A genetic linkage study in Brazil identifies a new locus for persistent developmental stuttering on chromosome 10.

Although twin, adoption, and family studies demonstrate that genetic factors are involved in the origins of stuttering, the mode of transmission of the disorder in families is not well defined and stuttering is considered a genetically complex trait. We performed a genome-wide linkage scan in a group of 43 Brazilian families, each containing multiple cases of persistent developmental stuttering. Linkage analysis under a dominant model of inheritance generated significant evidence of linkage in two Brazilian families, with a combined maximum single-point LOD score of 4.02 and a multipoint LOD score of 4.28 on chromosome 10q21. This demonstrated the presence of a novel variant gene at this locus that predisposes individuals to stuttering, which provides an opportunity to identify novel genetic mechanisms that underlie this disorder.

A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis.

Hereditary haemochromatosis (HH), which affects some 1 in 400 and has an estimated carrier frequency of 1 in 10 individuals of Northern European descent, results in multi-organ dysfunction caused by increased iron deposition, and is treatable if detected early. Using linkage-disequilibrium and full haplotype analysis, we have identified a 250-kilobase region more than 3 megabases telomeric of the major histocompatibility complex (MHC) that is identical-by-descent in 85% of patient chromosomes. Within this region, we have identified a gene related to the MHC class I family, termed HLA-H, containing two missense alterations. One of these is predicted to inactivate this class of proteins and was found homozygous in 83% of 178 patients. A role of this gene in haemochromatosis is supported by the frequency and nature of the major mutation and prior studies implicating MHC class I-like proteins in iron metabolism.

The genetic linkage map of the human X chromosome.

A database useful for mapping the human X chromosome has been established. The data consist of the genotypic characterizations obtained at more than 20 DNA marker loci from a set of 38 selected families. Multilocus linkage analysis has provided an initial genetic map completely spanning the distance from the distal short arm to the distal long arm of the chromosome, for a total genetic length of at least 185 recombination units. Analysis of the recombinational behavior of fully marked chromosomes suggests that the number of recombination events on the X chromosome may be nonrandom. Linkage studies of six families that carry the mutation which causes Duchenne muscular dystrophy were combined with linkage data from a large number of normal families. This permitted mapping of the locus for Duchenne muscular dystrophy with greater precision and statistical confidence than studies in which disease families alone provided the genotypic database. This observation suggests that the normal linkage map of this chromosome should be especially valuable in the mapping of rare X-linked diseases.

Genetic correlates of musical pitch recognition in humans.

We used a twin study to investigate the genetic and environmental contributions to differences in musical pitch perception abilities in humans. We administered a Distorted Tunes Test (DTT), which requires subjects to judge whether simple popular melodies contain notes with incorrect pitch, to 136 monozygotic twin pairs and 148 dizygotic twin pairs. The correlation of DTT scores between twins was estimated at 0.67 for monozygotic pairs and 0.44 for dizygotic pairs. Genetic model-fitting techniques supported an additive genetic model, with heritability estimated at 0.71 to 0.80, depending on how subjects were categorized, and with no effect of shared environment. DTT scores were only weakly correlated with measures of peripheral hearing. This suggests that variation in musical pitch recognition is primarily due to highly heritable differences in auditory functions not tested by conventional audiologic methods.

Molecular analysis of the GJB2, GJB6 and SLC26A4 genes in Korean deafness patients.

OBJECTIVES: Mutations in the GJB2, GJB6 and SLC26A4 genes are a frequent cause of hearing loss in a number of populations. However, little is known about the genetic causes of hearing loss in the Korean population. METHODS: We sequenced the GJB2 and GJB6 genes to examine the role of mutations in these genes in 22 hearing loss patients. We also sequenced the SLC26A4 gene in seven patients with inner ear malformations, including enlarged vestibular aqueduct (EVA) revealed by computer tomography. RESULTS: Coding sequence mutations in GJB2 were identified in 13.6% of the patients screened. Two different mutations, 235delC and T86R were found in three unrelated patients. The 235delC was the most prevalent mutation with an allele frequency of 6.9% in our patient group. No mutations, including 342-kb deletion, were found in GJB6 gene. Three different variants of SLC26A4 were identified in the EVA patients, including one novel mutation. Four EVA patients carried two mutant alleles of SLC26A4, and at least one allele in all patients was the H723R mutation, which accounted for 75% of all mutant alleles. CONCLUSIONS: Our results suggest that GJB2 and SLC26A4 mutations together make up a major cause of congenital hearing loss in the Korean population. Further studies may be able to identify other common variants that account for a significant fraction of hearing loss in the Korean population.

