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Human Molecular Genetics Advance Access originally published online on September 14, 2004
Human Molecular Genetics 2004 13(21):2607-2612; doi:10.1093/hmg/ddh293
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Human Molecular Genetics, Vol. 13, No. 21 © Oxford University Press 2004; all rights reserved

Reduced KIAA0471 mRNA expression in Alzheimer's patients: a new candidate gene product linked to the disease?

Lluïsa de Yebra1,*, Rosa Adroer1, Nuria de Gregorio-Rocasolano2, Rafael Blesa3, Ramon Trullas2 and Nicole Mahy1

1Unitat de Bioquímica, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Facultat de Medicina, Universitat de Barcelona, C/Casanova 143, 08036 Barcelona, Spain, 2Unitat de Neurobiologia, Institut d'Investigacions Biomèdiques de Barcelona, CSIC, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), C/Rosselló 161, 08036 Barcelona, Spain and 3Servei de Neurologia, Hospital de la Santa Creu i Sant Pau, C/St. Antoni M. Claret 167, 08025 Barcelona, Spain

Received May 13, 2004; Revised July 6, 2004; Accepted September 4, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Alzheimer's disease (AD) phenotype complexity raises the question whether genetic features remain unknown. Although a few percentage of patients are familial cases linked to mutations in amyloid precursor protein, presenilin 1 or presenilin 2 genes, the remainder are considered mainly sporadic late-onset cases with a complex etiology. However, changes in gene expression or other genetic features of the individual can clearly contribute to develop the illness. Consequently, in this paper we have focused on the identification of new genes, the expression of which is altered in AD. We used the technique of differential display reverse transcriptase–polymerase chain reaction (DDRT–PCR) in order to study the gene expression differences in brain tissue from patients in an advanced stage of AD. After studying medial septum and hippocampus brain areas, we found an inhibition of the KIAA0471 gene expression in three out of six AD patients, including one with a presenilin 1 gene mutation. This gene encodes for a large protein that presents, in its predicted form, 95% homology with IDN4-GGTR sequences. These results may provide significant clues for understanding the molecular mechanisms underlying septohippocampal neurodegeneration. In addition, they may open a new area of research for diagnostic and therapeutic tools, the relevance of which is also considered.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Although Alzheimer's disease (AD) is one of the most common illnesses of modern society, affecting ~10% of population over 65 years, molecular bases of the disease remain unclear. Familial forms that account for <5% of the total number of cases have been linked to mutations in three different genes: the amyloid precursor protein (APP) gene on chromosome 21 (1), the presenilin 1 (PS1) gene on chromosome 14 (2,3) and the presenilin 2 (PS2) gene on chromosome 1 (4,5). Nevertheless, it is well accepted that some genetic profiles predispose some individuals to suffer the illness more than others, as in case of apolipoprotein {varepsilon}4 (apoE) allele, which has been demonstrated to be a susceptibility gene for AD (6).

The septohippocampal system plays an important role in memory and cognition; how it is affected in AD patients is characterized by a reduction of cholinergic activity and a significant atrophy of the medial septum and diagonal band of Broca (MS–DBB) that correlates with the grade of cognitive and memory deficits (7). As pathological changes that arise in diseases are mostly believed to be driven by changes in gene expression (8–10), the aim of this study was to disentangle the different pattern of the genetic expression that characterize AD in the medial septum and hippocampus.

