Genetic abnormalities in sporadic parathyroid adenomas: loss of heterozygosity for chromosome 3q markers flanking the calcium receptor locus.

Inactivating mutations of the parathyroid cell calcium receptor (CaR) gene cause one form of familial benign/hypocalciuric hypercalcemia, and in homozygous form, cause neonatal severe primary hyperparathyroidism with parathyroid hyperplasia. Thus, we postulated that partial or total loss of CaR function might contribute to calcium insensitivity or even stimulate cell proliferation in sporadic parathyroid adenomas (PAds). To examine this possibility, we sought loss of heterozygosity (LOH) for markers flanking the CaR locus (3cen-3q21) in 35 PAds. We used 16 highly-polymorphic PCR-based markers in paired normal and tumor DNA, extracted from slices of archived surgical specimens. Nineteen to 24 of the DNA pairs were informative with at least one marker. In two informative pairs, we found LOH for markers D3S1303, D3S1267, or D3S1269, which are tightly-linked with and flank the CaR locus. In one tumor, deletion mapping confined the lost area between D3S1271 and D3S1238 (41.7 centimorgans, cM). In the other tumor, LOH spanned most of chromosome 3, ranging at least from D3S1307 to D3S1311 (271.4 cM). LOH was confirmed by repetition of the experiments and quantified by phosphorimaging. Thus, we found LOH encompassing the CaR locus in approximately 10% of sporadic PAds. These data are consistent with the hypothesis that loss of CaR function may occur in PAds, with functional consequences for calcium sensitivity and cell proliferation.

Clustered inactivating mutations and benign polymorphisms of the calcium receptor gene in familial benign hypocalciuric hypercalcemia suggest receptor functional domains.

The predominant variety of familial benign hypocalciuric hypercalcemia (FBHH) is FBHH(3q), which is associated with presumed inactivating mutations of the cell surface calcium receptor (CaR) gene on chromosome 3q13.3-q21. We sought mutations of the CaR gene in FBHH by direct sequencing of PCR-amplified genomic DNA from 14 affected families: 8 mapped to 3q13, 1 mapped to chromosome 19p, and 5 unmapped. We sequenced the entire coding region of the gene (exons 2-7) in one or two affected members of each family and found six point mutations that altered one amino acid, cosegregated with hypercalcemia, and were absent in more than 100 unaffected persons. Four mutations were unique (S53P, D215G, S657Y, and P748R), and two had been reported previously (P55L and R185Q). Of four mutant CaR proteins expressed in Xenopus oocytes, three were deficient in extracellular Ca2+-induced signaling. No CaR mutations were found in eight families, including the one mapped to chromosome 19p. Three benign polymorphisms occurred in the COOH-terminal region of the CaR protein in 10%, 15%, and 30% of more than 100 unaffected persons. Thus, FBHH-causing CaR mutations were clustered in the NH2-terminal extracellular and membrane-spanning regions of the receptor protein. We suggest that these are important functional domains, probably for calcium binding and signal transduction, respectively. Finally, mutations in regulatory or intronic regions of the CaR gene may also underlie many cases of FBHH.

Generation of variability at VNTR loci in human DNA.

Our laboratory has constructed linkage maps of the human chromosomes to use as a tool towards the goal of cloning by position the genes responsible for genetic disorders. Construction of the map required the development of polymorphic marker systems in the form of Restriction Fragment Length Polymorphisms (RFLPs). Work by Yusuke Nakamura in the laboratory led to the identification of more than 200 highly informative Variable Number Tandem Repeat (VNTR) markers. The hypervariable nature of these marker loci has allowed individualization at the DNA level. Techniques for individualization have subsequently been adopted by diverse fields including gene mapping, cancer genetics and forensic biology. These markers have also become a resource to test hypotheses as to how the VNTRs generate their intrinsic variability. We have demonstrated that the hypothesis that VNTRs generate their variability by unequal exchange between homologous chromosomes in incorrect (Wolff et al., 1988; Wolff et al., 1989). Our data are consistent with intrachromosomal models such as unequal sister chromatid exchange and replication slippage. Using DNA derived from nonhuman primate species, we have tested hypotheses that try to explain the sequence relationship at dispersed VNTR loci. Our data reveal that VNTR loci are most likely not related by transposition but rather arose independently at multiple loci.

Establishing paternity using minisatellite DNA probes when the putative father is unavailable for testing.

