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Center for Mineral Metabolism and Clinical Research, University of Texas Southwestern Medical Center, Dallas, Texas 75390-8885
Address all correspondence and requests for reprints to: Berenice Y. Reed, Ph.D., Center for Mineral Metabolism and Clinical Research, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390-8885. E-mail: . berenice.gitomer{at}utsouthwestern.edu
Abstract
Absorptive hypercalciuria (AH) is a kidney stone-forming condition frequently complicated by bone loss. Previously, we mapped the locus for an inherited form of AH to chromosome 1q23.3-q24. We have sequenced a putative gene (subsequently shown by others to be homologous with the rat soluble adenylate cyclase gene) in this region in 12 unrelated Caucasian AH patients. Eighteen base substitutions were identified in the soluble adenylate cyclase human homolog gene. All sequence variations were further evaluated in 3–68 additional unrelated AH patients and 19–132 normal subjects, and 1 additional base substitution was identified. Six of the identified sequence variations occurred with increased frequency in the AH population and tracked with the AH phenotype in AH families. Calculated odds ratios showed that the occurrence of any 4 of these individual base substitutions was associated with a 2.2- to 3.5-fold increase in estimated risk for AH (P < 0.02). In addition, 1 or more base changes was associated with a lower L2–L4 vertebral bone density. Sequence analysis of 3 other genes within the AH linkage interval showed no difference in the distribution of sequence variations between AH and normal populations. This is the first description of a specific gene defect associated with AH.
ABSORPTIVE HYPERCALCIURIA (AH) is a common cause of calcium oxalate nephrolithiasis (1). Clinically, AH is characterized by intestinal hyperabsorption of calcium in the presence of normal serum calcium and immunoreactive PTH (iPTH) (2, 3, 4). It is often accompanied by low bone mineral density (BMD), particularly of the lumbar spine (5, 6, 7). About 50% of patients with AH present with a family history of calcium oxalate nephrolithiasis and hypercalciuria. This high familial transmission of the disease indicates that AH is at least in part genetic in origin (8), and in some families with the AH phenotype an autosomal dominant pattern of inheritance has been reported (9, 10). Previously, we mapped the phenotypic presentation of intestinal hyperabsorption of calcium and hypercalciuria to chromosome 1q23–24 (11) in three well characterized and phenotypically similar families with AH. However, the nature of the genetic defect(s) in AH has not previously been reported.
The region defined by our linkage studies contained several known genes and a number of putative genes. After sequencing several known genes [Na+/K+ transporting, ß1-polypeptide adenosine triphosphatase (ATPase; ATP1B1), G protein-coupled receptor (RE2), and myelin protein zero-like 1 (MPZL1)] in this area and finding no difference in the frequency of occurrence of base substitutions in AH patients compared with volunteer study subjects, we focused on a potential new gene encoding a hypothetical protein (GenBank accession no. AL035122) in our search for the defective gene in AH. During the course of the work a rat gene that coded for a bicarbonate-sensitive, soluble adenylate cyclase (sAC) was shown to be homologous with this putative gene (12).
The present study was undertaken to elucidate the genomic structure of the hypothetical gene and to determine whether the presence of sequence variation in this gene was associated with an increased risk for AH and low spinal BMD.
Subjects and Methods
Study subjects and evaluation
All subjects gave informed consent to participate in the protocol, which was approved by the institutional review board of University of Texas Southwestern Medical Center (Dallas, TX). For the purpose of the genetic analyses, study subjects were drawn from the Caucasian population to eliminate confounding factors due to ethnic variation.
Clinical evaluation of AH patients
Eighty unrelated patients with AH were identified from our kidney stone clinic. The diagnosis of AH was made after either in-patient or out-patient evaluation.
