A Founder Mutation in EHD1 Presents with Tubular Proteinuria and Deafness.

BACKGROUND: The endocytic reabsorption of proteins in the proximal tubule requires a complex machinery and defects can lead to tubular proteinuria. The precise mechanisms of endocytosis and processing of receptors and cargo are incompletely understood. EHD1 belongs to a family of proteins presumably involved in the scission of intracellular vesicles and in ciliogenesis. However, the relevance of EHD1 in human tissues, in particular in the kidney, was unknown. METHODS: Genetic techniques were used in patients with tubular proteinuria and deafness to identify the disease-causing gene. Diagnostic and functional studies were performed in patients and disease models to investigate the pathophysiology. RESULTS: We identified six individuals (5-33 years) with proteinuria and a high-frequency hearing deficit associated with the homozygous missense variant c.1192C>T (p.R398W) in EHD1. Proteinuria (0.7-2.1 g/d) consisted predominantly of low molecular weight proteins, reflecting impaired renal proximal tubular endocytosis of filtered proteins. Ehd1 knockout and Ehd1R398W/R398W knockin mice also showed a high-frequency hearing deficit and impaired receptor-mediated endocytosis in proximal tubules, and a zebrafish model showed impaired ability to reabsorb low molecular weight dextran. Interestingly, ciliogenesis appeared unaffected in patients and mouse models. In silico structural analysis predicted a destabilizing effect of the R398W variant and possible inference with nucleotide binding leading to impaired EHD1 oligomerization and membrane remodeling ability. CONCLUSIONS: A homozygous missense variant of EHD1 causes a previously unrecognized autosomal recessive disorder characterized by sensorineural deafness and tubular proteinuria. Recessive EHD1 variants should be considered in individuals with hearing impairment, especially if tubular proteinuria is noted.

Glycine Amidinotransferase (GATM), Renal Fanconi Syndrome, and Kidney Failure.

Background For many patients with kidney failure, the cause and underlying defect remain unknown. Here, we describe a novel mechanism of a genetic order characterized by renal Fanconi syndrome and kidney failure.Methods We clinically and genetically characterized members of five families with autosomal dominant renal Fanconi syndrome and kidney failure. We performed genome-wide linkage analysis, sequencing, and expression studies in kidney biopsy specimens and renal cells along with knockout mouse studies and evaluations of mitochondrial morphology and function. Structural studies examined the effects of recognized mutations.Results The renal disease in these patients resulted from monoallelic mutations in the gene encoding glycine amidinotransferase (GATM), a renal proximal tubular enzyme in the creatine biosynthetic pathway that is otherwise associated with a recessive disorder of creatine deficiency. In silico analysis showed that the particular GATM mutations, identified in 28 members of the five families, create an additional interaction interface within the GATM protein and likely cause the linear aggregation of GATM observed in patient biopsy specimens and cultured proximal tubule cells. GATM aggregates-containing mitochondria were elongated and associated with increased ROS production, activation of the NLRP3 inflammasome, enhanced expression of the profibrotic cytokine IL-18, and increased cell death.Conclusions In this novel genetic disorder, fully penetrant heterozygous missense mutations in GATM trigger intramitochondrial fibrillary deposition of GATM and lead to elongated and abnormal mitochondria. We speculate that this renal proximal tubular mitochondrial pathology initiates a response from the inflammasome, with subsequent development of kidney fibrosis.

Mistargeting of peroxisomal EHHADH and inherited renal Fanconi's syndrome.

