Exome sequencing reveals cubilin mutation as a single-gene cause of proteinuria

In two siblings of consanguineous parents with intermittent nephrotic-range proteinuria, we identified a homozygous deleterious frameshift mutation in the gene CUBN, which encodes cubulin, using exome capture and massively parallel re-sequencing. The mutation segregated with affected members of this family and was absent from 92 healthy individuals, thereby identifying a recessive mutation in CUBN as the single-gene cause of proteinuria in this sibship. Cubulin mutations cause a hereditary form of megaloblastic anemia secondary to vitamin B(12) deficiency, and proteinuria occurs in 50% of cases since cubilin is coreceptor for both the intestinal vitamin B(12)-intrinsic factor complex and the tubular reabsorption of protein in the proximal tubule. In summary, we report successful use of exome capture and massively parallel re-sequencing to identify a rare, single-gene cause of nephropathy.     

Genotype/Phenotype Correlation in Nephrotic Syndrome Caused by WT1 Mutations

Background and objectives: The risk of developing Wilms tumor (WT) can be present or absent in patients with nephrotic syndrome (NS) caused by WT1 mutations. Here, the genotype/phenotype correlation regarding the outcome and risk for WT in 52 patients from 51 families with NS due to WT1 mutations is described.Design, setting, participants, & measurements: This study followed 19 patients with mutations in intron 9 splice donor site (KTS mutations), 27 patients with missense mutations, 4 patients with nonsense mutations, 1 patient with a splice site mutation in intron 8, and 1 patient with a deletion.Results: Twenty-four different WT1 mutations were detected. Sixteen of the 19 patients with KTS mutations were females. These patients had isolated NS if karyotype was 46,XX and Frasier syndrome if karyotype was 46,XY. Patients with KTS mutations presented at a significantly older age and with a slower progression toward chronic kidney disease (CKD) stage 5, compared with missense mutations. Patients with nonsense mutations presented initially with WT. Six patients with missense mutations developed WT after the diagnosis of NS (interval-range from NS onset to WT of 0.1 to 1.4 years).Conclusions: (1) KTS mutations cause isolated NS with absence of WT in 46,XX females. (2) KTS mutations cause Frasier syndrome with gonadoblastoma risk in 46,XY phenotypic females. (3) KTS mutations cause NS with a slower progression when compared with missense mutations. (4) Missense mutations can occur with and without WT. (5) WT1 analysis is important in young patients with NS for early detection and tumor prophylaxis.WT1 mutations can lead to three distinct clinical entities that are associated with the glomerular disease of steroid-resistant nephrotic syndrome (SRNS): (1) Denys-Drash syndrome (DDS) (1); (2) Frasier syndrome (FS) (2); and (3) isolated NS (3,4). DDS is classically characterized by the triad of infantile SRNS, ambiguous genitalia, and Wilms tumor (WT) (5,6). The characteristic renal biopsy finding in DDS is diffuse mesangial sclerosis (DMS) (6,7). Incomplete forms of DDS have been reported with early glomerulopathy associated with either major urogenital abnormalities or WT (6). FS is characterized by the association of SRNS with male pseudohermaphroditism. In FS, the onset of NS occurs later than in DDS with typical histologic findings of focal segmental glnomerulosclerosis (FSGS) (6). The term “isolated NS” is used here to denote NS without accompanying WT or major urogenital abnormalities. In more than 95% of cases with NS and WT1 mutations, the mutations are located in exon 8 or 9 of WT1 (4,8–10). FS is caused by mutations in the donor splice-site of intron 9. The predicted consequence of mutations in this site is the heterozygous loss of the three amino acid residues KTS (referred from here as KTS mutations) with a resulting reduction in the ratio of the isoforms +KTS/-KTS (11).There is still uncertainty about the appropriate management of these patients. An important clinical issue emerges in cases where the diagnosis of SRNS caused by WT1 mutation is made, yet without the diagnosis of a tumor. The known risk of WT in DDS patients and of gonadoblastoma in FS patients creates a challenge to the clinician in terms of surveillance and tumor prevention (12,13). This uncertainty led us to investigate the clinical outcome in a large cohort of 52 patients from 51 different families with WT1 mutations as the cause of NS. Our goal was to describe the genotype/phenotype correlation in this large group of patients. Our hypothesis was that specific mutations may predict isolated NS without the manifestation of WT or gonadoblastoma.     