For the in-patient evaluation, patients were admitted to the General Clinical Research Center for 4 d where they were maintained on a constant metabolic diet containing 100 mmol sodium, 10 mmol calcium, and 26 mmol phosphorous per day for the first 3 d (d 1–3). They were kept on an instructed diet of similar composition for 1 wk before admission. Fasting venous serum samples were drawn on d 1–4 and were analyzed for calcium and alkaline phosphatase (SmithKline Beecham, Dallas, TX). Fasting venous serum samples on d 1 and 4 were also analyzed for iPTH by immunoradiometric assay (Nichols Institute Diagnostics, San Juan Capistrano, CA), and 1,25-hydroxyvitamin D [1,25-(OH)2D] was analyzed by RRA. Calcium and creatinine were measured in three successive 24-h urine collections (d 1–3). On d 4, a 2-h fasting urine collection was obtained for measurement of calcium and creatinine, and a 4-h urine collection was obtained for the same tests after oral ingestion of a synthetic meal containing 1 g calcium. The calciuric response after the calcium load gave an indirect measure of intestinal calcium absorption (1, 3). Fractional calcium absorption (
) was determined either from the fecal recovery of 47Ca after ingestion of a synthetic test meal containing trace radiocalcium (2) or by using a double stable isotope technique (13) The two tests yielded equivalent results. The BMD of L2–L4 vertebrae was measured using dual energy x-ray absorptiometry (QDR-2000, Hologic, Inc., Waltham, MA). The z-score indicated deviation from the age- and sex-matched control value expressed in SD. The t-score represented deviation from the normal peak value expressed in SD. A t-score of less than -2.5 is defined as osteoporosis. A heparinized venous blood sample was obtained for lymphocyte isolation and immortalization, and an EDTA-treated venous blood sample was obtained for genomic DNA isolation (14).
Some patients underwent an out-patient evaluation (1) after 1 wk on an instructed diet designed to mimic the in-patient metabolic diet in sodium, calcium, and phosphorous content. This evaluation included a fasting venous serum for calcium, creatinine, iPTH, and 1,25-(OH)2D determination, a heparinized venous blood sample for lymphocyte isolation and immortalization, an EDTA-treated venous blood sample for genomic DNA isolation, a 24-h urine collection for calcium and creatinine determinations, a 2-h fasting urine collection for calcium and creatinine determinations, and a 4-h urine collection for the same tests after oral ingestion of a synthetic meal containing 1 g calcium (1, 3). In addition, BMD was measured as noted in the previous section.
Normal volunteers
Normal volunteers (n = 132) were recruited by advertisement in the daily newspaper, notices in the medical school complex, or word of mouth. Volunteers were matched by race with the AH patients. All participants completed a standardized questionnaire detailing personal and family history of kidney stone formation and osteoporosis. A sample of venous blood was obtained for lymphocyte transformation and DNA isolation and for the measurements of serum iPTH, 1,25-(OH)2D, and calcium. Any volunteer with a personal or family history of stone disease or osteoporosis or an abnormal serum PTH or calcium level was excluded from the study.
cDNA sequencing
The nucleotide sequence of the full-length cDNA encoded by the human homolog of the rat sAC gene was determined as follows. Normal human intestinal cDNA (Marathon ready cDNA) and normal human intestinal mRNA were purchased from CLONTECH Laboratories, Inc. (Palo Alto, CA). Rapid amplification of cDNA ends (RACE) was used to obtain 5' and 3' sequence data. RACE ready cDNA was prepared from the intestinal mRNA using a RLM RACE kit from Ambion, Inc. (Austin, TX), in accordance with the supplier’s instructions. Similarly, RACE analysis was performed directly on the Marathon ready cDNA using a SMART RACE cDNA amplification kit (CLONTECH Laboratories, Inc.). Forward and reverse primers (Table 1
) were designed, based on the cDNA sequence of the hypothetical protein (GenBank accession no. AL035122) or on predicted exon sequence obtained by analysis of the genomic PAC clones dJ313L4 and 295C6 using the gene-finding program GRAIL (15). DNA sequence analysis was performed using an ABI Big Dye cycle sequencing kit (PE Applied Biosystems, Foster City, CA) and analyzed on an ABI 377 automated DNA sequencer. Intron-exon boundary information was obtained by alignment of the cDNA sequence with the genomic PAC clones dJ313L4 and 295C6 using the BLASTN 2.1.1 computer program at National Center for Biotechnology Information.