A paternity case involving a putative father who had died a few years earlier in an automobile accident was referred to the laboratory for testing. The child and his mother, the deceased's parents, and nine of the deceased's siblings were available for analysis. As previously reported, paternity testing using red blood cell groups, human leukocyte antigens (HLA), red blood cell enzymes, serum proteins, and immunoglobulin allotypes gave a cumulative paternity index of 43,300 and a combined probability of paternity equal to 99.998%. RFLP analysis using Hinf I and Sau 3A single digests and the minisatellite deoxyribonucleic acid (DNA) probes 15.1.11.4 and 6.3 showed no exclusion of paternity and gave nearly conclusive evidence that the putative father was the biological father of the child.

A method for accurate amplification of polymorphic CA-repeat sequences.

Anomalous PCR products are often produced during the amplification of d(CA)n.d(TG)n sequences. Upon denaturing polyacrylamide gel electrophoresis, these products yield a ladder-like pattern that can complicate genotypic interpretation. We have developed two related techniques, referred to as two- and three-stage linear amplification (2-SLA and 3-SLA, respectively), which largely overcome this problem and yield readily interpretable banding patterns.

Dedifferentiation of mammalian myotubes induced by msx1.

The process of cellular differentiation culminating in terminally differentiated mammalian cells is thought to be irreversible. Here, we present evidence that terminally differentiated murine myotubes can be induced to dedifferentiate. Ectopic expression of msx1 in C2C12 myotubes reduced the nuclear muscle proteins MyoD, myogenin, MRF4, and p21 to undetectable levels in 20%-50% of the myotubes. Approximately 9% of the myotubes cleave to produce either smaller multinucleated myotubes or proliferating, mononucleated cells. Finally, clonal populations of the myotube-derived mononucleated cells can be induced to redifferentiate into cells expressing chondrogenic, adipogenic, myogenic, and osteogenic markers. These results suggest that terminally differentiated mammalian myotubes can dedifferentiate when stimulated with the appropriate signals and that msx1 can contribute to the dedifferentiation process.

Mammalian myotube dedifferentiation induced by newt regeneration extract.

Newts are capable of regenerating several anatomical structures and organs, including their limbs. This remarkable regenerative capacity is thought to depend on cellular dedifferentiation. Terminally differentiated mammalian cells, by contrast, are normally incapable of reversing the differentiation process. Several factors could explain the absence of cellular dedifferentiation in mammals: (i) inadequate expression of genes that initiate dedifferentiation; (ii) insufficient intracellular signaling pathways; (iii) irreversible expression of differentiation factors; and (iv) structural characteristics that make dedifferentiation impossible. To investigate the causes underlying the lack of cellular plasticity in mammalian cells, we examined the effect of an extract derived from newt regenerating limbs on terminally differentiated mouse C2C12 myotubes. Approximately 18% of murine myotubes reentered the cell cycle when treated with regeneration extract, whereas 25% of newt myotubes exhibited cell cycle reentry. The muscle differentiation proteins MyoD, myogenin, and troponin T were reduced to undetectable levels in 15-30% of treated murine myotubes. We observed cellular cleavage in 11% of the treated murine myotubes and approximately 50% of these myotubes continued to cleave to produce proliferating mononucleated cells. These data indicate that mammalian myotubes can dedifferentiate when stimulated with the appropriate factors and suggest that one mechanism preventing dedifferentiation of mammalian cells is inadequate spatial or temporal expression of genes that initiate dedifferentiation.

Template-switching during DNA synthesis by Thermus aquaticus DNA polymerase I.

Recombinant DNA molecules are often generated during the polymerase chain reaction (PCR) when partially homologous templates are available [e.g., see Pääbo et al. (1990) J. Biol. Chem. 265, 4718-4721]. It has been suggested that these recombinant molecules are a consequence of truncated extension products annealing to partially homologous templates on subsequent PCR cycles. However, we demonstrate here that recombinants can be generated during a single round of primer extension in the absence of subsequent heat denaturation, indicating that template-switching produces some of these recombinant molecules. Two types of template-switches were observed: (i) switches to pre-existing templates and (ii) switches to the complementary nascent strand. Recombination is reduced several fold when the complementary template strands are physically separated by attachment to streptavidin magnetic beads. This result supports the hypothesis that either the polymerase or at least one of the two extending strands switches templates during DNA synthesis and that interaction between the complementary template strands is necessary for efficient template-switching.

A gene for familial total anomalous pulmonary venous return maps to chromosome 4p13-q12.

