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Pkhdl1, a homolog of the autosomal recessive kidney disease gene

Pkhdl1, a homolog of the autosomal recessive kidney disease gene description/claims

This application is a continuation-in-part and claims benefit under 35 U.S.C. § 119(a) of International Application No. PCT/US2004/004300 having an International Filing Date of Feb. 12, 2004, which published in English as International Publication Number WO 2004/072268, and which claims priority to U.S. Provisional Application Ser. No. 60/446,860, filed Feb. 12, 2003.

Funding for the work described herein was provided in part by the federal government: National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) grants DK58816, DK59597, and DK59505. The federal government may have certain rights in the invention.

This invention relates to PKHDL1, a homolog of the autosomal recessive kidney disease gene, and more particularly, to PKHDL1 nucleic acids and polypeptides, and variants thereof.

Autosomal recessive polycystic kidney disease (ARPKD) is an important renal cause of death in the perinatal period and of childhood renal failure. Neonatal disease presentation is typical, and characterized by greatly enlarged kidneys due to fusiform dilation of collecting ducts; congenital hepatic fibrosis is often a major complication in older patients. Progress toward understanding this complex disorder has recently been made by the identification of the disease-causing gene, PKHD1, in chromosome region 6p12. PKHD1 is a very large gene (˜470 kb) containing 67 exons and an open reading frame (ORF) of 12,222 bp. PKHD1 has a tissue-specific expression pattern with the highest levels in fetal and adult kidney and lower levels in liver, pancreas and lung. The murine ortholog, Pkhd1, has recently been described.

A notable feature of both the human and murine genes is that multiple different splice forms may be generated. Visualization of PKHD1 transcripts by northern analysis has proved difficult with a smear of products often detected. These may represent multiple splice forms, unusual sensitivity of this transcript to degradation, or a combination of these factors. In situ hybridization of the murine transcript showed expression in the developing kidney and mature collecting ducts, plus ductal plate and bile ducts in the liver. Other sites of expression during development detected by in situ analysis were: large vessels, testis, sympathetic ganglia, pancreas and trachea with evidence that some sites of expression may be of specific splice forms.

The PKHD1 encoded protein, fibrocystin, is large (4074 aa) and predicted to be an integral membrane protein with a large extracellular region and a short cytoplasmic tail. Fibrocystin is not closely related to any other characterized protein, although it contains multiple copies of a defined domain and has regions of homology to other proteins; it seems to represent the founder member of a new protein family. The only well characterized domain in fibrocystin is the TIG/IPT (immunoglobulin-like fold shared by plexins and transcription factors) that is also found in the hepatocyte growth factor receptor (HGFR), plexins and other receptor molecules. Although fibrocystin has many more copies of this domain than these other proteins, the presence of the TIG domain, along with the structure of the protein, suggested that it may also act as a receptor.

The invention is based on the identification, cloning, and sequence analysis of PKHDL1 and Pkhdl1, human and murine homologs, respectively, of the ARPKD gene PKHD1. The PKHD1 homologs encode fibrocystin-L, a large receptor protein (approximately 466 kDa) that contains a signal peptide, a single transmembrane domain, and a short cytoplasmic tail. Fibrocystin-L has low, but highly significant, homology to fibrocystin over the entire length of the protein, except the extreme C-terminal region containing the predicted transmembrane domain and cytoplasmic tail. This level of homology is greater than that seen between polycystin homologs, establishing the fibrocystins as a new protein family. PKHDL1 expression is up-regulated specifically in T lymphocytes and may have a role in cellular immunity. PKHDL1 expression also is up-regulated in endometrial cancer and other cancers, including breast, ovarian, and colon cancers.