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A human multiple tissue expression array (CLONTECH Laboratories, Inc.) was screened using a 526-bp probe that spanned exons 17–20 of the human homolog of the rat sAC cDNA. The probe was generated by PCR using the following primers: 2168 forward, 5'-tggattcgaggtcctggagat; and 2694 reverse, 5'-aagtctcatgctatccagctggatc. Before hybridization the probe was labeled with digoxigenin-dUTP for chemiluminescent detection with Disodium 3-(4-methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.13,7decan}-4-yl)phenylphosphate according to the manufacturer’s protocol (Roche Molecular Biochemicals, Indianapolis, IN). Hybridization was carried out under stringent conditions using the standard protocol recommended for chemiluminescent detection. Following exposure and development, the negative was scanned before digital quantitation. After the initial hybridization, the blot was stripped and reprobed with a control ubiquitin probe (CLONTECH Laboratories, Inc.) labeled and detected as described above.
Mutation screening, PCR, and DNA sequencing
Human homolog of sAC gene analysis.
Genomic DNA was prepared from peripheral blood lymphocytes using a whole blood DNA extraction kit (QIAGEN, Chatsworth, CA). An initial screening for mutations was performed by sequencing all 33 exons in the genomic DNA from 12 AH patients. Primers were designed based on the DNA sequence from each flanking intron. PCR amplification was performed in a total volume of 50 µl with 50–100 ng DNA, 2.0 U AmpliTaq Gold DNA polymerase (Perkin-Elmer Corp., Norwalk, CT), 50 pmol of each primer, 1x PCR buffer (Perkin-Elmer Corp.), 200 µM dNTP, and MgCl2 between 1–2.5 mM as specified for individual primer sets. Amplification conditions were as follows: 10-min initial denaturation at 95 C, followed by 35 cycles of (10 sec at 95 C, 30 sec annealing at between 58–68 C as specified for individual primer sets, 45 sec at 72 C) with a final 10-min incubation at 72 C. All primer sets used to amplify genomic DNA are depicted in Table 2. Genomic DNA from a minimum of 19 control subjects and 15 nonrelated AH was examined for all sequence variations revealed in the initial mutation screen. Certain sequence variations were evaluated more extensively in the full population of 132 Caucasian normal subjects and 80 Caucasian AH patients. As certain base changes destroyed a restriction endonuclease recognition site, restriction fragment polymorphism analysis was performed on the following PCR fragments amplified from genomic DNA of study participants: BcgI (exon 7), AluI (exon 11 and intron 23), and HaeIII or MboII (exon 20) (New England Biolabs, Inc., Beverly, MA). All restriction enzyme digests were performed using the supplier-recommended protocol. Fragment analysis was performed by electrophoresis on 1.5% agarose gels or 4% NuSieve (FMC Bioproducts, Rockland, ME) when the resultant product size was less than 100 bp in length. All mutations detected were verified by DNA sequence analysis.
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Statistical analysis
Allele frequencies were calculated for each genotype, and the significant difference in allele frequencies between the AH and normal control populations was assessed using Fisher’s exact test. Odds ratios for both the variant alleles, compared with the wild-type, were calculated as a measure of the association between genotype and AH disease. These odds ratios were used as an estimate of relative risk. The association of mutation with bone density (L2–L4 BMD, z-score) was determined by multiple regression analysis with correction for body mass index (BMI). Significance was assessed using a two-tailed t test, as variance between groups was equal. The difference in the number of patients meeting the criteria for osteoporosis (t-score below -2.5) was assessed using Fisher’s exact test. In all analyses, P < 0.05 was considered significant. Statistical analyses were performed using SAS version 8.0 software (SAS Institute, Inc., Cary, NC).
Results
Characterization of AH patients
A total of 80 patients with AH were evaluated (63 men and 17 women; Table 3). The mean age of the group was 48 yr. All patients were Caucasian and had the key features of type 1 absorptive hypercalciuria. These features include having hypercalciuria on a restricted calcium diet, exaggerated calciuric response to an oral calcium load, and/or elevated intestinal calcium absorption (1, 2, 3). All patients were normocalcemic and had normal or low serum iPTH. Included in this group were the 3 probands from our original linkage study (11).