The differential display reverse transcriptase–polymerase chain reaction (DDRT–PCR) technique is quite unique in its potential to visualize the expressed genes in an eukaryotic cell in a systematic, non-biased and sequence-dependent manner by using multiple primer combinations (11). Thus, this technique allows searching genes differentially expressed in any specific disease. DDRT–PCR was used in our laboratory to identify new genes involved in AD and we now report that the KIAA0471 gene is differentially expressed in human brain of several AD patients, including one with a PS1 mutation.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The brain samples of six AD patients (including one with the PS1 mutation) and three age-matched controls were compared (Table 1). To observe one cDNA fragment differentially expressed, 15 arbitrary primers in combination with different 3' anchored primers were needed (Fig. 1). This represents the analysis of ~37% of the total mRNA, according to the formula P=1–(0.97)n where n corresponds to the number of arbitrary primers (12). The band disappeared in both hippocampus and septum of three out of the six AD patients (including one with the PS1 mutation) and was present in all the three analyzed controls. These results were confirmed after reproducing the experiments in duplicate from the RNA isolation step, validating the observed pattern of expression and suggesting that in-depth characterization of the identified gene is warranted. In addition, we found other differences in band intensities that could not be confirmed and therefore had been discarded.


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  		Table 1. Human brain samples
 


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  		Figure 1. Differential display analysis of gene expression in AD: Total RNA was extracted from hippocampus and septum of post-mortem AD and control patients. The autoradiogram shows the band pattern obtained from [{alpha}-33P]dATP-labeled differential display reactions performed in duplicate using 5'-AAGCTTTTTTTTTTTG-3' as an anchored primer (H-T11G) and 5'-AAGCTTCCTCTAT-3' as a random arbitrary primer (H-AP53). The arrow indicates a band corresponding to a cDNA fragment that is differentially expressed in some AD patients. A–I indicates Alzheimer's patients and controls. AD is for patients with Alzheimer's disease (AD*: patient with the mutation in the PS1 gene) and C for controls. Two brain areas were analyzed per case: hippocampus (1) and septum (3). The different lanes are numbered from 1 to 30, and every two lanes correspond to the same duplicated sample. Patient E is not included in the figure and gives results similar to patients B and D.

 
After excising bands of interest, reverse northern dot blot was performed and gave several positive clones that confirmed differences in expression (Fig. 2A). The nucleotide sequence of the most representative clones (1 and 4) was identical (Fig. 2B). To further confirm these differences, northern blot analysis was performed with some AD patients (Fig. 2C). On the basis of known sequences of GenBank database, the fragment sequence presented a high homology (98%, e-166) with the Homo sapiens gene product KIAA0471. The sequence of this mRNA is part of a human brain cDNA library included in the Japanese protein database HUGE (human unidentified gene-encoded) (13). HUGE is a database for human large proteins that aims to predict primary structures from sequences of human large cDNAs (>4 kb), in particular cDNA clones capable of coding for large proteins (>50 kDa) (14). KIAA0471 is a 6834 bp gene located on chromosome 1 that maps in the 1q24–q25 region, having a 5309 bp 3'-UTR.



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  		Figure 2. Confirmation of differentially expressed cDNAs. (A) Reverse northern dot blot: The cDNA from the differential band represented in Figure 1 (lane 5) (candidate cDNA) and another band not showing a different intensity in the DDRT gel (control cDNA) were excised, reamplified and ligated into the PCR–TRAP cloning vector. The PCR from five picked colonies (C1, C2, C3, C4 and C5) were blotted onto duplicated filters. One pair of these filters was hybridized with 32P-labeled cDNA from a control individual. The other pair of filters was hybridized with 32P-labeled cDNA from an AD patient. C1 and C4 clones corresponding to the candidate cDNA present marked differences after hybridization with control and AD mRNA probes. (B) Nucleotide sequence of candidate clone cDNA fragment. The cDNA inserts of the candidate 1 and 4 clones showing differential expression were sequenced. The nucleotide sequence of both clones was identical. The sequence of the primers used in the differential gene display analysis are underlined (H=HindIII site at the 5' end of the primers). (C) Northern blot analysis showing overexpression of KIAA0471 in some AD patients. Hippocampus and septum from different patients were used; the two lanes in each pair show hippocampus (H) and septum (S) from one patient. Equal amounts (20 µg/lane) of total RNA of septum and hippocampus were isolated for each studied brain area and run in a 1.3% agarose gel and transferred to nylon filters. The filters were hybridized with the 32P-labeled KIAA0471 cDNA fragment identified by differential display. The blot was also rehybridized to ß-actin probe as control.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Changes in gene expression may contribute to explain part of the high percentage of 95% of sporadic forms of Alzheimer which do not have a clear etiology. As DDRT–PCR method is a powerful technique for identifying differentially expressed transcripts between two or more tissues or cell types of interest (15), our main objective was to apply this technique to identify new genes responsible for expression changes involved in AD. In order to carry it out, we analyzed human brain samples of Alzheimer's patients and age-matched controls, whose medical history was fully documented. This fact limited the number of samples to a few but extremely well qualified patients. We followed this approach to avoid false results associated with variations in the patient conditions such as those related with agonal state (tissue pH and terminal medical conditions) (16). Differences in expression could not be due to differences in the cellular composition of the samples because all the blocks of hippocampus and septum were accurately obtained from the same area. As shown by cDNA bands, profiles displayed by all these samples, the limited differences of age and post-mortem interval did not affect the study.