Physical map of the human chromosome 8p12-p21 encompassing tumor suppressor and Werner's syndrome gene loci.

Detailed physical maps of the human genome are important resources for identification and isolation of genes responsible for diseases and for the study of their structure and function. We constructed a 2.0-Mb high-resolution physical map within the human chromosome 8p12-p21 region extending from marker D8S131 to D8S283. The map comprises a series of contigs mostly P1/PAC clones, which span the loci of potential tumor suppressor genes and the Werner's syndrome gene. Each P1/PAC DNA was defined by its size, restriction sites, terminal sequences, intermarker distances and location relative to major genes and markers. The genes on these P1/PAC DNAs were analyzed by an exon amplification method to determine their locations. The genes newly found by the exon amplification method together with other known genes, including those of glutathion reductase, a general transcription factor, protein phosphatase 2A beta subunit and Werner's syndrome, were precisely mapped within the contigs. These P1/PAC DNAs are useful reagents for the generation of new microsatellite markers to narrow the candidate region of the tumor suppressor gene(s) and/or genes responsible for other diseases, which are believed to exist in this region by linkage analysis.

A simple method for the preparation of single-stranded polydeoxynucleotides of desired length.

A method for the preparation of homogeneous, single-stranded polydeoxynucleotides of desired length up to 800 bases is described. The procedure entails 1) generation of double-stranded DNA of desired length by PCR using a pair of primers of which one is biotinylated and the other is either unlabeled or fluorescently labeled, 2) isolation of PCR products by agarose slab gel electrophoresis, 3) recovery of desired product by electroelution, 4) binding of the product to streptavidin-coated magnetic beads and is followed by 5) duplex denaturation and removal of the unbound single strand that is either unlabeled or fluorescently labeled. Final product characteristics were determined by capillary gel electrophoresis with fluorescence detection. Up to microgram quantities of homogeneous single-stranded DNA of a desired length were obtained. These can be used as single-stranded size standards in capillary gel electrophoresis experiments as well as in other techniques requiring such standards.

Genetics of human taste perception.

Genetic approaches are rapidly yielding new information about our sense of taste. This information comes from both molecular studies of genes encoding taste receptors and other taste-signaling components, and from studies of inherited variation in taste abilities. Our understanding of bitter taste has advanced by combined information from discovery and study of the TAS2R family of taste receptor genes, hand in hand with genetic linkage and positional cloning studies, notably on the ability to taste phenylthiocarbamide (PTC). Sweet and umami tastes, mediated by TAS1R receptors, are becoming well-characterized at the molecular genetic level, and these taste classes are now targets for linkage, positional cloning, and genetic association strategies. Salty and sour tastes are still poorly characterized in genetic terms, and represent opportunities for the future.

Genetics of individual differences in bitter taste perception: lessons from the PTC gene.

The ability or inability to taste the compound phenylthiocarbamide (PTC) is a classic inherited trait in humans and has been the subject of genetic and anthropological studies for over 70 years. This trait has also been shown to correlate with a number of dietary preferences and thus may have important implications for human health. The recent identification of the gene that underlies this phenotype has produced several surprising findings. This gene is a member of the T2R family of bitter taste receptor genes. It exists in seven different allelic forms, although only two of these, designated the major taster and major non-taster forms, exist at high frequency outside sub-Saharan Africa. The non-taster allele resides on a small chromosomal region identical by descent, indicating that non-tasters are descended from an ancient founder individual, and consistent with an origin of the non-taster allele preceding the emergence of modern humans out of Africa. The two major forms differ from each other at three amino acid positions, and both alleles have been maintained at high frequency by balancing natural selection, suggesting that the non-taster allele serves some function. We hypothesize that this function is to serve as a receptor for another, as yet unidentified toxic bitter substance. At least some of the remaining five haplotypes appear to confer intermediate sensitivity to PTC, suggesting future detailed studies of the relationships between receptor structure and taste function.