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5' and 3' cDNA fragments were generated by RACE using the primers 1921R and 1896 F. Subsequent primers were designed based on the 5'- and 3'-termini, which allowed generation of the complete 5085-bp cDNA (GenBank accession no. AF331033). Sequenced fragments were aligned, and an open reading frame was predicted using the computer program Sequence Navigator (PE Applied Biosystems, Foster City, CA). The predicted protein contained 1518 amino acids (176.5 kDa) with a predicted pI of 7.61. The mRNA was coded for by 33 exons with a corresponding gene size of 104 kb. Molecular modeling predicted 1 adenyl/guanylyl cyclase catalytic subunit and 2 34-amino acid tetratricopeptide-like regions (17). The predicted protein showed 77% homology to the predicted rat sAC sequence (Fig. 1) (12).
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Screening of a human multiple tissue array with a 526-bp probe complementary to exons 17–20 of the gene indicated low level expression in multiple human tissues, including kidney and jejunum (Fig. 2). The mRNA of the gene was also detected in human bone by RT-PCR, although increased sensitivity of this method may indicate lower expression in this tissue (Fig. 3).
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The gene structure was determined by aligning the sequenced cDNA structure with the GenBank sequences of two PAC clones (dJ313L4, Z99943.1 and 295C6, Z97876.1) that contained the gene. The intron-exon boundaries were then confirmed by sequencing of genomic DNA in the sense and antisense directions. The gene was found to consist of 33 exons covering approximately 104 kb of genomic DNA. The exons ranged in length from 39–291 base pairs. The intron-exon boundaries and the length of each of the exons are given in Table 4. All intron junctions conformed to the 5'gt ... ag3' rule, with the exception of intron 2, where gc replaces gt at the 5' junction. The first ATG of the open reading frame is present in exon 5 at base 499.
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Sequence analysis of the ATP1B1, RE2, and MPZL1 genes.
No sequence variations were detected in the RE2 gene. Two single-base substitutions were identified in the ATP1B1 gene (c.353–22T
A and c.1516 GT), and one base substitution (c.224-27 CT) was found in the MPZL1 gene. Comparison of the frequency distribution of these sequence variations within the normal and AH populations revealed no significant differences in one population relative to the other (Table 5). All sequence variations are described relative to the cDNA position based on current nomenclature recommendations (18).
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). Of these 19 base substitutions, 6 were found to occur significantly more frequently in our AH patient population compared with volunteer study subjects (Table 6). The distribution of all sequence variations occurring in the volunteer study subject population was shown to be in Hardy-Weinberg equilibrium.
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T in exon 7 that changed a threonine to a methionine residue and destroyed a BcgI restriction site, a c.1438+ 30 TC in intron 11 that destroyed an AluI restriction site, and a c.2660-13 CT in intron 19 that destroyed a HaeIII restriction cleavage site. Two substitutions were found separated by 72 bases, c.3532–580 AT and c.3532–508 GT, which destroyed an AluI restriction cleavage site. The latter substitution was linked to sequence variation c.3532–33 AT. In addition, a rare silent mutation within exon 20, c.2787 GA that destroyed an MboII restriction site was present with increased frequency in the AH population, although this difference was not significant. The positions of these base changes are indicated in Fig. 4. Six of these base substitutions were identified in the probands of our linkage families and segregated with the affected phenotype within the respective families. The number of base substitutions occurring within each family differed. The segregating alleles were c.3532–580 AT, c.3532–508 GT, and c.3532–33 AT in family 1; and c.923 CT, c.1438+30 TC, c.2660-13 CT, and c.3532–580 AT] in family 2, although the affected mother of the proband also carried the c.3532–508 GT and linked c.3532–33 AT sequence variations that were not inherited by any of the three evaluated children in family 2. A more complex pattern was found in family 3, the proband and his affected brother both have four sequence variations (c.2660-13 CT, c.3532–580 AT, c.923 CT, and c.1438+30 TC). The affected father carries only the first two sequence variations, which indicates that the latter two derive from the maternal chromosome. As the mother of the proband was deceased, no phenotypic evaluation was possible.