It is important to consider that besides the working problems of human samples and the fact that the human brain is thought to have the greatest complexity of gene expression of any region of the body (17), we obtained clear and sharp DDRT gels, improving the image of the bands compared with previously reported studies (18,19).

We have identified one cDNA that is not expressed in septum or hippocampus of several Alzheimer's patients (including the one with the PS1 mutation). As it was necessary to screen >35% of total mRNA to observe a difference in AD gene expression, this transcript could be important among the total genes potentially associated with AD. The differential mRNA was identified as KIAA0471. At this stage, this KIAA0471 mRNA cannot be directly associated with any known protein, but based on its predicted coding region it would be translated into a soluble large protein with a length of 370 amino acids (20) and a molecular weight of 43 kDa. This predicted protein presents at least 95% homology with H. sapiens aminoacid sequences of the IDN4-GGTR membrane protein, a protein associated with precancerous lesions of hepatocellular carcinoma (EMBL Bioinformatic Harvester). Recent experiments by DDRT–PCR reported an upregulation of KIAA0471 gene products in 7 out of 12 cases with esophageal tumors (21). Studies of predicted KIAA proteins indicate that having multiple domains they may participate in an intricate framework of assembly protein complexes. Their predicted functions are mainly implied in cell communication/signaling, cell structure/motility or nucleic acid management (22). This would be in agreement with an increased KIAA0471 expression in proliferative cells and its reduction in the neuronal loss associated with neurodegeneration. If true, the absence of KIAA0471 expression evidenced by our results may be directly involved in the pathogenesis of septohippocampal lesion presented in AD. At present, studies are being directly conducted to determine entire sequences of human KIAA proteins together with their biological functions and will help to better understand its involvement in a variety of diseases (23).

Lack of expression of KIAA0471 gene in the PS1 mutation patient could be an additional factor that contributes to explain its very early onset dementia and/or early death. A possible genetic association of presenilin 2 and KIAA0471 with AD cannot be discarded as both genes map in the long arm of chromosome 1 (1q24–q25 KIAA0471 and 1q31–q42 PS2). AD mutations in this chromosome result in individuals with a wide range in age of onset (24) and the same is also shown by our results. A special instability of the 1q chromosome is also suggested by the presentation of genetic abnormalities leading to gliomas (oligodendrogliomas and astrocytomas) (25) and ependymomas (26). On the other hand, well-characterized diseases sometimes appear linked to genetic mutations in the same loci where KIAA0471 maps (1q24–q25). This is the case of type 2 diabetes (27), immunoglobulin A nephropathy (28) and hereditary prostate cancer 1 (29). At present, no direct relationship with AD has been established for most of these diseases, except the recently recognized association among hyperinsulinemia, diabetes and AD (30) and the insulin-degrading enzyme activity on ß-amyloid peptides (31–33). Therefore, knowledge of the function of KIAA0471 protein in the future could elucidate their possible relationship. This deficiency in KIAA0471 only showed up in male individuals and, though only one AD woman was included in this study, further study will be necessary to disentangle whether there is a link to gender.