Genetic studies on the mechanism of chemical and physical inactivation of reovirus.

The three serotypes of reovirus differ markedly in their response to a variety of chemical inactivating agents. We used intertypic recombinants containing various combinations of genes derived from the parental serotypes to study the basis of these differences. In addition to recombinants derived from types 1 and 3, and 2 and 3, we were able to isolate recombinants derived from types 1 and 2, suggesting that these two serotypes also undergo unrestricted reassortment. The intertypic recombinants behaved like one parent or the other in the presence of the inactivating agents and allowed us to determine the genes responsible for each difference. Recombinants derived from crosses between wild-type parental serotypes produced straightforward results, while recombinants derived from mutagenized, temperature-sensitive parents often did not. Sensitivity to 2.5 M-guanidine-HCl and pH 11 was determined by the S1 gene, sensitivity to 55 degrees C and 1% SDS was determined by the S4 gene, and sensitivity to 33% ethanol and to 1% phenol was determined by the M2 gene. Thus, relatively nonspecific chemical agents appear to have their predominant effect on specific proteins of the reovirus virion.

Biochemical studies on the mechanism of chemical and physical inactivation of reovirus.

We have examined the effects of heat and several chemical inactivating agents on the buoyant density, particle-associated polypeptides and ultrastructure of reovirus particles. Treatment at pH 11 removed polypeptide sigma 1 from the outer capsid of reovirus type 2 but not from type 1; resultant particles were unchanged in their buoyant density and morphology. Treatment of reovirus types 2 and 3 with 2.5 M-guanidine-HCl produced particles with unchanged polypeptide content but an increased buoyant density, and caused aggregation of type 3 but not type 2. Treatment with 1% SDS removed polypeptide sigma 3 from both types 1 and 2 and increased the buoyant density of the virus particles. The outer capsid of SDS-treated virions was greatly altered and often indistinct. Treatment of type 3 with either 1% phenol or 33% ethanol produced particles that had a full complement of polypeptides, were unaltered in buoyant density, but were greatly aggregated. Thus, these inactivating agents affect reovirus particles in specific and distinct ways. The differential effects of such treatments can thus be used to study the structure and function of the reovirus capsid components.

Activation and characterization of the reovirus transcriptase: genetic analysis.

We studied the ability of chymotrypsin to activate the transcriptases of the three serotypes of reovirus. When we used conditions that reproducibly caused the activation of type 3 transcriptase by chymotrypsin alone, type 2 transcriptase was sometimes activated, and type 1 transcriptase was never activated. Using intertypic recombinants containing various combinations of genome segments from reovirus types 3 and 1, we showed that the M2 segment determined this difference. Biochemical experiments indicated that the digestion of reovirus type 1 by chromotrypsin was blocked at an intermediate stage in uncoating. We found conditions which reproducibly activated the transcriptases of all three serotypes. This allowed us to compare the biochemical properties of the three transcriptases. Although the monovalent cation preferences, divalent cation preferences and optima, and temperature optima of type 1, 2, and 3 transcriptases were indistinguishable, the pH activity curves were reproducibly different. The largest difference was between type 2 and 3 transcriptases; the pH optimum of type 2 transcriptase was lower than the pH optimum of type 3 transcriptase. Using intertypic recombinants containing various combinations of genome segments from reovirus types 2 and 3, we demonstrated that the L1 segment specified this difference.

DNA polymorphism at the locus for human cholesteryl ester transfer protein (CETP) is associated with high density lipoprotein cholesterol and apolipoprotein levels.

Cholesteryl ester transfer protein (CETP) is a protein involved in "reverse cholesterol transport" and it could play an important role in facilitating the removal of cholesteryl esters from peripheral tissues for transport to the liver or for transfer of cholesterol between plasma lipoprotein particles. Both functions may be relevant to susceptibility or resistance to atherosclerotic disease. We have studied 149 and 146 unrelated persons, respectively, for the A and B polymorphism at the CETP locus detectable with the restriction enzyme TaqI. The B system is by far the more polymorphic. A search for association with risk or "anti-risk" factor levels was conducted with the following quantitative parameters: total cholesterol, HDL cholesterol, triglycerides, apolipoprotein AI (apoA-I), apolipoprotein B (apoB) and Lp(a) lipoprotein levels. Highly significant differences in apoA-I concentration were found between the two categories of homozygotes in the B polymorphism. The association observed remained significant after multiplying the p value by the number of quantitative parameters used for the association tests. There was a dosage effect on the apoA-I level of genes in the B polymorphism. We conclude that the associations observed are likely to reflect true biological phenomena. The effect of CETP genes appeared to be limited to non-smokers.