BACKGROUND: In renal Fanconi's syndrome, dysfunction in proximal tubular cells leads to renal losses of water, electrolytes, and low-molecular-weight nutrients. For most types of isolated Fanconi's syndrome, the genetic cause and underlying defect remain unknown. METHODS: We clinically and genetically characterized members of a five-generation black family with isolated autosomal dominant Fanconi's syndrome. We performed genomewide linkage analysis, gene sequencing, biochemical and cell-biologic investigations of renal proximal tubular cells, studies in knockout mice, and functional evaluations of mitochondria. Urine was studied with the use of proton nuclear magnetic resonance ((1)H-NMR) spectroscopy. RESULTS: We linked the phenotype of this family's Fanconi's syndrome to a single locus on chromosome 3q27, where a heterozygous missense mutation in EHHADH segregated with the disease. The p.E3K mutation created a new mitochondrial targeting motif in the N-terminal portion of EHHADH, an enzyme that is involved in peroxisomal oxidation of fatty acids and is expressed in the proximal tubule. Immunocytofluorescence studies showed mistargeting of the mutant EHHADH to mitochondria. Studies of proximal tubular cells revealed impaired mitochondrial oxidative phosphorylation and defects in the transport of fluids and a glucose analogue across the epithelium. (1)H-NMR spectroscopy showed elevated levels of mitochondrial metabolites in urine from affected family members. Ehhadh knockout mice showed no abnormalities in renal tubular cells, a finding that indicates a dominant negative nature of the mutation rather than haploinsufficiency. CONCLUSIONS: Mistargeting of peroxisomal EHHADH disrupts mitochondrial metabolism and leads to renal Fanconi's syndrome; this indicates a central role of mitochondria in proximal tubular function. The dominant negative effect of the mistargeted protein adds to the spectrum of monogenic mechanisms of Fanconi's syndrome. (Funded by the European Commission Seventh Framework Programme and others.).

HNF1B-associated clinical phenotypes: the kidney and beyond.

Mutations in HNF1B, the gene encoding hepatocyte nuclear factor 1ß are the most commonly identified genetic cause of renal malformations. HNF1B was first identified as a disease gene for diabetes (MODY5) in 1997, and its involvement in renal disease was subsequently noted through clinical observations in pedigrees affected by MODY5. Since then, a whole spectrum of associated phenotypes have been reported, including genital malformations, autism, epilepsy, gout, hypomagnesaemia, primary hyperparathyroidism, liver and intestinal abnormalities and a rare form of kidney cancer. The most commonly identified mutation, in approximately 50 % of patients, is an entire gene deletion occurring in the context of a 17q12 chromosomal microdeletion that also includes several other genes. Some of the associated phenotypes, especially the neurologic ones, appear to occur only in the context of this microdeletion and thus may not be directly linked to HNF1B. Here we review the spectrum of associated phenotypes and discuss potential implications for clinical management.

Cyclophosphamide and rituximab in frequently relapsing/steroid-dependent nephrotic syndrome.

BACKGROUND: Steroid-sensitive nephrotic syndrome is the most common form of nephrotic syndrome in childhood, defined by the response to treatment with glucocorticoids with consequent remission. While most children eventually experience spontaneous resolution of the disease, some have a difficult course with frequent relapses or steroid dependence nephrotic syndrome (FRSDNS). The consequent steroid toxicity often prompts administration of other immunosuppressive drugs, traditionally cyclophosphamide. Recently, rituximab has been reported as effective in this disorder, but long-term experience is lacking. METHODS: Retrospective note review of all children with FRSDNS treated with a first course of cyclophosphamide and/or rituximab in our center between December 2006 and April 2015. We reviewed time to first relapse after treatment, co-medications, and side effects. RESULTS: A total of 102 children were treated with cyclophosphamide (79) and/or rituximab (42). Of these, 34 received cyclophosphamide prior to rituximab. Median time to first relapse was 7 months after cyclophosphamide and 14 months after rituximab. Documented side effects of cyclophosphamide included neutropenia, hair loss, and hemorrhagic cystitis (1). Rituximab was associated with an allergic reaction at infusion in two patients. CONCLUSIONS: Rituximab was used in children with the most difficult to treat FRSDNS, yet was associated with longer remission time and less side effects than cyclophosphamide. A randomized controlled trial is needed to directly compare these drugs.

Nephrocalcinosis (enamel renal syndrome) caused by autosomal recessive FAM20A mutations.

BACKGROUND/AIMS: Calcium homeostasis requires regulated cellular and interstitial systems interacting to modulate the activity and movement of this ion. Disruption of these systems in the kidney results in nephrocalcinosis and nephrolithiasis, important medical problems whose pathogenesis is incompletely understood. METHODS: We investigated 25 patients from 16 families with unexplained nephrocalcinosis and characteristic dental defects (amelogenesis imperfecta, gingival hyperplasia, impaired tooth eruption). To identify the causative gene, we performed genome-wide linkage analysis, exome capture, next-generation sequencing, and Sanger sequencing. RESULTS: All patients had bi-allelic FAM20A mutations segregating with the disease; 20 different mutations were identified. CONCLUSIONS: This autosomal recessive disorder, also known as enamel renal syndrome, of FAM20A causes nephrocalcinosis and amelogenesis imperfecta. We speculate that all individuals with biallelic FAM20A mutations will eventually show nephrocalcinosis.