Mild Recessive Mutations in Six Fraser Syndrome–Related Genes Cause Isolated Congenital Anomalies of the Kidney and Urinary Tract

Congenital anomalies of the kidney and urinary tract (CAKUT) account for approximately 40% of children with ESRD in the United States. Hitherto, mutations in 23 genes have been described as causing autosomal dominant isolated CAKUT in humans. However, >90% of cases of isolated CAKUT still remain without a molecular diagnosis. Here, we hypothesized that genes mutated in recessive mouse models with the specific CAKUT phenotype of unilateral renal agenesis may also be mutated in humans with isolated CAKUT. We applied next-generation sequencing technology for targeted exon sequencing of 12 recessive murine candidate genes in 574 individuals with isolated CAKUT from 590 families. In 15 of 590 families, we identified recessive mutations in the genes FRAS1, FREM2, GRIP1, FREM1, ITGA8, and GREM1, all of which function in the interaction of the ureteric bud and the metanephric mesenchyme. We show that isolated CAKUT may be caused partially by mutations in recessive genes. Our results also indicate that biallelic missense mutations in the Fraser/MOTA/BNAR spectrum genes cause isolated CAKUT, whereas truncating mutations are found in the multiorgan form of Fraser syndrome. The newly identified recessive biallelic mutations in these six genes represent the molecular cause of isolated CAKUT in 2.5% of the 590 affected families in this study.     

Rapid Detection of Monogenic Causes of Childhood-Onset Steroid-Resistant Nephrotic Syndrome

Background and objectives

In steroid-resistant nephrotic syndrome (SRNS), >21 single-gene causes are known. However, mutation analysis of all known SRNS genes is time and cost intensive. This report describes a new high-throughput method of mutation analysis using a PCR-based microfluidic technology that allows rapid simultaneous mutation analysis of 21 single-gene causes of SRNS in a large number of individuals.

Design, setting, participants, & measurements

This study screened individuals with SRNS; samples were submitted for mutation analysis from international sources between 1996 and 2012. For proof of principle, a pilot cohort of 48 individuals who harbored known mutations in known SRNS genes was evaluated. After improvements to the method, 48 individuals with an unknown cause of SRNS were then examined in a subsequent diagnostic study. The analysis included 16 recessive SRNS genes and 5 dominant SRNS genes. A 10-fold primer multiplexing was applied, allowing PCR-based amplification of 474 amplicons in 21 genes for 48 DNA samples simultaneously. Forty-eight individuals were indexed in a barcode PCR, and high-throughput sequencing was performed. All disease-causing variants were confirmed via Sanger sequencing.

Results

The pilot study identified the genetic cause of disease in 42 of 48 (87.5%) of the affected individuals. The diagnostic study detected the genetic cause of disease in 16 of 48 (33%) of the affected individuals with a previously unknown cause of SRNS. Seven novel disease-causing mutations in PLCE1 (n=5), NPHS1 (n=1), and LAMB2 (n=1) were identified in <3 weeks. Use of this method could reduce costs to 1/29th of the cost of Sanger sequencing.

Conclusion

This highly parallel approach allows rapid (<3 weeks) mutation analysis of 21 genes known to cause SRNS at a greatly reduced cost (1/29th) compared with traditional mutation analysis techniques. It detects mutations in about 33% of childhood-onset SRNS cases.     

ARHGDIA mutations cause nephrotic syndrome via defective RHO GTPase signaling

Nephrotic syndrome (NS) is divided into steroid-sensitive (SSNS) and -resistant (SRNS) variants. SRNS causes end-stage kidney disease, which cannot be cured. While the disease mechanisms of NS are not well understood, genetic mapping studies suggest a multitude of unknown single-gene causes. We combined homozygosity mapping with whole-exome resequencing and identified an ARHGDIA mutation that causes SRNS. We demonstrated that ARHGDIA is in a complex with RHO GTPases and is prominently expressed in podocytes of rat glomeruli. ARHGDIA mutations (R120X and G173V) from individuals with SRNS abrogated interaction with RHO GTPases and increased active GTP-bound RAC1 and CDC42, but not RHOA, indicating that RAC1 and CDC42 are more relevant to the pathogenesis of this SRNS variant than RHOA. Moreover, the mutations enhanced migration of cultured human podocytes; however, enhanced migration was reversed by treatment with RAC1 inhibitors. The nephrotic phenotype was recapitulated in arhgdia-deficient zebrafish. RAC1 inhibitors were partially effective in ameliorating arhgdia-associated defects. These findings identify a single-gene cause of NS and reveal that RHO GTPase signaling is a pathogenic mediator of SRNS.     