Total anomalous pulmonary venous return (TAPVR) is a cyanotic congenital heart defect that, without surgical correction, has a high mortality rate in the first year of life. It usually occurs without a family history and has a low recurrence risk. However, we recently reported a large Utah-Idaho family in which TAPVR segregates as an autosomal dominant trait with decreased penetrance. Linkage mapping with highly polymorphic microsatellite markers localizes the disease locus in this pedigree to the centromeric region of chromosome 4 (maximum lod = 6.51 at theta = .00). Apparent genetic anticipation in the pedigree prompted a search for expanded trinucleotide repeats by using repeat expansion detection. We have found no evidence for a trinucleotide repeat expansion that segregates with TAPVR. A vascular endothelial growth-factor receptor that is thought to have a role in vasculogenesis maps near the pericentric region of chromosome 4 and is a candidate gene for both familial and sporadic cases of TAPVR.

Characterization of eight VNTR loci by agarose gel electrophoresis.

Allelic frequencies and their confidence intervals were obtained for eight independent VNTR loci from a sample of more than 75 Utah Caucasians. Using high-resolution agarose gel electrophoresis, we were able to resolve alleles at the D17S5 locus that differed by only one repeating unit; it was therefore possible to name the alleles according to the number of repeating units each contained. Two a priori probabilities were calculated for each VNTR locus separately and for all eight loci jointly: (i) the "power of exclusion" for an alleged father/mother/child trio and for an alleged parent/child duo, and (ii) the "probability of matching" when two unrelated individuals or two siblings are genotyped.

Loss of chromosome 17 loci in prostate cancer detected by polymerase chain reaction quantitation of allelic markers.

Using a polymerase chain reaction/microsatellite marker system, we demonstrated that 6 of 22 (27%) clinical stage B (early) primary prostate tumors showed loss of heterozygosity at one or more of five loci on chromosome 17. The sensitivity of this study was increased by use of a PhosphorImager and statistical analysis of replicate tumor-normal DNA pairs. Two patients showed tumor-specific interstitial loss at a locus in close proximity to the familial breast cancer gene BRCA1. These findings suggest that genes on the proximal long arm of chromosome 17 play a pivotal role in the early development of at least a subset of prostatic tumors.

An extended genetic linkage map and an "index" map for human chromosome 17.

Our previous genetic map for chromosome 17 has been expanded to include 72 loci defined by 90 RFLP markers and four microsatellite markers assayed by the polymerase chain reaction. Forty-one of these loci were ordered with odds greater than 1000:1 against local inversion, and the other 31 were ordered within 95% confidence limits. From the set of 41 unambiguously mapped loci, 14 well-spaced "index markers" can be extracted for efficient genetic studies. The complete map spans 173 cM (136 cM in males and 214 cM in females); average spacing between markers is 4.2 cM.

DNA deletion associated with hereditary neuropathy with liability to pressure palsies.

Hereditary neuropathy with liability to pressure palsies (HNPP) is an autosomal dominant disorder that causes episodes of focal demyelinating neuropathy following minor trauma to peripheral nerves. We assign the HNPP locus to chromosome 17p11.2 and demonstrate the presence of a large interstitial deletion associated with this disorder in three unrelated pedigrees. De novo deletion is documented in one pedigree. The deleted region appears uniform in all pedigrees and includes the gene for peripheral myelin protein 22 (PMP-22), suggesting that underexpression of PMP-22 may cause HNPP. The deletion in HNPP spans approximately 1.5 Mb and includes all markers that are known to map within the Charcot-Marie-Tooth neuropathy type 1A (CMT1A) duplication. Furthermore, the breakpoints in HNPP and CMT1A map to the same intervals in 17p11.2, suggesting that these genetic disorders may be the result of reciprocal products of unequal crossover.

LIM-kinase1 hemizygosity implicated in impaired visuospatial constructive cognition.

To identify genes important for human cognitive development, we studied Williams syndrome (WS), a developmental disorder that includes poor visuospatial constructive cognition. Here we describe two families with a partial WS phenotype; affected members have the specific WS cognitive profile and vascular disease, but lack other WS features. Submicroscopic chromosome 7q11.23 deletions cosegregate with this phenotype in both families. DNA sequence analyses of the region affected by the smallest deletion (83.6 kb) revealed two genes, elastin (ELN) and LIM-kinase1 (LIMK1). The latter encodes a novel protein kinase with LIM domains and is strongly expressed in the brain. Because ELN mutations cause vascular disease but not cognitive abnormalities, these data implicate LIMK1 hemizygosity in imparied visuospatial constructive cognition.