Hereditary hypophosphatemic rickets with hypercalciuria and nephrolithiasis-identification of a novel SLC34A3/NaPi-IIc mutation.

Hereditary hypophosphatemic rickets with hypercalciuria (HHRH) is characterized by rickets, hyperphosphaturia, hypophosphatemia, elevated 1,25-dihydroxyvitamin-D, increased gastrointestinal calcium absorption and hypercalciuria. Serum calcium, 25-hydroxyvitamin-D and PTH levels are normal. Here we describe a boy with HHRH, nephrolithiasis, and compound heterozygosity for one previously described mutation (g.4225_50del) and a novel splice mutation (g.1226G>A) in SLC34A3, the gene encoding the renal sodium-phosphate co-transporter NaPi-IIc. The patient's mother and grandmother are carriers of g.4225_50del, and both have a history of nephrolithiasis associated with hypercalciuria and elevated 1,25-dihydroxyvitamin-D. His three siblings (2-6 years old), who are also carriers of g.4225_50del, have hypercalciuria but so far their renal ultrasounds are normal. Thus, SLC34A3/NaPi-IIc mutations appear to be associated with variable phenotypic changes at presentation, which can include recurrent nephrolithiasis.

Renal FMD may not confer a familial hypertensive risk nor is it caused by ACTA2 mutations.

Renal fibromuscular dysplasia (FMD) can cause hypertension, and previous reports suggest that FMD is familial. We hypothesized that, in families containing an individual with proven FMD, relatives of index cases would have an increased risk of hypertension. ACTA2 mutations cause a spectrum of extra-renal arteriopathy, leading to our second hypothesis that mutations are implicated in FMD. The blood pressure of first-degree relatives was measured using standard devices and, when indicated, with 24-h ambulatory monitoring. Leucocyte DNA was obtained from FMD index cases and ACTA2 sequenced. Thirteen unrelated index cases, aged 2-32 (median 15) years, were recruited. Blood pressure was assessed in 40 first-degree relatives, comprising 22 parents aged 28-58 (median 44) years and 18 siblings aged 3-30 (median 13) years. Hypertension was evident in six (27%) parents but in none of the eight adult siblings. Of the ten screened siblings aged less than 18 years, one teenager was pre-hypertensive (90th-95th centile), the remainder being normotensive. No ACTA2 mutations were found in 13 index cases. Hypertension was evident in 20% of all assessed adult first-degree relatives and is therefore not increased relative to 25% of the adult population. Although hypertensive parents did not undergo angiography to assign FMD status, this observation, together with the lack of hypertension in 18 siblings, indicates that FMD is unlikely to confer an excess hypertension risk in first-degree relatives up to middle-age. Furthermore, in our cohort, FMD was not caused by ACTA2 mutations.

Quantification of FAM20A in human milk and identification of calcium metabolism proteins.

BACKGROUND: FAM20A, a recently discovered protein, is thought to have a fundamental role in inhibiting ectopic calcification. Several studies have demonstrated that variants of FAM20A are causative for the rare autosomal recessive disorder, enamel-renal syndrome (ERS). ERS is characterized by defective mineralization of dental enamel and nephrocalcinosis suggesting that FAM20A is an extracellular matrix protein, dysfunction of which causes calcification of the secretory epithelial tissues. FAM20A is a low-abundant protein that is difficult to detect in biofluids such as blood, saliva, and urine. Thus, we speculated the abundance of FAM20A to be high in human milk, since the secretory epithelium of lactating mammary tissue is involved in the secretion of highly concentrated calcium. Therefore, the primary aim of this research is to describe the processes/methodology taken to quantify FAM20A in human milk and identify other proteins involved in calcium metabolism. METHOD: This study used mass spectrometry-driven quantitative proteomics: (1) to quantify FAM20A in human milk of three women and (2) to identify proteins associated with calcium regulation by bioinformatic analyses on whole and milk fat globule membrane fractions. RESULTS: Shotgun MS/MS driven proteomics identified FAM20A in whole milk, and subsequent analysis using targeted proteomics also successfully quantified FAM20A in all samples. Combination of sample preparation, fractionation, and LC-MS/MS proteomics analysis generated 136 proteins previously undiscovered in human milk; 21 of these appear to be associated with calcium metabolism. CONCLUSION: Using mass spectrometry-driven proteomics, we successfully quantified FAM20A from transitional to mature milk and obtained a list of proteins involved in calcium metabolism. Furthermore, we show the value of using a combination of both shotgun and targeted driven proteomics for the identification of this low abundant protein in human milk.

