Type 2 Diabetes Locus on 12q15
Further Mapping and Mutation Screening of Two Candidate Genes
Arsun Bektas,
Jennifer N. Hughes,
James H. Warram,
Andrzej S. Krolewski, and
Alessandro Doria
From the Section on Genetics and Epidemiology (A.B., J.N.H., J.H.W.,
A.S.K., A.D.), Research Division, Joslin Diabetes Center; and the Department
of Medicine (A.B., A.S.K., A.D.), Harvard Medical School, Boston,
Massachusetts.
Address correspondence and reprint requests to Alessandro Doria, MD, PhD,
Section on Genetics and Epidemiology, Joslin Diabetes Center, One Joslin
Place, Boston, MA 02215. E-mail:
adoria{at}joslin.harvard.edu
.
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ABSTRACT
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We recently reported evidence of a novel type 2 diabetes locus placed on
chromosome 12q15 between markers D12S375 and D12S1684
(Diabetes 48:2246-2251, 1999). Four multigenerational families having
logarithm of odds (LOD) scores >1.0 in the original analysis were genotyped
for 11 additional markers in this interval to refine this mapping; this
allowed us to narrow the linked region to the interval between markers
D12S1693 and D12S326. In a multipoint parametric analysis
using the VITESSE software, the LOD score for linkage at this location reached
3.1 in one of these families. This interval contains the gene for protein
tyrosine phosphatase receptor type R (PTPRR)a protein that may
be involved in both insulin secretion and action. After determining
PTPRR exon-intron structure, we identified several polymorphisms in
this gene but no mutation segregating with diabetes. The search for mutations
was also negative for carboxypeptidase M (CPM)another
candidate gene mapped to this region. In summary, our data provide further
evidence for the existence of a type 2 diabetes locus on chromosome 12q15.
This locus, however, does not appear to correspond to the PTPRR or
CPM, although a contribution of mutations in regulatory regions
cannot be excluded at this time.
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INTRODUCTION
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It is becoming increasingly clear that the etiology of type 2 diabetes is
much more heterogeneous than previously thought. Several attempts to identify
type 2 diabetes genes by linkage studies have led to conflicting results,
indicating that distinct genes are probably involved in different populations
(1,2).
Even within the same ethnic group, different genes may be involved in
different families (2). The
situation does not appear to be simpler for monogenic forms of diabetes, such
as those transmitted with an autosomal-dominant pattern of inheritance. Six
distinct genes have been described for the best-known form of this
groupmaturity-onset diabetes of the young (MODY)
(3,4)and
several additional loci probably exist. In France and England,
25% of
MODY families have diabetes unaccounted for by known MODY genes, but this
proportion seems to be much higher among families with an older age of
diabetes diagnosis than classical MODY
(5,6,7).
We recently described a novel locus for autosomal-dominant type 2 diabetes on
chromosome 12q15, 50 cM from the previously described locus NIDDM2
(8). All of the evidence of
linkage came from four families that had individual LOD scores >1.0. By
analyzing obligate recombinants in the two families with highest LOD scores
(2.35 and 2.1), we assigned this putative locus to the 6 cM between markers
D12S375 and D12S1684. Here we report the further mapping of
this diabetes locus and the results of the study of two candidate genes placed
in the critical region.
