2016. Age of onset was restricted to <25 years of age. Exome data were evaluated for 33 known monogenic steroid-resistant nephrotic syndrome genes. RESULTS: In 74 of 300 families (25%), we identified a causative mutation in one of 20 genes known to cause steroid-resistant nephrotic syndrome. In 11 families (3.7%), we detected a mutation in a gene that causes a phenocopy of steroid-resistant nephrotic syndrome. This is consistent with our previously published identification of mutations using a panel approach. We detected a causative mutation in a known steroid-resistant nephrotic syndrome gene in 38% of consanguineous families and in 13% of nonconsanguineous families, and 48% of children with congenital nephrotic syndrome. A total of 68 different mutations were detected in 20 of 33 steroid-resistant nephrotic syndrome genes. Fifteen of these mutations were novel. NPHS1, PLCE1, NPHS2, and SMARCAL1 were the most common genes in which we detected a mutation. In another 28% of families, we detected mutations in one or more candidate genes for steroid-resistant nephrotic syndrome. CONCLUSIONS: Whole exome sequencing is a sensitive approach toward diagnosis of monogenic causes of steroid-resistant nephrotic syndrome. A molecular genetic diagnosis of steroid-resistant nephrotic syndrome may have important consequences for the management of treatment and kidney transplantation in steroid-resistant nephrotic syndrome.

Exome Sequencing Discerns Syndromes in Patients from Consanguineous Families with Congenital Anomalies of the Kidneys and Urinary Tract.

Congenital anomalies of the kidneys and urinary tract (CAKUT) are the leading cause of CKD in children, featuring a broad variety of malformations. A monogenic cause can be detected in around 12% of patients. However, the morphologic clinical phenotype of CAKUT frequently does not indicate specific genes to be examined. To determine the likelihood of detecting causative recessive mutations by whole-exome sequencing (WES), we analyzed individuals with CAKUT from 33 different consanguineous families. Using homozygosity mapping and WES, we identified the causative mutations in nine of the 33 families studied (27%). We detected recessive mutations in nine known disease-causing genes: ZBTB24, WFS1, HPSE2, ATRX, ASPH, AGXT, AQP2, CTNS, and PKHD1 Notably, when mutated, these genes cause multiorgan syndromes that may include CAKUT as a feature (syndromic CAKUT) or cause renal diseases that may manifest as phenocopies of CAKUT. None of the above monogenic disease-causing genes were suspected on clinical grounds before this study. Follow-up clinical characterization of those patients allowed us to revise and detect relevant new clinical features in a more appropriate pathogenetic context. Thus, applying WES to the diagnostic approach in CAKUT provides opportunities for an accurate and early etiology-based diagnosis and improved clinical management.

Recent advances in the pathogenesis of hypertension in children.

This commentary discusses current knowledge of the pathogenesis of pediatric hypertension, focusing on the fact that adult health and, to a large extent, adult diseases, are deeply rooted in both hereditary and environmental factors that originate in childhood. Understanding these factors and their pathogenesis not only allows us to appreciate the etiology of pediatric hypertension, but also helps us to target our treatment and better manage the risk factors in the very young that may result in less cardiovascular and kidney disease among adults.

Tuberous sclerosis complex and renal angiomyolipoma: case report and review of the literature.

A 5-year-old boy with a known diagnosis of tuberous sclerosis complex was found to have an enlarging renal mass on routine ultrasound. He was diagnosed with an angiomyolipoma (AML) and scheduled for close observation. Follow-up magnetic resonance imaging demonstrated the AML to be significantly enlarged and hypervascular. Selective arterial embolization of the tumor was performed, which resulted in an appropriate decrease in tumor size. Angiomyolipoma is a known and well-described complication of the tuberous sclerosis complex that is usually found among patients in their adolescent and adult years. The case presented here illustrates the need for early and repeated renal imaging of younger pediatric patients with tuberous sclerosis. Our experience adds to the literature on young pediatric patients requiring embolization for treatment of large renal angiomyolipomas.