Further work is needed to determine what causes this lack of expression. Among these, it could be due to a mutation in the KIAA0471 gene that results in an altered or missing mRNA (upstream effect) or simply to an ulterior suppresion of mRNA production by another factor (downstream effect). At this moment, we are beginning new assays using microchips with DNA probes and specific antibodies against the predicted KIAA047 protein; these new attempts are aimed to understand the difference of gene expression found in this study using DDRT–PCR technique. The study of the incidence of KIAA0471 mRNA deficit in Alzheimer's patients in a wide population sample, both in brain tissue and in blood, will give a key information for a better diagnosis of the disease in future.

In conclusion, we found for the first time a strong reduction of the KIAA0471 gene expression in several AD patients, including one with the PS1 mutation. The predicted defective protein presents 95% homology with IDN4-GGTR sequences. This identification of a new genetic defect may give new clues to explain the pathogenesis of the AD process and be a new starting point for the design of rational diagnostic and therapeutic tools.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Tissue samples
Brain post-mortem septal and hippocampal samples (Table 1) from nine patients were obtained from our local Neurological Tissue Bank (Serveis Científico-Tècnics, Universitat de Barcelona, Spain) with the approval of the appropriate Medical Ethic Committee. Neuropathological assessment was established by the Bank Neuropathologist, according to Braak and Newel criteria (34,35). All the blocks of hippocampus and septum were extracted from the same area by our Brain Bank, which performed their histological characterization to ensure their similarity. Post-mortem intervals (PMI) ranged between 4.15 and 15 h. Three of these patients (two men, one woman; mean age of death 54.7±7.9 years; PMI 11±2.6 h) did not present any brain pathology and they were used as controls. Six patients (five men, one woman; mean age of death 67.8±3.6 years; PMI 6.7±0.9 h) suffered AD. One of these AD patients presented a PS1 mutation (V89L) (36). In all cases, the cerebrospinal fluid pH was measured at autopsy with indicator strips, as an index of the agonal state. The mean result was 7 and the range was 6–8 and considered appropriate. Pieces of the septal and hippocampal regions were frozen separately at –80°C until RNA extraction.

RNA extraction
RNA was extracted from about 100 mg of each studied brain area (hippocampus and septum) using the Tripure isolation reagent (Boehringer Manheim, Germany) and treated with DNase I (GenHunter, Nashville, TN, USA). Briefly, tissue samples were sliced and homogenized in Tripure and aqueous and organic phases were separated by centrifugation after addition of chloroform (Merck, Darmstadt, Germany). The aqueous RNA solution was precipitated with isopropyl alcohol (Scharlau Chemie, Spain) rinsed in 75% ethanol and resuspended in water. After quantification of their concentrations by spectrophotometry, total RNA samples were kept frozen at –80°C as ethanol precipitates.

Gene expression analysis
We used the differential display technique (DDRT–PCR) described by Liang and Pardee (11,12) to study gene expression. First strand cDNA synthesis and PCR amplification with labeled [{alpha}-33P]dATP labeled were performed using the RNA image kit from GenHunter. Basically, 0.2 µg of total RNA was reverse transcribed using an anchored oligo-dT: H-T11V (V=A, G or C). An aliquot of the generated cDNA corresponding to 1 ng of total RNA was amplified by PCR with the anchored oligo-dT used in the cDNA reaction and an arbitrary primer. The PCR reactions were performed always in duplicate. PCR products were resolved by electrophoresis using a 6% denaturing polyacrylamide gel. The gel was dried in 3MM Whatman paper for 2 h under vacuum at 80°C and exposed overnight to X-Omat AR film (Eastman Kodak Co., Rochester, NY, USA). The film was put on top of the dried gel and both were marked with needle punches in order to locate in the gel the bands identified in the film after the film was developed. cDNA displays that showed differences in band intensities were repeated from RNA isolation step in order to ensure the same results and avoid false positives. The cDNA bands of interest corresponding to human samples were excised from the gel, reamplified by PCR with the same set of primers and PCR conditions used in the mRNA display but with a higher concentration of dNTPs and ligated into the PCR–TRAP cloning vector (GenHunter). The PCR–TRAP vector includes a tetracycline-dependent positive selection of plasmids with DNA inserts; thus, only recombinant plasmids show antibiotic resistance. Ligated plasmids were transformed in GH-competent cells and plated on LB plates containing 20 µg/ml tetracycline.

