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Volume 65, Issue 6, Pages (June 2004)

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1 Volume 65, Issue 6, Pages 2030-2040 (June 2004)
A large tandem duplication within the COL4A5 gene is responsible for the high prevalence of Alport syndrome in French Polynesia  Christelle Arrondel, Georges Deschênes, Yannick Le Meur, Amandine Viau, Christophe Cordonnier, Alain Fournier, Stephane Amadeo, Marie-Claire Gubler, Corinne Antignac, Laurence Heidet  Kidney International  Volume 65, Issue 6, Pages (June 2004) DOI: /j x Copyright © 2004 International Society of Nephrology Terms and Conditions

2 Figure 1 Electron microscopy from a 33-year-old Tahitian male patient with hematuria, proteinuria and renal failure. A regular thinning of the glomerular basement membrane is observed in one preserved glomerulus (uranyl acetate-lead citrate, ×5000). Kidney International  , DOI: ( /j x) Copyright © 2004 International Society of Nephrology Terms and Conditions

3 Figure 2 Pedigree of family I and haplotypes of some genotyped family members from one branch of family I. (A) Open squares/circles represent unaffected individuals, black squares/circles represent affected individuals, and gray squares/circles represent individuals with an unknown clinical status. Open circles with a central dot represent obligate carriers with an unknown clinical status. (B) The branch of family I (branch 13) descends from the affected son of S-L indicated by the arrowhead. Only genotyped individuals from branch 13 are shown. Haplotypes are indicated for 4 polymorphic microsatellite loci, covering a ∼6 megabases interval at Xq22. The marker 2B6 is located within the COL4A5 gene (20). All affected individuals carry the same haplotype at the COL4A5 locus. However, some unaffected individuals also carry that haplotype (arrows). Kidney International  , DOI: ( /j x) Copyright © 2004 International Society of Nephrology Terms and Conditions

4 Figure 3 Analysis of the restriction fragment patterns found in a control individual (c) and a Polynesian patient (p), restriction map of the region and illustration of the proposed mechanisms for the recombination event resulting in the mutated allele. (A) After digestion with NruI, NotI, or SacII and hybridization with any COL4A5 cDNA probe, restriction fragments were ∼150kb larger in Polynesian patients than in control DNA. After digestion with SfiI and hybridization with JZ4, a cDNA probe covering exons 1 to 9, a ∼65kb fragment was observed in a Polynesian patient in addition to the normal restriction fragments. (B) The long range restriction map around the COL4A5 gene is from (23); Note: NotI, Nru: NruI, Sac: SacII, X: XhoI, F: SfiI. COL4A5 exons 1, 2, 3, 9, 36, 37, and 51 are shown by black boxes. For clarity, the other exons are not indicated. The cross schematizes unequal crossing-over between COL4A5 introns 1 and 36. (C) Schematic representation of the mutant COL4A5 allele resulting from unequal recombination, containing a tandem gene duplication of ∼130kb, extending from intron 1 to intron 36. We used several probes located at regular intervals in intron 1 and in intron 36 to hybridize SfiI-digested DNA, and thereby mapped the most proximal region of intron 1 hybridizing the abnormal 65kb fragment to between nt 70,960 and 73,957 of intron 1, and the most distal region of intron 36 hybridizing the abnormal 65kb fragment to between nt 14,259 and 25,934 of intron 36. This was consistent with the size of an SfiI junction fragment bounded by the SfiI site in intron 35 (5′) (F2), and the SfiI site in exon 3 (3′) (F1). After XhoI digestion, a probe covering nt 74,556 to 75,175 of intron 1 and a probe containing exon 36 hybridized, in addition to the normal fragments, an abnormal 40kb fragment (not shown), in agreement with a junction fragment bounded by XhoI sites in intron 31 (5′) (X2) and in intron 1 (3′) (X1). Kidney International  , DOI: ( /j x) Copyright © 2004 International Society of Nephrology Terms and Conditions

5 Figure 4 Schematic representation of the 3′ end of the normal (top) and mutated (bottom) COL4A5 RNAs, reverse transcription-polymerase chain reaction (RT-PCR) amplification of patients (p) and control (c) COL4A5 fibroblast cDNA with a forward primer located in exon 34 and a reverse primer in exon 2, sequencing analysis of the amplified cDNA in a Polynesian patient, Northern blot analysis of fibroblast RNAs from control (c) and from a male Polynesian patient (p) after hybridization with a COL4A5 cDNA probe. (A) COL4A5 exons are represented by boxes and are numbered. In the mutant RNA, a splicing occurred between exon 36 and exon 2. The locations of the primers used for RT-PCR amplification of the mutant cDNA shown in (B) are indicated by arrows. (B) RT-PCR amplification of fibroblast RNA from a male patient generated a 303bp mutant specific product. RT+ with reverse transcriptase; RT- without reverse transciptase. (C) Sequencing of this product showed that nt 3246 in the patient COL4A5 transcript was followed by nt 82, corresponding to the fusion of exon 36 and exon 2. (D) Northern blot analysis showed a transcript of ∼6.5kb in the control and a transcript of ∼9.6kb in the patient. Kidney International  , DOI: ( /j x) Copyright © 2004 International Society of Nephrology Terms and Conditions

6 Figure 5 EcoRI restriction pattern of genomic DNA of a patient (p) and control (c), polymerase chain reaction (PCR) amplification of genomic DNA and DNA sequencing analysis of the COL4A5 mutation in a male Polynesian patient. (A) After digestion with EcoRI and hybridization with the COL4A5 int A probe, located at nt 74,556 to 75,175 of intron 1, a 7.2kb junction fragment was observed in the patient's DNA in addition to the expected 8.3kb fragment. (B) Multiplex PCR amplification of genomic DNA with a forward primer located at nt 14,993 of intron 36 and a reverse primer located at nt 71,896 of intron 1 yielded a 1136bp product in affected individual (p) but not in control (c), whereas the ∼200bp microsatelite marker D17S1843 was amplified in patient and control. (C) Sequencing analysis showed that nt 16,073 of intron 36 is followed by intron 1 sequence found 27,699 nt upstream of exon 2. Sequence of the mutant allele and the corresponding regions of the normal allele in introns 36 and 1 are aligned. The box shows the 4bp sequence that is identical between intron 36 and intron 1, where the recombination occurred. Kidney International  , DOI: ( /j x) Copyright © 2004 International Society of Nephrology Terms and Conditions

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