To narrow the linked interval, the four families that had a LOD score
>1.0 in the original analysis (families 8, 19, 24, and 32
[8]) were genotyped for 11
microsatellite markers located between D12S375 and D12S1684
(Fig. 1). Included were 27
affected and 25 nonaffected family members. The 11 markers were chosen from
contig WC12.4 of the WI/MIT integrated map
(9). Seven of the
microsatellites were also in the Marshfield genetic map
(Fig. 1). Within each family,
all affected members shared at least one portion of this region identically by
descent (Fig. 2A),
although the shared haplotype differed among families. In family 8, which had
a maximum LOD score (Zmax) of 2.35 in the original
analysis, the shared haplotype went from D12S1693 on the centromeric
side to D12S326 on the telomeric side
(Fig. 2A). The LOD
score for linkage at this location peaked to 3.1
(Fig. 2B). The shared
haplotype in family 24, which had a Zmax of 2.15, further
moved the telomeric boundary, narrowing the linked region to the interval
between D12S1693 and D12S1347
(Fig. 2A and
B). No additional information was provided by family 19,
in which no recombinants were identified. The affected haplotype of family 32
did not overlap with the critical interval defined by families 8 and 24, but
extended instead from D12S1693 toward the centromere, with the LOD
score peaking between D12S1702 and D12S375
(Zmax = 1.5). A possible explanation for this finding is
that there are two diabetes loci in this region: one operating in family 8,
placed between D12S1693 and D12S326, and the other in family
32, centromeric to D12S1693, with diabetes in families 19 and 24
being attributable to either locus. Indeed, it is not unusual that two
adjacent loci responsible for the same disorder are initially mapped as a
single locus (10). Another
possibility is that the recombinants defining either the centromeric boundary
in family 8 or the telomeric boundary in family 32 are actually phenocopies.
Finally, because family 32 has a Zmax of only 1.5, it is
also possible that the linkage observed in this family is entirely due to
chance.

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FIG. 1. Microsatellite markers that were genotyped in the study. On the left are
markers that were used for the original mapping
(8); on the right are those
used in the new analysis. Markers that were used in both the original and new
analyses are indicated in bold. The marker order is that of contig WC12.4 of
the WI/MIT integrated map.
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FIG. 2. A: Haplotypes shared by all affected individuals within each
family. The minus signs indicate regions that did not segregate with diabetes
in all affected family members. The allele numbering is arbitrary, but
consistent across families. B: Multipoint parametric linkage
analysis. Marker distances are those indicated in the Marshfield map. For this
analysis, markers that are not included in the map (D12S1703, D12S1693,
D12S1025, and D12S1347) were placed at equal distances from the
closest mapped markers.
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One gene placed in the D12S1693-D12S326 interval that caught our
attention is protein tyrosine phosphatase receptor type R (PTPRR,
also known as NC-PTPCOM1). Tyrosine phosphorylation is a key
component of the cascade of reactions linking the insulin receptor to insulin
action in peripheral tissues
(11). Furthermore, a tyrosine
phosphatase of the same class as PTPRR has been shown to be
associated with insulin-containing vesicles in ß-cells
(12). Another interesting
aspect was that the major site for PTPRR expression was the brain,
followed by pancreatic islets and lung
(Fig. 3). This distribution
pattern is remarkably similar to that of another gene (NEUROD1) that
has been found to be mutated in MODY and type 2 diabetes
(4). Thus, PTPRR was a
plausible candidate gene for diabetes in these families. The PTPRR
gene had been assigned to YAC clones 959-C-8 and 788-E-12, which also contain
D12S1025, but its exon-intron structure had not been resolved. After
isolating and sequencing three overlapping BAC clones containing the whole
gene, we identified 14 exons ranging in size between 80 and 473 bp
(Fig. 4A and Table A1,
which can be found in an on-line appendix at
www.diabetes.org/diabetes/appendix.htm
). Mutation screening of the coding sequence of probands from the four linked
families and other 12 families in which linkage could not be excluded (LOD
score > -2.0) revealed several polymorphisms, one of which changed the
amino acid sequence (Arg314
Lys
[Table 1 and Table A2, which can
be found in an on-line appendix at
www.diabetes.org/diabetes/appendix.htm
]). However, no mutations segregating with diabetes were identified.
Furthermore, the Arg314
Lys polymorphism was similarly
frequent among the 32 original family probands (0.266), 173 subjects with type
2 diabetes (0.266), and 181 nondiabetic control subjects (0.226), making
PTPRR unlikely as a type 2 diabetes locus in these families.