"Variability gene" effect of cholesteryl ester transfer protein (CETP) genes.

Cholesteryl ester transfer protein (CETP) may have important roles in transfer of lipids from cells to serum lipoproteins or between circulating lipoprotein particles. Restriction fragment length polymorphisms (RFLPs) in DNA at the CETP locus have been detected. In the present study we have used RFLPs detectable with the restriction enzyme TaqI to examine if CETP influences serum lipid variability (as opposed to absolute lipid levels). We have compared within-pair difference in serum lipid and apolipoprotein levels in monozygotic twin pairs of various genotypes in the B polymorphism at the CETP locus and uncovered significant differences between genotypes. We conclude that the CETP locus has "variability genes" (as opposed to "level genes") with respect to total and LDL cholesterol variability. A person's total genetic risk for coronary heart disease may depend on his or her combination of "level genes" and "variability genes". The method of analysis applied may be the best available for the study of gene - environment interaction.

A new MspI restriction fragment length polymorphism in the hemophilia B locus.

Using a partial cDNA probe for human coagulation factor IX, we have detected a new restriction fragment length polymorphism in human DNA digested with MspI. The frequency of the minor allele is 0.20 +/- 0.05 and average heterozygosity is about 0.32. The MspI RFLP is in strong linkage disequilibrium with the TaqI RFLP previously described, but should nevertheless be useful in segregation analysis in case of homozygosity for the TaqI minor allele.

Restriction site polymorphism at the LPA (Lp(a) apolipoprotein; apolipoprotein (a)) locus.

A restriction site polymorphism in the Lp(a) apolipoprotein gene (the LPA gene) is reported. The basis for the polymorphism is presence or absence of an MspI restriction site that appears to be 3' to the last kringle IV structure of the gene. The "1" gene (presence of the restriction site) has a frequency of 0.316 and the "2" gene (absence of the restriction site) has a frequency of 0.684. Both members of each of 67 monozygotic (MZ) twin pairs had the same genotype and there was Mendelian segregation of the DNA variants in 40 families with a total of 75 children. There was a lower proportion of people with genotype 1-1 in the top quartile than in the 3 bottom quartiles of the population distribution of Lp(a) lipoprotein levels but the difference did not reach statistical significance.

A genetic analysis of the Werner syndrome region on human chromosome 8p.

Werner syndrome (WRN) is an inherited disorder that produces symptoms of premature aging. This disease is caused by a recessive mutation that has previously been mapped to chromosome 8p. We have now used genetic linkage analysis to map the WRN gene relative to chromosome 8 reference loci, to screen candidate genes, and to identify a novel dinucleotide repeat polymorphic marker closely linked to WRN. The WRN locus was mapped relative to the marker loci, PLAT, ANK1, D8S135, and D8S87 of the comprehensive chromosome 8 linkage map. The heregulin (HRG) and the fibroblast growth factor receptor 1 genes (FGFR1) have been mapped to chromosome 8p and are involved in cellular growth. Recombination events were detected between WRN and the HRG and FGFR1 genes, excluding them as candidates for the WRN gene. A polymorphic marker generated in this study, WT251, is linked to WRN at a recombination fraction of 0.006, with a lod score of 16.5.

A cytological map of the human X chromosome--evidence for non-random recombination.

The cytological location of six cloned DNA sequences on the human X chromosome has been determined to a high resolution by direct hybridisation 'in situ' to metaphase chromosomes. Each locus has been identified using clones which also detect restriction fragment length polymorphisms by Southern hybridisation. The six loci identified are spaced along the chromosome from Xp22 to Xq28. By combining data obtained using this powerful sequence localisation technique with that from hybrid cell panels and from family studies, it is possible to compare physical and genetic distances, and to demonstrate that the frequency of reciprocal genetic exchange is not uniform along the chromosome length.