Nephrin-mutations in congenital nephrotic syndrome of the Finnish type and cell lineage specific gene regulation
Author: Beltcheva, Olga
Date: 2005-05-12
Location: Hillarpsalen, Retzius väg 8, Karolinska Institutet
Time: 10.00
Department: Institutionen för medicinsk biokemi och biofysik (MBB) / Department of Medical Biochemistry and Biophysics
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Thesis (475.7Kb)
Abstract
Kidneys, the main excretory organs in vertebrates, play central role in removing water soluble metabolic waste products from the organism. Many acquired and inherited renal diseases in man lead to kidney dysfunction and nephrotic syndrome - life-threatening condition affecting increasing number of people in the modem society. Congenital nephrotic syndrome of the Finnish type (CNF) is one such disease of the renal filter. CNF is an authosomal, inherited disease characterised by development of nephrotic syndrome shortly after birth. In 1998 Kestilä et al. reported the positional cloning of the gene defective in CNF patients. The gene product was termed nephrin due to its nephron specific expression. In Finnish patients two common mutations were identified, Finmajor and Finnminor, which account for about 94% of the CNF cases. In contrast, most of the patients outside Finland have unique, individual mutations".
This thesis work presents the summarized results from the mutation screening done for more than 30 non-Finnish families. In the course of the study 20 novel disease causing nucleotide changes were identified including missense, nonsense, frameshift and splice mutations, as well as small deletions and insertions. Missense mutations are the most common and account for almost half of all reported CNF causing nucleotide changes in the nephrin gene.
Nephrin is a transmembrane protein belonging to the lg super family. In man it is found only in podocytes, where it is localised in the slit diaphragm and has central role for organisation and maintenance of the renal ultrafilter. In rodents nephrin is expressed not only in kidney, but also in central nervous system and pancreas.
One of the aims of the presents work was to study the cis- and trans-regulatory elements directing this tissue specific expression of the gene in mice. For that purpose five different sequences from the nephrin upstream region were cloned in front of betagalactosidase as a reporter gene and the resulting constructs were used for generation of transgenic mice. Several independent transgenic lines were generated with each construct. The tissue expression of LacZ in each line was studied by histochemistry. The data from the transgenic mice indicated presence of kidney and brain specific regulatory elements in the region -4 kb; -2.1 kb (where the first nucleotide of the ATG codon of nephrin is +1). The sequence between -1.9 and -1.2 kb was found to be important for spinal cord and pancreas expression.
Further studies of the -4; -2.1 kb podocyte specific enhancer region based on homology between mouse and man led to the identification of a region specifically recognised and bound by putative transcription factor(s) expressed in podocytes. A stretch of six guanines was shown to be essential for the binding of that protein(s) from podocyte nuclear extract. This, as yet, unknown transcription factor is a zincfinger protein other than WT1. It was found to be expressed by mouse podocytes and human embryonic kidney cells.
The transgenic mouse experiments resulted also in the identification of a novel nephrin isoform, expressed in rodent brains along with the regular nephrin. This brain specific nephrin has an alternative exon 1, exon 1B, situated approximately 1.5 kb upstream of the original first exon, now designated 1A. Exon 1B is spliced with exons 2 through 30, thus giving rise to a protein differing from the regular nephrin only in the sequence coding for the signal peptide. A detailed study of the expression of nephrin in brain of wild type and nephrin knockout mice was carried out for the first time as a part of this thesis work.
The summarized results from the nephrin gene regulation studies presented in this thesis showed that the differential expression of nephrin is controlled by both alternative splicing in the 5' end of the gene, giving rise to alternative promoters and existence of tissue specific enhancer elements.
This thesis work presents the summarized results from the mutation screening done for more than 30 non-Finnish families. In the course of the study 20 novel disease causing nucleotide changes were identified including missense, nonsense, frameshift and splice mutations, as well as small deletions and insertions. Missense mutations are the most common and account for almost half of all reported CNF causing nucleotide changes in the nephrin gene.
Nephrin is a transmembrane protein belonging to the lg super family. In man it is found only in podocytes, where it is localised in the slit diaphragm and has central role for organisation and maintenance of the renal ultrafilter. In rodents nephrin is expressed not only in kidney, but also in central nervous system and pancreas.
One of the aims of the presents work was to study the cis- and trans-regulatory elements directing this tissue specific expression of the gene in mice. For that purpose five different sequences from the nephrin upstream region were cloned in front of betagalactosidase as a reporter gene and the resulting constructs were used for generation of transgenic mice. Several independent transgenic lines were generated with each construct. The tissue expression of LacZ in each line was studied by histochemistry. The data from the transgenic mice indicated presence of kidney and brain specific regulatory elements in the region -4 kb; -2.1 kb (where the first nucleotide of the ATG codon of nephrin is +1). The sequence between -1.9 and -1.2 kb was found to be important for spinal cord and pancreas expression.
Further studies of the -4; -2.1 kb podocyte specific enhancer region based on homology between mouse and man led to the identification of a region specifically recognised and bound by putative transcription factor(s) expressed in podocytes. A stretch of six guanines was shown to be essential for the binding of that protein(s) from podocyte nuclear extract. This, as yet, unknown transcription factor is a zincfinger protein other than WT1. It was found to be expressed by mouse podocytes and human embryonic kidney cells.
The transgenic mouse experiments resulted also in the identification of a novel nephrin isoform, expressed in rodent brains along with the regular nephrin. This brain specific nephrin has an alternative exon 1, exon 1B, situated approximately 1.5 kb upstream of the original first exon, now designated 1A. Exon 1B is spliced with exons 2 through 30, thus giving rise to a protein differing from the regular nephrin only in the sequence coding for the signal peptide. A detailed study of the expression of nephrin in brain of wild type and nephrin knockout mice was carried out for the first time as a part of this thesis work.
The summarized results from the nephrin gene regulation studies presented in this thesis showed that the differential expression of nephrin is controlled by both alternative splicing in the 5' end of the gene, giving rise to alternative promoters and existence of tissue specific enhancer elements.
List of papers:
I. Beltcheva O, Martin P, Lenkkeri U, Tryggvason K (2001). Mutation spectrum in the nephrin gene (NPHS1) in congenital nephrotic syndrome. Hum Mutat. 17(5): 368-73.
Pubmed
II. Beltcheva O, Kontusaari S, Fetissov S, Putaala H, Kilpelainen P, Hokfelt T, Tryggvason K (2003). Alternatively used promoters and distinct elements direct tissue-specific expression of nephrin. J Am Soc Nephrol. 14(2): 352-8.
Pubmed
III. Beltcheva O, Hjorleifsdottir E, Kontusaari S, Trggvason K (2005). Characterization of a nephrin gene regulatory element active in podocytes. [Manuscript]
I. Beltcheva O, Martin P, Lenkkeri U, Tryggvason K (2001). Mutation spectrum in the nephrin gene (NPHS1) in congenital nephrotic syndrome. Hum Mutat. 17(5): 368-73.
Pubmed
II. Beltcheva O, Kontusaari S, Fetissov S, Putaala H, Kilpelainen P, Hokfelt T, Tryggvason K (2003). Alternatively used promoters and distinct elements direct tissue-specific expression of nephrin. J Am Soc Nephrol. 14(2): 352-8.
Pubmed
III. Beltcheva O, Hjorleifsdottir E, Kontusaari S, Trggvason K (2005). Characterization of a nephrin gene regulatory element active in podocytes. [Manuscript]
Issue date: 2005-04-21
Rights:
Publication year: 2005
ISBN: 91-7140-351-5
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