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Journal Reading

Journal Reading. 2012-1-11 Present: F1 侯羿州 指導醫師：MA 張明揚. Brenner 9th ed. Chapter 73: Malformation of the kidney: structural and function consequences Kidney international January 2012. Introduction. Congenital abnormalities of the kidney and urinary tract (CAKUT)

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Journal Reading

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  1. Journal Reading • 2012-1-11 • Present: F1 侯羿州 • 指導醫師：MA 張明揚

  2. Brenner 9th ed. Chapter 73: Malformation of the kidney: structural and function consequences • Kidney international January 2012

  3. Introduction • Congenital abnormalities of the kidney and urinary tract (CAKUT) • 3–6 per 1000 live births and constitute 20–30% of all anomalies identified in the neonatal period. • 50% of all cases(ESRD in children).

  4. CAKUT • Multiple structures within one or both K-U tract be affected. • Mutation of particular gene in different affected individuals. • Mutation of different gene results in similar phenotype.

  5. Classification of CAKUT • Aplasia(agenesis) • Simple hypoplasia: < 2 SD • Dysplasia with or without cysts • Isolated dilation of the renal pelvis or ureter or both • Anomaly of position

  6. Epidemiology of CAKUT • 3–6 per 1000 live births50%: lower UT; 30% renal • Vesicluoureteral reflux: 25%Ureteropelvic junctional obstruction11% • Unilateral agenesis: 1/1000 autopsyUnilateral dysplasia: 1/3000 • Duplication of renal collecting system: the most common.

  7. Pathogenesis of CAKUT • Variable penetrance and phenotypic heterogenecity. • No clear mendelian pattern of inheritance. • Single-nucleotide polymorphism. eg:RET.

  8. Pathogenesis of CAKUT • Molecular:Ureter Budding: Double collecting if overgrowth ROBO2-> GDNF 增加 ->ectopic BMP4 -> GDNF-RET 抑制 -> renal dysgenesis and VU refluxUreter Branching: PAX2:mutation-> hypoplasia,VU reflux GDNF-RET: homozygous deletion->agenesis

  9. Pathogenesis of CAKUT • Control of GDNF in metanephron mesenchyme: SALL1 SIX1 EYA1-> GDNF supression-> dysgenesis • Hedgehog Signaling: GLI3 repressor • Medulla: Glypican-3 induce proliferation increased branching-> medullar destruction. • TCL2

  10. Environmental in Utero and CAKUT

  11. Functional Consequences of CAKUT • Nephron number decrease • Tubule number, cross-sectional area and cell maturation abnormal. • Insufficient elongation of Henle-> loss of concentration. • Loss of Na absorption and K excretion

  12. Clinical manifestation of CAKUT • Screening ultrasound: 16-20wks. Ureter visualized: bladder obstruction or VU reflux. • Amniotic fluid: 9th week. Oligohydramnios: bilateral ureter obstruction or renal dysplasia. • If absent after 20 months: lung maturation fails.

  13. Clinical Manifestation in the fetus. • Urine B2M, osmolarity -> decrease. Na-> increase Urine osmolar<210-> poor prognosis

  14. Clinical Manifestation of specific forms. • Renal agenesis->Unilateral and asymptomatic. Associated with VU reflux. • Renal dysplasia->Bilateral: easily found. Complicated with UTI. • Double collecting system:Most common Partial>complete

  15. Clinical Manifestation of specific forms. • Renal ectopy: Decreased renal function by DMSA. VU reflux 30%. • Renal fusion: Horseshoe kidneyLoin pain and hematuria. Renal calculi 20%. Wilm tumor(?)

  16. Overall approach to management of CAKUT • In utero: Reduce damage from UT obstruction Rescue pulmonary development->bladder-amniotic shunt • After delivery: Respiration, abdominal mass, ear abnormality, single umbilical a.