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FIG. 3. PTPRR expression in different tissues. The presence of
PTPRR mRNA was determined by standard reverse transcriptase-PCR using
GENEPAIR primer #31640 (Research Genetics). The arrow indicates the 220-bp
band that was amplified from the PTPRR cDNA. Is, islets; B, brain; K,
kidney; L, liver; Lg, lung; H, heart.
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Because there was evidence that the linked interval might extend
centromeric to D12S1693, or that another diabetes gene might be
located beyond this marker, we also analyzed a candidate gene in the vicinity
of D12S375. In the National Center for Biology Information GeneMap 99
(13), the carboxypeptidase
(CP) M gene was mapped to this region, and we confirmed this
location by assigning it to CEPH YAC clones 916-C-11 and 883-H-12, which also
contain D12S375. CPM is a membrane-bound enzyme belonging to
the regulatory carboxypeptidase subfamilya class of proteins that have
been implicated in the processing and sorting of polypeptide hormones, either
at the site of hormone production or in target tissues
(14,15).
Of note, a mutation in a member of this family (CPE) has been shown
to be responsible for diabetes in the fat/fat mouse model
(16). After isolating BAC
clones containing the CPM gene, we identified nine exons ranging in
size from 98 to 240 bp (Fig.
4B and Table A3, which can be found in an online appendix
at
www.diabetes.org/diabetes/appendix.asp
). In the same 16 families that were screened for PTPRR, we
identified several silent polymorphisms together with a private
Tyr99
His mutation that showed incomplete segregation with
diabetes in one family (Table 1
and Table A4, which can be found in an online appendix at
www.diabetes.org/diabetes/appendix.asp
). No mutations, however, were identified in the four linked families,
suggesting that CPM is unlikely to be the diabetes locus on
12q15.
In summary, we have narrowed the diabetes locus on 12q15 to the region
between D12S1693 and D12S326. The LOD score for this
location is 3.1 in one family, but it is still possible that the critical
interval extends beyond these boundaries. It is unlikely that this locus
corresponds to the PTPRR or CPM genes, although a role of
mutations in regulatory regions cannot be excluded at this time. New clues are
expected from the upcoming completion of the Human Genome Project, which will
help us define the exact size of the linked interval, and will provide an
inclusive list of the genes placed in this region.
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RESEARCH DESIGN AND METHODS
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Marker genotyping and linkage analysis. The ascertainment and
clinical characteristics of the families with autosomal-dominant type 2
diabetes have been previously described
(7). Marker genotypes were
determined by 32P-labeled polymerase chain reaction (PCR) followed
by denaturing PAGE and autoradiography. To avoid misreading of genotype
results, either a standard sequencing marker (Research Genetics, Huntsville,
AL) or PCR products of known genotypes were run together with the samples.
Genotypes were read separately by two individuals and ambiguous results were
repeated. Multipoint parametric linkage analysis was performed using the
Vitesse software (17). Because
of this software's limitations on the number of markers that can be run at a
time, multiple analyses were performed using overlapping sets of four adjacent
markers. Individual family LOD scores were calculated assuming an
autosomal-dominant mode of inheritance with a disease allele frequency of
0.001, consistent with the rarity of families segregating these forms of
diabetes. Similar to previous linkage analyses of MODY, four age-related
liability classes (0-10, 11-25, 26-40, and >40 years of age) were assumed.
Penetrance of type 2 diabetes in the four age-groups was set at 0.30, 0.50,
0.70, and 0.90, respectively, for the susceptible genotypes DD and Dd. On the
basis of the risk of diabetes in the general population, penetrances for the
nonsusceptible genotype dd were set to 0.001, 0.005, 0.01, and 0.10. The
marker order was that indicated in the Whitehead Institute/Massachusetts
Institiue of Technology physical map
(http://carbon.wi.mit.edu:8000/cgi-bin/contig/phys_map
). Intermarker distances (sex-averaged) were those indicated in the Marshfield
map
(http://www.marshmed.org/genetics/
). Markers that were not included in the Marshfield map were placed at equal
distance from the closest mapped markers. Founder haplotypes were inferred by
means of the Genehunter software
(18).