A patient with hypophosphatemia, a femoral fracture, and recurrent kidney stones: report of a novel mutation in SLC34A3.

OBJECTIVE: To determine if there was a genetic contribution to our patient's unusual clinical presentation of nephrolithiasis and nonhealing stress fracture. METHODS: We describe a 31-year-old man who had rickets as a child and developed a femur insufficiency fracture and recurrent nephrolithiasis as an adult after moving to the United States from India. The patient's clinical course and results from radiographic and biochemical analyses are described. Analysis of the SLC34A3 gene was performed using genomic DNA samples from the patient and his family members. RESULTS: Before referral to the Yale Bone Center, the patient was treated with calcitriol, ergocalciferol, and phosphate. Changing therapy to phosphate alone led to clinical improvement. Genetic analysis revealed that the patient is a compound heterozygote for mutations in the SLC34A3 gene. On 1 allele, he has a previously described missense mutation in exon 7: c.575C>T (p.Ser192Leu). The other allele carries a novel nonsense mutation in exon 3: c.145C>T (p.Gln49X). One unaffected sibling is a carrier of the missense mutation and 1 sister with a history of flank pain is a carrier of the novel mutation. CONCLUSIONS: Hereditary hypophosphatemic rickets with hypercalciuria is a rare metabolic disorder associated with mutations in SLC34A3, the gene that encodes the renal sodium phosphate cotransporter NaPi-IIc. Although hypercalciuria is a distinguishing feature of the disease, nephrolithiasis is rarely described. The patient's atypical clinical presentation illustrates that both environmental and genetic factors potentially affect phenotypic expression of SLC34A3 mutations.

A novel missense mutation in SLC34A3 that causes hereditary hypophosphatemic rickets with hypercalciuria in humans identifies threonine 137 as an important determinant of sodium-phosphate cotransport in NaPi-IIc.

The present study describes two novel compound heterozygous mutations, c.410C>T(p.T137M) (T137M) on the maternal and g.4225_50del on the paternal allele of SLC34A3, in a previously reported male with hereditary hypophosphatemic rickets with hypercalciuria (HHRH) and recurrent kidney stones (Chen C, Carpenter T, Steg N, Baron R, Anast C. Pediatrics 84: 276-280, 1989). For functional analysis in vitro, we generated expression plasmids encoding enhanced green fluorescence protein (EGFP) concatenated to the NH2 terminus of wild-type or mutant human type IIc Na-Pi cotransporter (NaPi-IIc), i.e., EGFP-hNaPi-IIc, EGFP-[M137]hNaPi-IIc, or EGFP-[Stop446]hNaPi-IIc. The V446Stop mutant showed complete loss of expression and function when assayed for apical patch expression in opossum kidney (OK) cells and sodium-dependent 33P uptake into Xenopus laevis oocytes. Conversely, EGFP-[M137]hNaPi-IIc was inserted into apical patches of OK cells and into oocyte membranes. However, when quantified by confocal microscopy, surface fluorescence was reduced to 40% compared with wild-type. After correction for surface expression, the rate of 33P uptake by oocytes mediated by EGFP-[M137]hNaPi-IIc was decreased by an additional 60%. The resulting overall reduction of function of this NaPi-IIc mutant to 16%, taken together with complete loss of expression and function of g.4225_50del(V446Stop), thus appears to be sufficient to explain the phenotype in our patient. Furthermore, the stoichiometric ratio of 22Na and 33P uptake was increased to 7.1 +/- 3.65 for EGFP-[M137]hNaPi-IIc compared with wild-type. Two-electrode studies indicate that EGFP-[M137]hNaPi-IIc is nonelectrogenic but displayed a significant phosphate-independent inward-rectified sodium current, which appears to be insensitive to phosphonoformic acid. M137 thus may uncouple sodium-phosphate cotransport, suggesting that this amino acid residue has an important functional role in human NaPi-IIc.