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Abstract |
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Three kindreds were evaluated in a systematic autosomal genome-wide
linkage analysis study. The AH phenotype, characterized by
hyperabsorption of calcium and hypercalciuria, was linked to only one
chromosomal locus, 1q23.3-q24. A 2-point logarithm of odds score
of 3.3 was obtained with markers D1S318 and D1S196 at a recombination
frequency of 
= 0. Nonparametric multipoint linkage analysis
yielded a peak nonparametric linkage Zall-score of
12.7, P = 6 x 10-6. Analysis of
key recombinants within the families studied localized the gene to a
4.3-megabase region between markers D1S2681 (centromere) and
D1S2815.
A trait associated with intestinal hyperabsorption of calcium in a severe form of absorptive hypercalciuria has been mapped to chromosome 1q23.3-q24.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All participants gave informed consent to a protocol approved by
the institutional review board. Three kindreds with severe AH
participated in this study. The probands were identified from patients
in our kidney stone clinic. In the first kindred, AH-01 (Fig. 1
), 22 family members and 4 unrelated
spouses were evaluated. In the second kindred, AH-02 (Fig. 2a), 5 individuals were evaluated. In the
third kindred, AH-03 (Fig. 2b), 4 family members were evaluated. All
kindreds were North American Caucasians of Western European descent.
The number of subjects evaluated and the scope of investigation
depended on the willingness and cooperation of the subjects. Either an
in-patient or out-patient evaluation was performed on consenting study
participants. Some individuals agreed to undergo only a partial
out-patient evaluation.
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) was determined either from the
fecal recovery of 47Ca after ingestion of a
synthetic test meal containing trace radiocalcium (18) or by using a
double stable isotope technique (19). The two tests yielded equivalent
results. Bone mineral densities of L2–L4 vertebrae, femoral neck, and
radial shaft were measured using dual energy x-ray absorptiometry
(QDR-2000, Hologic, Inc., Waltham, MA). A heparinized
venous blood sample was obtained for lymphocyte isolation and
immortalization, and an ethylenediamine tetraacetate-treated venous
blood sample was obtained for genomic DNA isolation (20). Out-patient evaluation. Subjects underwent an out-patient evaluation (2) after 1 week on an instructed diet designed to mimic the inpatient metabolic diet in sodium, calcium, and phosphorous contents. This evaluation included fasting venous serum for calcium, creatinine, iPTH, and 1,25-(OH)2D, heparinized venous blood for lymphocyte isolation and immortalization, ethylenediamine tetraacetate-treated venous blood for genomic DNA isolation, a 24-h urine collection for calcium and creatinine, a 2-h fasting urine collection for measurement of calcium and creatinine, and a 4-h urine collection for the same tests after oral ingestion of a synthetic meal containing 1 g calcium (2, 3). Each participant completed a standardized questionnaire that included kidney stone and dietary history.
Phenotype assignment. Phenotype assignment in kindreds AH-01
and AH-02 was based on four criteria: 1) evidence of hyperabsorption of
calcium, either a calciuric response to an oral calcium load greater
than 0.05 mmol Ca/L glomerular filtrate or greater than
61%, 2) elevated fasting urinary calcium (>0.027 mmol Ca/L glomerular
filtrate, 3) hypercalciuria (>5 mmol Ca/day on a
calcium-restricted diet), and 4) a low or normal serum PTH (<65 ng/L)
(4). Individuals who satisfied at least three of the four criteria were
assigned the affected phenotype. Those with intestinal hyperabsorption
of calcium (criterion 1) who met only one additional criterion were
classified as unknown phenotype. If an unrelated spouse had either an
AH phenotype or was not evaluated, their progeny, who would otherwise
have an affected or unknown phenotype, were assigned unknown phenotype.
All others were classified as unaffected.