YAC library screening. Plates 805-984 of the CEPH Human YAC library
were screened by PCR using the CEPH "B" human YAC DNA pools from
Research Genetics and gene-specific primers.
Definition of exon-intron boundaries. BAC clone 2015-A-4 was
identified as containing the 3' portion of the PTPRR gene by
means of a basic local alignment search tool (BLAST) search of the GenBank
Genome Survey Sequence division, using the PTPRR cDNA sequence as a
probe. Genomic BAC clones containing the remaining portions of the
PTPRR gene and the whole CPM gene were identified by PCR
screening of the CITB human genomic BAC library (Release IV; Research
Genetics) using primers placed in the 3' end of BAC 2015-A-4 and in the
3' untranslated region of the CPM cDNA. Direct sequencing of
the BAC clones was performed using the Sequiterm Excel II DNA sequencing kit
(Epicentre, Madison, WI) with 32P-dATP. Intron lengths were
determined by long-range PCR (Advantage GC Genomic Polymerase Mix; Clontech,
Palo Alto, CA) with primers placed in adjacent exons.
Mutation screening. The coding sequences of PTPRR and
CPM were screened for sequence differences by dideoxy fingerprinting
(17). DNA fragments covering
the exons and exon-intron boundaries were amplified by PCR from the DNA of two
affected members of families 8 and 24, and one affected member of families 19
and 32, and 12 other pedigrees for which linkage could not be excluded (LOD
score > -2.0). Primer sequences and annealing temperatures for each
amplification are reported in Tables A2 and A4, which can be found in an
on-line appendix. PCR products were purified and subjected to Sanger's dideoxy
chain termination reaction using dideoxy GTP in a 10-µl reaction as
described by Sarkar et al.
(19). To increase the
sensitivity, reactions were performed twice, with the forward and the reverse
primer. After adding 20 µl stop/denaturing solution (7 mol/l urea, 50%
formamide, 0.5% bromophenol blue, and 0.5% xylene cyanol) and heating the
samples at 95°C for 5 min, 4 µl were electrophoresed overnight in a
nondenaturing 0.75 x MDE (mutation detection enhancement) gel in 0.5
x TBE (Tris borate EDTA) on a sequencing apparatus at a constant power
of 6 watts at room temperature. Dried gels were autoradiographed overnight.
Allele frequencies were determined in the 32 original family probands, 173
Joslin's patients with type 2 diabetes, and 181 non-diabetic control subjects
by PCR, dot-blotting, and allele-specific hybridization. The recruitment of
the type 2 diabetic individuals and nondiabetic control subjects has been
previously described (20). The
clinical features of these subjects are reported in Table A5, which can be
found in an on-line appendix at
www.diabetes.org/diabetes/appendix.htm
.
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ACKNOWLEDGMENTS
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This study was supported by National Institutes of Health grants DK-55523
(A.D.) and DK-47475 (A.S.K.), and Joslin's Diabetes and Endocrinology Research
Center Grant DK-36836 (Genetics Core). Human islets for RNA extraction were
provided by the Juvenile Diabetes Foundation Center for Islet Transplantation
at Harvard Medical School.
Part of this work was presented at the 59th Annual Scientific Sessions
(June 2000) of the American Diabetes Association in San Antonio, Texas.
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FOOTNOTES
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The nucleotide sequences reported in this article have been submitted to
the GenBank Data Bank with accession numbers AF262940-AF262947 and
AF263016-AF263029.
Additional information can be found in an online appendix at
www.diabetes.org/diabetes/appendix.asp
.
CP, carboxypeptidase; LOD, logarithm of odds; MODY, maturity-onset diabetes
of the young; PCR, polymerase chain reaction; PTPRR, protein tyrosine
phosphatase receptor type R; Zmax, maximum LOD score.
Received for publication May 10, 2000
and accepted in revised form August 25, 2000
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