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T was not found in 264 normal chromosomes screened, whereas 7 base substitutions were found among 160 AH chromosomes. Similarly, the linked sequence variation c.3532–33 AT was not found in 158 normal chromosomes screened, whereas 6 base substitutions occurred among 146 AH chromosomes screened (DNA from 1 additional patient screened for c.3532–508 was not available for this analysis). In the case of sequence variation 2787 GA, only 1 substitution was found of 264 normal chromosomes screened, whereas 3 were found among 160 AH patient chromosomes. The remaining 4 substitutions occurred in more than 1% of the normal population as indicated in Table 6
. The distribution of all 4 sequence variations occurred with marginally or significantly elevated frequency in the AH population compared with the control population. Human sAC genotype and risk for AH
The presence of the c.923 CT substitution in one or both alleles was associated with a significant 3.1-fold increase in estimated risk for stone formation and occurrence of the AH phenotype [hypercalciuria, elevated intestinal calcium absorption; 95% confidence interval (CI), 1.33–7.12]. Similar calculation of the odds ratio for c.1438+ 30 TC, c.2660-13 CT, and c.3532–580 AT revealed an increase in estimated risk of 3.2 (95% CI, 1.36–7.52), 2.3 (95% CI, 1.19–4.27), and 2.63 (95% CI, 1.43–4.82) for one or both base substitutions of each mutation, respectively (Table 7).
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As low bone density is frequently found in AH (7) and was disclosed in 3 probands of our original study kindreds (11), we examined the association between L2–L4 BMD and the presence of the 6 sequence variations. L2–L4 vertebral BMD (z-score) was compared between 35 AH patients without any substitution and 45 patients with 1 or more substitutions. Although the mean BMD was not significantly different from normal for the whole group (Table 3), there was a trend toward decreasing vertebral BMD with increasing number of substitutions (Fig. 5). The difference from the group without base changes was significant for the group with at least 3 or at least 4 base changes. For patients with at least 4 base changes, the decline in L2–L4 BMD was 2-fold greater than that in patients without base changes (z-score, -1.79 ± 0.85 vs. -0.89 ± 1.00; P = 0.006). The significant differences were maintained after correction for BMI. In addition, BMD corrected for BMI was significantly lower among those with 3 or more mutations than in those with 0–2 mutations (P < 0.02).
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; P = 0.006). There was no association between low lumbar BMD and well established risk factors for osteoporosis (alcohol abuse, smoking, or hyperthyroidism) in the AH patients. Osteopenia was present in several family members of our study families based on bone density measurement as reported previously (11). However, no consistent record of familial osteoporosis was identified in our AH patient population based on patients verbal histories.
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Data from 35 AH patients without any base change were compared with those from 45 patients with at least 1 base change. There were no significant differences in age, height, weight, serum alkaline phosphatase, iPTH, calcitriol, urinary calcium, or intestinal calcium absorption. Urinary calcium excretion was not routinely determined in our normal volunteer group; however, 24-h calcium excretion data were available for 41 individuals from this group. Within this evaluated group, no sequence variations were detected in 26 individuals, and urinary calcium excretion greater than 5.0 mmol Ca/d was present in 3 subjects from this group (11.5%). One or more base substitutions were present in the remaining 15 individuals, and 4 subjects from this group (26.7%) were identified with urinary calcium excretion greater than 5.0 mmol/d.
Discussion
In this report we have sequenced several known genes and a hypothetical gene within the chromosome 1q 23.3–24 region that we previously identified as the AH locus (11). Of the known genes in the region of interest, we sequenced the ATP1B1, RE2, and MPZL1 genes. There were no significant differences in the frequency distribution of sequence variations between controls and AH patients within these genes (Table 5). This shows that there was no association between sequence variation within these genes and AH. Therefore, one putative gene from this region that coded for a hypothetical protein (GenBank accession no. AL035122) was chosen as the initial target in the search for the genetic basis of AH.