In kindred AH-03, affected phenotype assignment was based on the
satisfaction of criteria 3 and 4 alone, as fasting urinary calcium,
calciuric response to an oral calcium load, and were
determined only in the proband. An unknown status was assigned when
only criterion 3 was met. All affected members from all three kindreds
had normocalcemia. Bone density was not used in the definition of AH
phenotype, as only a limited number of subjects were available for this
measurement.
DNA analysis
Genomic DNA was prepared from peripheral blood lymphocytes (QIAGEN, Chatsworth, CA). DNA genotyping was performed using fluorescently labeled primers available from Perkin-Elmer Corp. PE Applied Biosystems (Foster City, CA), or Research Genetics, Inc., on an PE Applied Biosystems model 377 automated DNA sequencer with GENESCAN 2.0 software. A total of 178 randomly spaced markers [10–30 centiMorgans (cM) spacing] were analyzed in the initial low density screening. Regions where a 2-point logarithm of odds (lod) score was more than 0.3 were screened using high density markers. Fifty-five additional markers were used in this secondary screening. All PCR amplification reactions were performed in a Perkin-Elmer Corp. thermal cycler (model 9600) following suppliers’ protocols. Samples were analyzed on 4% polyacrylamide gels. Data analysis was performed using GENOTYPER software (PE Applied Biosystems).
Linkage analysis
A simulation study was performed using SIMLINK (21) to determine whether the study pedigrees contained enough information to detect linkage. Two-point lod scores were calculated using the computer program Linkage 5.1 (22). The AH trait was assumed to be dominant with a penetrance of 80% and a disease frequency of 0.02. Nonparametric and parametric multipoint linkage analysis was performed using GENEHUNTER software (Whitehead Institute for Biomedical Research) (23). Analyses were run on a 180-MHz Pentium Pro computer using max bits = 20. All affected individuals were included in the analysis. Initial 2-point lod scores were calculated using published values for allele frequencies, and map distances were taken from the literature (24, 25). Critical 2-point lod scores and multipoint linkage analyses were also calculated using allele frequency from the study kindreds. Homogeneity testing was performed using the program HOMOG (26).
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Results |
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The proband of the kindred AH-01 (Fig. 1
, III-14) was a 37-yr-old
white male who underwent an out-patient evaluation. He had a history of
recurrent kidney stone formation; elevated 24-h urinary calcium,
fasting urinary calcium, and calcium load response; and a low serum
iPTH. The proband of the kindred AH-02 (Fig. 2a, III-2) was a 47-yr-old
white female who underwent an in-patient evaluation. She had elevated
24-h urinary calcium, fasting urinary calcium, calcium load response,
and and a normal iPTH. The proband of the kindred AH-03 (Fig. 2b, III-4) was a 32-yr-old white male who underwent an in-patient
evaluation. He had a history of recurrent kidney stone formation;
elevated 24-h urinary calcium, calcium load response, and ; high
normal fasting urinary calcium; and normal iPTH. All three probands had
no history of bowel disease, primary hyperparathyroidism, primary
hyperoxaluria, renal tubular acidosis, gout or cystinuria. They all
satisfied the diagnostic criteria of AH (4, 6).
Families
Kindred AH-01 (Fig. 1). Twenty-six blood samples, including
the proband, were collected for genotype analysis. Twenty-four members
of the family underwent clinical evaluation using the out-patient
protocol. Bone density measurements were obtained for eight family
members and three unrelated spouses. Biochemical and physiological
characteristics of family members with affected phenotype are presented
in Table 1.
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). There were 7 stone-formers in
the family (Fig. 1) and 1 stone-forming spouse (III-2). Stones that
were analyzed were calcium oxalate and/or calcium phosphate in
composition. Six of the 7 stone-formers had the affected phenotype
(Fig. 1), whereas the remaining stone-former (IV-I) was of uncertain
phenotype. Based on clinical evaluation, 5 additional nonstone-forming
family members and 1 spouse had the AH phenotype (Fig. 1). Individuals
III-18 and III-19 were monozygotic twins and were treated as a single
entity for purposes of linkage analysis.
