The human polycystic kidney disease 2-like (PKDL) gene: exon/intron
structure and evidence for a novel splicing mechanism
Lei Guo, Minghua Chen, Nuria Basora, Jing Zhou
Renal Division, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA
Received: 26 July 1999 / Accepted: 16 September 1999
Abstract. Polycystin-L is a member of the expanding family of
polycystins. Mutations in polycystin-1 or -2 cause human autoso-
mal dominant polycystic kidney disease (ADPKD). The mouse
ortholog of PKDL, Pkdl, is deleted in a mouse line with renal and
retinal defects. We recently have shown that polycystin-L has
calcium channel properties. In the current study, we determined
the exon/intron organization of the PKDL gene and its alternative
splicing. We show that PKDL has 16 exons. All splice acceptor/
donor sites for these exons conform to the GT-AG rule. The po-
sitions of introns and the sizes of exons in the PKDL gene are very
similar to those of PKD2, except for the last two 3Ј end exons.
RT-PCR demonstrates the existence of at least three polycystin-L
splice variants: PKDL(⌬5), PKDL(⌬456), and PKDL(⌬15) that
are expressed in a tissue-specific manner. In addition, we have
localized polymorphic marker D10S603 to intron 4 and exon 5 of
PKDL. Elucidation of the gene structure, exact location, and al-
ternative splicing patterns of PKDL will facilitate its evaluation as
a candidate gene in cystic or other genetic disorders.
Human autosomal dominant polycystic kidney disease (ADPKD)
is one of the most common genetic diseases, affecting approxi-
mately 600,000 individuals and accounting for 10% of end-stage
renal disease cases in the United States. ADPKD is genetically
heterogeneous, with loci mapped to Chromosome (Chr) 16p13.3
(PKD1) and 4q21 (PKD2), and with at least one additional un-
mapped locus (Daoust et al. 1995; de Almeida et al. 1995). Poly-
cystin-1, the PKD1 gene product, is a 4303-amino acid membrane
glycoprotein containing a ∼2500-amino acid N-terminal extracel-
lular domain, 11 membrane-spanning regions, and a short intra-
cellular tail (International PKD consortium 1995; Hughes et al.
1995). Polycystin-2, encoded by PKD2, is a 968-amino acid mem-
brane protein with six membrane-spanning domains and intracel-
- and COOH-termini (Mochizuki et al. 1996). There is
∼25% identity and ∼50% similarity between polycystin-2 and an
∼450-amino acid portion of polycystin-1. These two proteins are
critical in organ development, because targeted mouse mutants
show that deficiency in either polycystin-1 or -2 leads to polycystic
kidney disease and perinatal lethality (Lu et al. 1997; Wu et al.
1998a). Both proteins are thought to be part of the same or parallel
signal transduction pathways that may be involved in tubular mor-
phogenesis. Interaction between the C-termini of the two proteins
has been demonstrated (Qian et al. 1997; Tsiokas et al. 1997).
PKDL, which encodes a new member of the polycystin protein
family designated polycystin-L, was recently identified by homol-
ogy with PKD1 and PKD2 (Nomura et al. 1998; Wu et al. 1998b).
Polycystin-L has 50% amino acid sequence identity and 71% ho-
mology to polycystin-2. Both polycystins have significant homol-
ogy to the ␣
subunits of voltage-activated Ca
channel and the
mammalian transient receptor potential channels (TRPC). PKDL is
not yet known to be mutated in ADPKD or other human genetic
disorders, but its murine ortholog, Pkdl, is deleted in Krd (kidney
and retinal defects) mice with renal and retinal defects (Nomura et
al. 1998). We have recently demonstrated that polycystin-L is a
calcium-regulated cation channel that is permeable to calcium ions
(Chen et al. 1999). It follows that disruption of pathways for ion
permeation may account primarily for the features of polycystic
PKDL has been assigned to 10q24 by radiation hybrid map-
ping and fluorescent in situ hybridization (Nomura et al. 1998).
Several inherited disease loci, including partial epilepsy (Ottman et
al. 1995), infantile-onset spinocerebellar ataxia (IOSCA; Nikali et
al. 1995, 1997), and urofacial syndrome (UFS; Wang et al. 1997)
have been mapped to this region. Therefore, we set out to deter-
mine the exon/intron structure of PKDL for the design of intronic
primers as a means of developing reagents necessary for muta-
Materials and methods
PCR and sequencing.
Genomic clone PAC346c12 was obtained from
the RPCI human P1-derived artificial chromosome library. PCR was per-
formed with exonic primers on PAC346c12 or human genomic DNA by
using HotstarTaq DNA polymerase (Qiagen, Hilden, Germany), and the
products were purified and sequenced directly with PCR primers on Ap-
plied Biosystems 377 automated DNA sequencers (Perkin-Elmer).
To obtain the sequence of intron 2 of PKDL, we performed inverse
PCR. Human genomic DNA (2 g) was digested by ApaI and ligated with
T4 DNA ligase. Forward and reverse primers derived from the known
exonic sequences (f104: 5ЈTGACTGAGAACACAGCTGAGAACC and
r102: 5ЈCATCTTCAGGCTTCTTGGGTTG; r104: 5ЈAGGAGACTCCA-
GTGTCTGATGGA and f106: 5ЈAGCTAAAGGTCCGCAATGAC),
which face away from each other, were used to generate PCR products that
were extracted from agarose gels, purified, and then directly sequenced
with PCR primers.
RT-PCR analysis of alternative splicing.
Total RNA from human
transformed lymphoblasts, a human hepatocellular carcinoma cell line
(HepG2), and various human fetal tissues was isolated using the TRIzol
protocol (GIBCO BRL/Life Technologies) and treated with DNase. cDNA
templates for amplification were synthesized by reverse transcription of 5
g total RNA using reverse transcriptase Superscript II (GIBCO BRL/Life
Technologies) with Oligo-dT primer. PCR was performed with two pairs of
exonic primers (f106: 5ЈAGCTAAAGGTCCGCAATGAC and r108:
5ЈGGCGAGGAACTCAAAGTCTG; f115: 5ЈGTCCCAGATTGATGCT-
GTAGGC and r115: 5ЈTGATAGCCACCATGGAAACC). PCR primers
f106 and r108 are located in exon 4 and exon 7, respectively. PCR primers
f115 and r115 are located in exon 14 and exon 16, respectively.
Correspondence to: J. Zhou at Harvard Institutes of Medicine, Room 520,
77 Louis Pasteur Ave., Boston, MA 02115, USA.
The nucleotide sequence data reported in this paper have been submitted to
GenBank and have been assigned the accession numbers AF153459–
Mammalian Genome 11, 46–50 (2000).
© Springer-Verlag New York Inc. 2000