COQ6 mutations in human patients produce nephrotic syndrome with sensorineural deafness

Steroid-resistant nephrotic syndrome (SRNS) is a frequent cause of end-stage renal failure. Identification of single-gene causes of SRNS has generated some insights into its pathogenesis; however, additional genes and disease mechanisms remain obscure, and SRNS continues to be treatment refractory. Here we have identified 6 different mutations in coenzyme Q10 biosynthesis monooxygenase 6 (COQ6) in 13 individuals from 7 families by homozygosity mapping. Each mutation was linked to early-onset SRNS with sensorineural deafness. The deleterious effects of these human COQ6 mutations were validated by their lack of complementation in coq6-deficient yeast. Furthermore, knockdown of Coq6 in podocyte cell lines and coq6 in zebrafish embryos caused apoptosis that was partially reversed by coenzyme Q10 treatment. In rats, COQ6 was located within cell processes and the Golgi apparatus of renal glomerular podocytes and in stria vascularis cells of the inner ear, consistent with an oto-renal disease phenotype. These data suggest that coenzyme Q10-related forms of SRNS and hearing loss can be molecularly identified and potentially treated.     

Late complications of radiotherapy for nasopharyngeal carcinoma

Objective

To evaluate and assemble late complications of radiotherapy in cases of nasopharyngeal cancer.

Methods

From October 2003 to January 2005, a prospective cohort study was done in a tertiary center, Chiang Mai University Hospital. Two hundred patients were evaluated for late complication according to the RTOG/EORTC late radiation morbidity scoring criteria.

Results

Of 200 patients, 131 were male (65.5%) and 69 female (34.5%). The mean age was 49.7 ± 13.5 years (11–78). The mean pre- and post-treatment body mass indexes (BMI) were 22.5 ± 4 (15–35.6), and 19.8 ± 3.2 (12.9–34.5; P < 0.05). Mean post-radiation period was 3.6 ± 3.4 years (0.3–18.6 years). The radiation dosage ranged from 60 to 76 Gy (mean 69 Gy). Most of the patients (92%) had undifferentiated (50.5%) and poorly differentiated (41.5%) squamous cell carcinoma. Eighty-eight percent of the patients were in Stage III and IV. Chemotherapy was given to 145 patients (72.5%). The mean post-radiation period in the added chemotherapy group was lower than the group treated with radiation alone (2.9 ± 2.7 years vs. 5.4 ± 4.4 years, P < .05). The most common complication was dryness of mouth (97.5%); followed by hearing impairment (inner ear 82.5%). Added chemotherapy increased the complication severity significantly for the skin (P < 0.05). The mean number of complications was 6.3 ± 2.2 (range from 1 to 12).

Conclusion

In this study, every patient had a more or less adverse reaction to radiation. Doctors need to be aware of these complications in order to prevent serious ones and to improve the patients’ quality of life in the long term.     

Identification of two novel CAKUT-causing genes by massively parallel exon resequencing of candidate genes in patients with unilateral renal agenesis

Congenital abnormalities of the kidney and urinary tract (CAKUT) are the most frequent cause of chronic kidney disease in children, accounting for about half of all cases. Although many forms of CAKUT are likely caused by single-gene defects, mutations in only a few genes have been identified. In order to detect new contributing genes we pooled DNA from 20 individuals to amplify all 313 exons of 30 CAKUT candidate genes by PCR analysis and massively parallel exon resequencing. Mutation carriers were identified by Sanger sequencing. We repeated the analysis with 20 new patients to give a total of 29 with unilateral renal agenesis and 11 with other CAKUT phenotypes. Five heterozygous missense mutations were detected in 2 candidate genes (4 mutations in FRAS1 and 1 in FREM2) not previously implicated in non-syndromic CAKUT in humans. All of these mutations were absent from 96 healthy control individuals and had a PolyPhen score over 1.4, predicting possible damaging effects of the mutation on protein function. Recessive truncating mutations in FRAS1 and FREM2 were known to cause Fraser syndrome in humans and mice; however, a phenotype in heterozygous carriers has not been described. Thus, heterozygous missense mutations in FRAS1 and FREM2 cause non-syndromic CAKUT in humans.     

Spontaneous extradural hematoma as a presentation of sinusitis: case report and literature review

Spontaneous extradural hematoma is rarely mentioned in literature as intracranial complications of sinusitis. The authors presented a girl with spontaneous extradural hematoma secondary to pansinusitis and reviewed the literature. In a child with spontaneous extradural hematoma without a history of head trauma, sinusitis should be excluded.     

Clinical Outcomes in Pediatric Renal Transplant Recipients Who Received Steroid-Based Immunosuppressive Regimen

Background

Although the clinical outcomes of pediatric renal transplantation (RT) in developed countries have improved significantly, the data on clinical outcomes in developing countries are wildly different.