Kindred AH-02 (Fig. 2a). Five individuals underwent phenotypic
evaluation and genotype analysis. Three members of the kindred had
in-patient evaluations with the determination of , whereas the
remaining two had out-patient evaluations. Bone density measurements
were obtained for three family members. All three affected family
members, including the proband (Fig. 2a), met the phenotype diagnostic
criteria and had evidence of severe AH, with fasting hypercalciuria and
low bone density (Table 1). One member was assigned unknown phenotype
(III-1). There was a family history of stone formation on the maternal
side of the family (I-3 and II-4).
Kindred AH-03 (Fig. 2b). Four members of the kindred underwent
phenotypic evaluation and genotype analysis. The proband had an
in-patient evaluation, whereas three family members (II-1, III-2, and
III-5) had partial out-patient evaluations. It was not possible to
obtain fasting urinary calcium and the calciuric response to a calcium
load data for these individuals. Bone density measurements were
obtained in three family members. The three affected individuals,
including the proband (II-1, III-2, and III-4; Fig. 2b), had
biochemical features compatible with severe AH (Table 1). They had
marked hypercalciuria and low bone density. One member (III-5) was
assigned an unknown phenotype. Of the subjects evaluated, only the
proband had nephrolithiasis, although a paternal cousin (III-7) also
reported a history of nephrolithiasis.
Linkage analysis
A simulation study performed using SIMLINK and 350 replicates for
each kindred revealed average lod scores of 1.7, 0.3, and 0.2 at
= 0 for families AH-01, -02, and –03, respectively. Although
the maximum lod scores E(Zmax) was 4.7, 0.9 and
0.6 were predicted at the same recombination fraction. Kindred
AH-01 was first tested for linkage at potential candidate gene loci,
which included genes coding for the vitamin D receptor,
1-hydroxylase, plasma membrane calcium, adenosine triphosphatase
(PMCa1), calbindin 28K, PTH-related peptide, human renal type 1
Na+-phosphate eotransporter, human renal type 2
Na+-phosphate cotransporter,
osteocalcin, interleukin-1 (IL-1), IL-1ß,
and IL-1 receptor. No evidence for linkage was found at any of these
loci. Candidate genes located on the X-chromosome, such as CLCN5 and
calbindin 9K, were eliminated, because male to male transmission was
present in kindreds AH-01 and AH-03 ruling out a sex-linked gene defect
(Figs. 1 and 2b). After elimination of these candidate gene loci, an
autosomal genomewide screening was undertaken. Strong evidence of
linkage was found only on the q-arm of chromosome 1 after analyzing 178
markers randomly distributed at 10- to 30-cM intervals within the
genome. An additional 55 high density markers were analyzed in regions
where a lod score greater than 0.3 was obtained.
The maximum parametric 2-point lod scores calculated for chromosomes
2–22 are shown in Table 2. None exceeded
1.3. However, on chromosome 1, a positive 2-point lod score of 2.7 was
obtained for kindred AH-01 between marker D1S196 and the AH phenotype
at = 0 (Table 3). Combination of
the three kindreds gave a 2-point lod score of 3.3 (Table 3). As
initial linkage calculations were performed using the literature allele
frequency, these data were recalculated using family allele frequencies
to confirm that positive linkage did not result from incorrect
frequency specification. Identical lod scores were obtained using
familial allele frequency.
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), whereas a parametric multipoint lod
score of 3.3 was obtained for the combined families in the same region.
This locus corresponded to a region contained in 1q23.3-q24, based on
current mapping location for these markers (24). As the phenotype
work-up of the third kindred was not as complete as those for the other
two kindreds, a multipoint analysis using only the first 2 kindreds was
performed. An NPL Z-score of 15.2 (P = 7 x
10-6) and a multipoint parametric lod of 3.0
were obtained for kindreds AH-01 and AH-02.