In one aspect, the invention features an isolated nucleic acid that includes a sequence encoding a fibrocystin-L polypeptide. The fibrocystin-L polypeptide can be encoded by SEQ ID NO:1 or SEQ ID NO:2, and can include the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4. The fibrocystin-L polypeptide can include an amino acid sequence variant at a position selected from the group consisting of: position 702, position 1192, position 1199, position 1223, position 1514, position 1607, position 1638, position 3050, position 3607, and position 4220 of SEQ ID NO:3. For example, the amino acid sequence variant can be selected from the group consisting of: Pro at position 702, Ala at position 1192, Ser at position 1199, Val at position 1223, Ser at position 1514, Ile at position 1607, Cys at position 1638, Gln at position 3050, Glu at position 3607, and Ile at position 4220. The isolated nucleic acid can include a sequence variant with respect to SEQ ID NO:1, e.g., a sequence variant at a position selected from the group consisting of: position 1227, position 1404, position 1920, position 1965, position 2105, position 3574, position 3599, position 3668, position 4540, position 4819, position 4913, position 6621, position 9084, position 9150, position 10821, and position 12658 of SEQ ID NO:1. The sequence variant can be selected from the group consisting of: A at position 1227, T at position 1404, C at position 1920, G at position 1965, C at position 2105, G at position 3574, C at position 3599, T at position 3668, A at position 4540, A at position 4819, G at position 4913, G at position 6621, T at position 9084, G at position 9150, A at position 10821, and A at position 12658.

In another aspect, the invention features an isolated nucleic acid encoding a fibrocystin polypeptide, wherein the nucleic acid includes at least 300 contiguous nucleotides of SEQ ID NO:1 or a sequence variant thereof. The invention also features a vector that includes such isolated nucleic acids and host cells including the vector.

The invention also features an isolated nucleic acid 10 to 1700 nucleotides in length, the nucleic acid including a sequence, the sequence including one or more sequence variants relative to the sequence of SEQ ID NO:1, wherein the sequence is at least 80% identical over its length to the corresponding sequence in SEQ ID NO:1. The sequence variant can be at a position selected from the group consisting of: position 1227, position 1404, position 1920, position 1965, position 2105, position 3574, position 3599, position 3668, position 4540, position 4819, position 4913, position 6621, position 9084, position 9150, position 10821, and position 12658 of SEQ ID NO:1.

In yet another aspect, the invention features a plurality of oligonucleotide primer pairs (e.g., at least three, 13, 16, or 23 primer pairs), wherein each primer is 10 to 50 nucleotides in length, and wherein each primer pair, in the presence of mammalian genomic DNA and under polymerase chain reaction conditions, produces a nucleic acid product corresponding to a region of an PKHDL1 nucleic acid molecule, wherein the product is 30 to 1700 nucleotides in length. The nucleic acid product can include a nucleotide sequence variant relative to SEQ ID NO:1.

The invention also features a composition that includes a first oligonucleotide primer and a second oligonucleotide primer, wherein the first oligonucleotide primer and the second oligonucleotide primer are each 10 to 50 nucleotides in length, and wherein the first and second primers, in the presence of mammalian genomic DNA and under polymerase chain reaction conditions, produce a nucleic acid product corresponding to a region of a PKHDL1 nucleic acid molecule, wherein the product is 30 to 1700 nucleotides in length. The nucleic acid product can include a nucleotide sequence variant relative to SEQ ID NO:1. Isolated nucleic acids that include the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:2, or the complement of SEQ ID NO:1 or SEQ ID NO:2, also are featured.

In yet another aspect, the invention features an antibody having specific binding affinity for a fibrocystin-L polypeptide. Such antibodies can be used to detect fibrocystin-L in biological samples.

The invention also features a method for determining if a subject has altered cellular immunity. The method includes providing a nucleic acid sample (e.g., genomic DNA) from the subject, and determining whether the nucleic acid sample contains one or more sequence variants within the PKHDL1 gene of the subject relative to a wild-type PKHDL1 gene, wherein the presence of the one or more sequence variants is associated with altered cellular immunity in the subject. The determining step can be performed by denaturing high performance liquid chromatography or direct sequencing. The variant can be at position 2105, position 3574, position 3599, position 3668, position 4540, position 4913, or position 9150 of SEQ ID NO:1, or other positions. The method further can include identifying the sequence variant by DNA sequencing.

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