Reverse northern dot blot
Reverse northern dot blot was used to verify the differences in expression of the cDNA fragments identified previously by differential display. Tetracycline-resistant colonies were randomly picked from each plate and lysed with 50 µl of lysis buffer (TE buffer pH 8.0 with 0.1% Tween-20). The cloned cDNA fragments were amplified using primers flanking the cloning site of the vector and the PCR products were individually dot blotted onto duplicate nylon membranes (Hybond-XL, Amersham Pharmacia Biotech, UK) using a microfiltration system. After UV-crosslinking of the membranes for 2 h at 80°C, these were probed with total [32P]cDNA. The probes were prepared with 20 µg of total RNA isolated from the septum of an Alzheimer's patient without the band of interest and septum of a control that presents the band. The probes were labeled with [{alpha}-32P]dCTP (3000 Ci/mmol) (Amersham Pharmacia Biotech). Equal counts [(5–10)x106 c.p.m.)] of the cDNA probes from each case, Alzheimer's patients and controls, were heat denatured and used to probe the duplicate blots.

Northern blot
Once cDNA overexpression was confirmed by reverse northern dot blot, northern blots were performed to verify whether the selected cDNAs represent overexpression of a single mRNA. Total RNA was isolated using Tripure isolation reagent (Boehringer) without the DNAse I treatment. Denatured RNA (20 µg of total RNA) from brain samples was electrophoresed in 1.3% agarose gels, transferred to nylon membrane (Hybond-XL, Amersham Pharmacia Biotech), and the RNA was fixed to the membrane by baking for 2 h at 80°C. Hybridization with 32P-labeled probes and washing conditions were performed following the membrane manufacturer indications. Filters were exposed to BioMax films (Amersham Pharmacia Biotech) with intensifying screens for 12–48 h at –80°C.

Preparation of KIAA0471 and ß-actin probes
The KIAA0471 probe used for northern blot analysis corresponds to a fragment of 456 bp obtained by PCR amplification of a positive clone identified by reverse northern dot blot. The PCR product was directly purified using the QIAquick PCR purification kit (Qiagen), with previous confirmation of its size in agarose gel. The ß-actin probe was obtained by digestion of a pUC19 vector containing a 1.9 kb pair human ß-actin insert between BamHI sites. The digested product was electrophoresed in agarose and purified using the QIAquick gel extraction kit (Qiagen). Both probes were 32P-labeled using Ready-To-Go DNA labeling beads (Amersham Pharmacia Biotech).

Sequencing of cDNA, database searches and nucleotide alignment
cDNA for sequencing was obtained by miniprep purification and sequencing reactions were performed with the Big-Dye terminator kit (ABI Prism, Applied Byosistems, USA). DNA sequencing was done using an ABI Prism 377 fluorescent sequencing instrument at the Serveis Científico-Tècnics (Universitat de Barcelona). Database searches and sequence comparisons were performed using BLAST and FASTA search servers of the National Center for Biotechnology Information (NCBI) and the European Bioinformatics Institute (EBI), respectively.


   ACKNOWLEDGEMENTS
 
We acknowledge Banc de Teixits Neurològics (Serveis Científico-Tècnics, Universitat de Barcelona). This research was supported by DURSI 2001SGR00380, Ministerio de Sanidad V-2003-REDG167B-O and Red CIEN IDIBAPS-ISCIII RTIC C03/06.


   FOOTNOTES
 
* To whom correspondence should be addressed. Tel: +34 934024525; Fax: +34 934035882; Email: lluisadeyebra{at}ub.edu


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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