This gene was shown to consist of 33 exons, which encompassed a chromosomal region of approximately 104 kb. Sequencing of cDNA prepared from intestinal mRNA showed that the mRNA was 5085 nucleotides in length, with the ATG initiation codon starting at nucleotide 499 of the cDNA. The open reading frame continued to nucleotide 5053. The protein coded for was predicted to contain 1518 amino acids. During this work, Buck et al. (12) reported a soluble adenylate cyclase isolated from rat testis that was homologous with our gene. Based on analysis of the genomic DNA sequence, these workers and others proposed a sequence for the human gene (GenBank accession no. AF176813, AF271058, and XM001890). The rat gene and proposed human intestinal sAC gene are quite similar. However, the cDNA isolated from human intestine has several differences, most notably an inclusion of an additional 37 bases at the beginning of exon 5. This inclusion changes the initiation codon of the human cDNA and results in a protein that is 92 amino acids shorter than that predicted for the human gene by others. Also, an additional 20 bases were noted at the 3'-terminus in the intestinal cDNA compared with the predicted sequence. The human intestinal gene is 77% homologous to the rat sAC (Fig. 1) (12). The main difference in the two proteins is that the human intestinal protein contains only one predicted adenylyl/guanylyl cyclase catalytic domain, whereas the rat protein contains two (Fig. 1). This high degree of homology between the two genes makes it likely that the gene we isolated from human intestine is also a sAC.
Sequence analysis of genomic DNA coding for the intestinal gene revealed 6 base substitutions that were found predominantly or uniquely among patients with nondietary-dependent AH. The presence of four of these substitutions, c.923 CT, c.1438+30 CT, c2660-13 CT, and c.3532–580 AT, was shown to significantly increase the relative risk for AH. The original hypothetical protein (GenBank accession no. AL035122) contained an alternate exon coded by a portion of intron 23, where two of the base variations (c.3532–580 AT and c. 3532–508 GT) were found. It is interesting to note that the latter substitution is linked to the c.3532–33 substitution, which may influence splicing. Five of the observed substitutions were present in some of our normal volunteers. However, we did not routinely screen our normal volunteers for hypercalciuria and therefore cannot exclude the possibility of asymptomatic hypercalciuria among this group. This is supported by the fact that a higher proportion (26%) of normal volunteers with base substitutions were hypercalciuric than in the corresponding normal subjects without base substitutions (11%). Hypercalciuria is undoubtedly heterogeneous in origin; hence, our results relating relative risk for AH to the occurrence of specific DNA changes are pertinent only to nondietary AH.
As ethnicity may affect allele frequency, we carefully matched our patient and normal volunteer groups. This fact is underlined by the finding of similar distribution frequency for 13 of the identified sequence variations in the sAC gene and all of the sequence variations in the other genes studied (Tables 5 and 6) in both the patient and normal volunteer groups. This emphasizes the association between the 6 specific substitutions within the sAC gene and AH.
Low bone density has also been described as a complication of AH (7). We found that patients with AH with substitutions c.923 CT, c.1438+ 30 TC, c.2660-13 CT, c.2787 GA, c.3532–580 AT, and/or c.3532–508 GT in the sAC gene had a greater decline in L2–L4 BMD than those without these base changes (Table 8 and Figs. 5 and 6). These findings are supported by other studies. In mice, linkage was reported between bone density and a region on chromosome 1q23 that includes our region of interest (19). The genes in this region of mouse chromosome 1 closely corresponded to the location of the genes on human chromosome 1 (20). Furthermore, a human linkage study has shown an association between a gene(s) related to vertebral bone density and a broad region on chromosome 1q (21). However, based on the current study we cannot determine whether the observed association between low spinal BMD and sAC sequence variation is specific for AH alone.
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Acknowledgments
Footnotes
This work was supported by USPHS Grants PO1-DK-20543 and MO1-RR-00633 and the Robert T. Hayes Center for Mineral Metabolism Research.
Abbreviations: AH, Absorptive hypercalciuria; ATPase, adenosine triphosphatase; BMD, bone mineral density; BMI, body mass index; CI, confidence interval; iPTH, immunoreactive PTH; MPZL1, myelin protein zero-like 1; 1,25-(OH)2D, 1,25-hydroxyvitamin D; RACE, rapid amplification of cDNA ends; sAC, soluble adenylate cyclase.
Received January 30, 2001.
Accepted November 15, 2001.
References
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1, at least one base substitution (n = 43); 