Screening for Mitochondrial Disorders in Children with SRNS

Based on the available evidence, patients collected in the Registry will be screened for:
Mutations in the COQ2 and PDSS2 genes if they meet any of the following criteria:

• SRNS associated with neuro-muscular symptoms
• SRNS with abnormal SDH and/or COX by renal histochemistry
• SRNS with high urinary lactate and 3-OH butyrate excretion
• SRNS with abnormal mitochondria on electron micrography (all clinical groups of the consortium will be asked to carefully review renal electron micrographies to select cases with potential primary or secondary mitochondrial damage).
• Siblings of patients with proven CoQ10 biosynthesis defects

Preferably, ubiquinone levels will be measured on kidney or muscle specimens or in cultured fibroblasts, prior to the genetic analysis.
B. tRNALeu A3243G mutation if they meet any of the following criteria:

•   SRNS/proteinuria with hearing loss (reported frequency: 80-90%)
•   SRNS/proteinuria with neuro-muscular involvement (reported frequency: 10-20%)
•   SRNS/proteinuria with diabetes mellitus (reported frequency: 20-30%)
•   SRNS/proteinuria with cardiomyopathy or retinopathy (reported frequency: rare)

Current data suggests that ubiquinone biosynthesis defects involve primarily congenital and early onset SRNS, whereas tRNA Leu mutations are detected mostly in SRNS and glomerular proteinurias, developing during adolescence and early adulthood.

The analysis will be performed by direct sequencing or pre-screening with DHPLC analysis followed by direct sequencing, according to already established protocols in the Rome and Genova laboratories. If no COQ2-PDSS2 mutation is detected but a mitochondrial disorder highly likely, biochemical analyses will extended on muscle biopsies or cultured fibroblasts, to diagnose other forms of mitochondrial cytopathies. If ubiquinone depletion is present, other known CoQ10 biosynthesis genes will be screened.

Whenever possible histochemical analysis will also be performed. Unlike biochemical studies, these tests can be performed on 5 mm frozen renal sections incubated with cytochrome C in the presence of diaminobenzidine or with succinate in the presence of nitroblutetrazoline, to assess COX and SDH activities respectively. Each section will be analysed together with a control renal specimen. This method is well established and regularly used in the Rome laboratory. Generating semi-quantitative results, it allows for rapid detection of mitochondrial abnormalities which will also be used to screen biopsy samples of patients with proven mutations in the NPHS1 or NPHS2 genes.

Finally, the project will promote prospective therapeutic interventions in children with suspected CoQ10 deficiency with high doses of ubiquinone (30 mg/Kg), until the results of the genetic tests are obtained. These patients include all patients that meet the inclusion criteria reported above. The evolution of the nephrotic syndrome and renal function, as well of other extrarenal symptoms will be carefully monitored.