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). Key
recombinational events in individuals III-18 and IV-7 from kindred
AH-01 delineated the AH gene locus between markers D1S2681 (centromere)
and D1S2815 (telomere; Fig. 1). This locus corresponded to a physical
map distance of approximately 4.3 megabases based on the current
Linkage Data Base composite map (24). Six additional individuals (II-2,
III-3, III-15, IV-1, IV-2, and IV-8) from kindred AH-01 carried the
affected genotype based on haplotype analysis. Five of these
individuals, including one stone-former, had previously been classified
as uncertain phenotype, whereas the sixth individual had not undergone
a clinical evaluation. All phenotypically affected individuals carried
the same haplotype for markers D1S318, D1S196, D1S431, D1S2750, and
D1S2799 (Fig. 1). Possible genetic heterogeneity with respect to
potential disease loci was tested using the program HOMOG. The
conditional probability that each of the three study kindreds was of
linked type was more than 99%. However, although the support interval
for kindred AH-01 was good (0.99–1.00), the precision of the estimates
for the smaller kindreds, AH-02 and AH-03, was poorer, being 0.47–1.00
for the second family and 0.46–1.00 for the third family. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The clinical presentations of AH in all three kindreds were compatible with a severe form of AH. Thus, their characteristic features were moderate to marked hypercalciuria, low bone density, and fasting hypercalciuria. However, there is some evidence that the molecular abnormality disclosed here may be more generalized. Fasting hypercalciuria is not an uncommon finding in AH and may be present in a substantial number of patients, especially in those with marked intestinal hyperabsorption of calcium and parathyroid suppression (5, 6, 27, 28). In addition, low spinal bone density (29, 30) was present in AH patients with normal fasting urinary calcium (7) as well as the subgroup with fasting hypercalciuria.
Despite a rich family history of kidney stone formation in patients with AH, controversy persists concerning the mode of inheritance of this disease (8, 9, 31). We, therefore, used a nonparametric model-independent method of analysis (Genehunter) (23) as well as a parametric method of analysis that assumed an autosomal dominant mode of inheritance (8, 9). The results of the autosomal genomewide screening, using both methods of analysis, indicated that only one region of the genome met the criteria for linkage. Although no evidence of genetic heterogeneity was found among the study kindreds (P > 99%), the wide confidence interval associated with the conditional probability values for AH-02 and AH-03 should be noted. Thus, based on the results of our linkage screen, we conclude that AH is inherited as an autosomal dominant trait with suggestive evidence of linkage to chromosome 1q23.3-q24. Based on the most recent chromosome 1 map, no candidate genes of known function have been identified in this region. This lack of a known calcium regulatory gene at this chromosomal locus leads to the intriguing possibility that an as yet unreported gene may be involved in the regulation of intestinal calcium absorption and possibly bone loss.
Prior pathogenetic mechanisms for AH have implicated an abnormality in
either vitamin D metabolism or the vitamin D receptor (6, 10, 13, 32).
However, no evidence for linkage to the vitamin D receptor and
1-hydroxylase gene loci was found in the present study. Similarly,
no evidence for linkage was found for several other candidate gene
loci, including PMCA1, PMCA4, the 28K and 9K calbindins, and the
sodium/phosphate cotransporter genes, NPT1 and NPT2 (33, 34), which
have been implicated in the regulation of either the cellular transport
of calcium or the renal excretion of calcium. Some family members
without stones also had the common genotype at chromosome 1q23.3-q24.
The incomplete penetrance of stone formation is probably due to the
influence of environmental factors or possibly to other
disease-modifying genes. We chose not to use stone formation as part of
the phenotype to prevent other factors from complicating the analysis.
Identification of the specific gene and mutations contained therein
will be necessary to determine both the relationship of this gene
defect to the clinical features associated with AH and the prevalence
of this gene defect in the AH patient population. We are currently
pursuing this goal by positional cloning.
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Acknowledgments |
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Footnotes |
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1 This work was supported by USPHS Grants PO1-DK-20543 and
MO1-RR-00633 and the Robert T. Hayes Center for Mineral Metabolism
Research. 
Received April 26, 1999.
Revised July 16, 1999.
Accepted July 30, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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