  17. Management of CAKUT in utero and postnatal period • Abdominal echo within 24 hrs. • Serum creatinine after 1 day >1mg/dL • Renal dysplasia:Voiding cystourethrography and DMSA.Treat HTN if (+) • Renal ectopy and fusion: Prophylactic antibiotics if VU reflux

  18. Identification of two novel CAKUT-causing genes by massively parallel exon resequencing of candidate genes in patients with unilateral renal agenesis • Kidney International 2012-01

  19. Introduction • CAKUT may be caused in many instances by single-gene defect.

  20. Introduction • Only few causative genes have been identified so far. • In multiethnic cohort of 538 patients from 456 different families with non- syndromic CAKUT, we detected mutations of four known dominant CAKUT genes, TCF2/HNF1b, PAX2, UMOD, and EYA1, in only 12 families (1.9%).

  21. Introduction • novel approach of high- throughput mutation analysis to be performed in pooled genomic DNA of 40 individuals and 30 candidate genes simultaneously using exon PCR and massively parallel exon resequencing. • The CAKUT candidate genes were genes that have been implicated in the severe developmental phenotype of unilateral renal agenesis in humans or mice.

  22. Result

  23. 40 DNA samples of individuals with CAKUT (29 with unilateral renal agenesis and 11 with other forms of CAKUT). • Massively parallel exon resequencing of 342 different exon PCR products generated was carried out on an Illumina/Solexa GAII platform • 75% of all reads mapped back to one of the 342 target sequences that contained the exons plus 100-bp adjacent intronic sequence.

  24. On average, about 95% of nucleotides in targeted coding regions had at least 400-fold coverage depth. • 20% fold is sufficient to detect heterozygous change.

  25. Mutation Carrier identification by Sanger Sequencing • 114 variants from normal reference sequence within the coding regions • 47 of which were known single-nucleotide polymorphisms (SNPs) • Eight additional variants were present in a cohort of 96 Caucasian healthy control individuals and are thought to be either as yet unannotated SNPs or false calls

  26. 11 were predicted to truncate the protein products and 16 others had a PolyPhen score higher than 1.4(probably damaged) • direct Sanger sequencing to identify the mutation carrier(s) • 10 of the 27 variants are confirmed. • 3 out of 10 variants were not segregating.(DNA of relatives was not available)

  27. The remaining seven variants were heterozygous missense mutations in four genes

  28. Finding • 4 of 7 heterozygous mutation: FRAS1, FREM2, RET, BMP4. • FRAS1, FREM2: never reported in nonsyndromic CAKUT. • 2 FRAS1, 1 FREM: from Arabic and India respectively • Only A2381 II21 has nonsense mutation.

  29. Finding • A2381 II2:FRAS1-R3273H maternal FRAS1-R2621X Paternal • RET, BMP4: novel mutation

  30. Discussion • Fraser syndrome, a rare multi-organ disorder characterized by cryptophthalmos, cutaneous syndactyly, and renal agenesis. • Recessive mutation • Fras1, a protein containing repeats of the chondroitin sulfate proteoglycan core domain, the function of which is to maintain epithelial cell integrity

  31. FRAS1 • Homozygous Fras1 mutation leads to premature stop codon in blebbed mice. • Single heterozygous missense mutations of FRAS1 in non-syndromic unilateral renal agenesis. • SALL1 in human Townes–Brocks syndrome: Heterozygous mutation. Eya1 : both heterozygous and homozygous

  32. FRAS1 • (R3273H and R2621X): as this individual had only unilateral renal agenesis, but not full-spectrum Fraser syndrome. • The mutations were in trans, this might be compound heterozygous inheritance. • Variable expression: rare, but reported in other disease, such as ARPKD, congenital hepatic fibrosis, etc.

  33. FREM2 • Frem2, a FRAS1-related extracellular matrix protein 2 gene. • Frem2 was expressed in mesonephric and metanephric epithelium, especially in the ureteric bud. • Homozygous mutation in FREM2 in two families with Fraser syndrome not linked to FRAS1. • Heterozygous: unilateral renal agenesis.

  34. Criteria of rare genetic variant • The absence of the allele from 50 ethnically matched control individuals • ‘allelic strength’ as supported by the finding that the variant leads to truncation or absence of the gene product • previous publication of the allele as disease causing • functional studies in cell-based systems or animal models, if available

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