Two long QT syndrome loci map to chromosomes 3 and 7 with evidence for further heterogeneity.

Cardiac arrhythmias cause sudden death in 300,000 United States citizens every year. In this study, we describe two new loci for an inherited cardiac arrhythmia, long QT syndrome (LQT). In 1991 we reported linkage of LQT to chromosome 11p15.5. In this study we demonstrate further linkage to D7S483 in nine families with a combined lod score of 19.41 and to D3S1100 in three families with a combined score of 6.72. These findings localize major LQT genes to chromosomes 7q35-36 and 3p21-24, respectively. Linkage to any known locus was excluded in three families indicating that additional heterogeneity exists. Proteins encoded by different LQT genes may interact to modulate cardiac repolarization and arrhythmia risk.

Stable antibody-producing murine hybridomas.

A method is described for obtaining antibody-producing hybridomas that are preferentially retained in cultures of fused mouse spleen and myeloma cells. Hybridomas are produced by fusing mouse myeloma cells that are deficient in adenosine phosphoribosyltransferase (APRT) with mouse spleen cells containing Robertsonian 8.12 translocation chromosomes. The cell fusion mixtures are exposed to a culture medium that can be utilized only by APRT-positive cells, which results in the elimination of both unfused APRT-deficient myeloma cells and non-antibody-producing APRT-deficient hybridomas that arise by segregation of the 8.12 translocation chromosomes containing the APRT genes and the active heavy chain immunoglobulin gene.

Genetic control of the eighth component of complement.

Using isoelectric focusing in polyacrylamide gel and a hemolytic assay for development of patterns, extensive, structural polymorphism in human C8 has been delineated. Two alleles, C8A and C8B, have been identified in orientals, with gene frequencies of 0.655 and 0.345. In blacks, what appears to be a third common allele was found, so that frequencies were 0.692, 0.259, and 0.049 for C8A, C8B, and C8A1. In whites, C8A1 was rare with a frequency of 0.003, and frequencies for C8A and C8B were 0.649 and 0.349. Inheritance was autosomal codominant in family studies and the distribution of types in random unrelated populations fit the Hardy-Weinberg equilibrium in all groups. C8 allotypes have been determined for two previously studied families, each with a homozygous C8-deficient propositus. This study suggests that C8 deficiency is a silent or null allele of the C8 structural locus, and that half normal levels of C8 cannot be used as a single criterion for the establishment of heterozygous C8 deficiency. C8 allotypes, as well as 18 other autosomal markers, were also determined for 24 families. The C8 structural locus is not closely linked to these markers, including the human histocompatibility loci complex.

T wave "humps" as a potential electrocardiographic marker of the long QT syndrome.

OBJECTIVES: This study attempted to determine the prevalence and electrocardiographic (ECG) lead distribution of T wave "humps" (T2, after an initial T wave peak, T1) among families with long QT syndrome and control subjects. BACKGROUND: T wave abnormalities have been suggested as another facet of familial long QT syndrome, in addition to prolongation of the rate-corrected QT interval (QTc), that might aid in the diagnosis of affected subjects. METHODS: The ECGs from 254 members of 13 families with long QT syndrome (each with two to four generations of affected members) and from 2,948 healthy control subjects (age > or = 16 years, QTc interval 0.39 to 0.46 s) were collected and analyzed. Tracings from families with long QT syndrome were read without knowledge of QTc interval or family member status (210 blood relatives and 44 spouses). RESULTS: We found that T2 was present in 53%, 27% and 5% of blood relatives with a "prolonged" (> or = 0.47 s, "borderline" (0.42 to 0.46 s) and "normal" (< or = 0.41 s) QTc interval, respectively (p < 0.0001), but in only 5% and 0% of spouses with a borderline and normal QTc interval, respectively (p = 0.06 vs. blood relatives). Among blood relatives with T2, the mean [+/- SD] maximal T1T2 interval was 0.10 +/- 0.03 s and correlated with the QTc interval (p < 0.01); a completely distinct U wave was seen in 23%. T2 was confined to leads V2 and V3 in 10%, whereas V4, V5, V6 or a limb lead was involved in 90% of blood relatives with T2. Among blood relatives with a borderline QTc interval, 50% of those with versus 20% of those without major symptoms manifested T2 in at least one left precordial or limb lead (p = 0.05). A T2 amplitude > 1 mm (grade III) was observed, respectively, in 19%, 6% and 0% of blood relatives with a prolonged, borderline and normal QTc interval with T2 in at least one left precordial or limb lead. Among the 2,948 control subjects, 0.6% exhibited T2 confined to leads V2 and V3, and 0.9% had T2 involving one or more left precordial lead (but none of the limb leads). Among 37 asymptomatic adult blood relatives with QTc intervals 0.42 to 0.46 s, T2 was found in left precordial or limb leads in 9 (24%; 5 with limb lead involvement) versus only 1.9% of control subjects with a borderline QTc interval (p < 0.0001). CONCLUSIONS: These findings are consistent with the hypothesis that in families with long QT syndrome, T wave humps involving left precordial or (especially) limb leads, even among asymptomatic blood relatives with a borderline QTc interval, suggest the presence of the long QT syndrome trait.

Age-gender influence on the rate-corrected QT interval and the QT-heart rate relation in families with genotypically characterized long QT syndrome.

OBJECTIVES: We sought to analyze age-gender differences in the rate-corrected QT (QTc) interval in the presence of a QT-prolonging gene. BACKGROUND: Compared with men, women exhibit a longer QTc interval and an increased propensity toward torsade de pointes. In normal subjects, the QTc gender difference reflects QTc interval shortening in men during adolescence. METHODS: QTc intervals were analyzed according to age (< 16 or > or = 16 years) and gender in 460 genotyped blood relatives from families with long QT syndrome linked to chromosome 11p (KVLQT1; n = 199), 7q (HERG; n = 208) or 3p (SCN5A; n = 53). RESULTS: The mean QTc interval in genotype-negative blood relatives (n = 240) was shortest in men, but similar among women, boys and girls. For genotype-positive blood relatives, men exhibited the shortest mean QTc interval in chromosome 7q- and 11p-linked blood relatives (n = 194), but not in the smaller 3p-linked group (n = 26). Among pooled 7q- and 11p-linked blood relatives, multiple regression analysis identified both genotype (p < 0.001) and age-gender group (men vs. women/children; p < 0.001) as significant predictors of the QTc interval; and heart rate (p < 0.001), genotype (p < 0.001) and age-gender group (p = 0.01) as significant predictors of the absolute QT interval. A shorter mean QT interval in men was most evident for heart rates < 60 beats/min. CONCLUSIONS: In familial long QT syndrome linked to either chromosome 7q or 11p, men exhibit shorter mean QTc values than both women and children, for both genotype-positive and -negative blood relatives. Thus, adult gender differences in propensity toward torsade de pointes may reflect the relatively greater presence in men of a factor that blunts QT prolongation responses, especially at slow heart rates.

Genomic structure of the human unconventional myosin VI gene.

Mutations in myosin VI (Myo6) cause deafness and vestibular dysfunction in Snell's waltzer mice. Mutations in two other unconventional myosins cause deafness in both humans and mice, making myosin VI an attractive candidate for human deafness. In this report, we refined the map position of human myosin VI (MYO6) by radiation hybrid mapping and characterized the genomic structure of myosin VI. Human myosin VI is composed of 32 coding exons, spanning a genomic region of approximately 70 kb. Exon 30, containing a putative CKII site, was found to be alternatively spliced and appears only in fetal and adult human brain. D6S280 and D6S284 flank the myosin VI gene and were used to screen hearing impaired sib pairs for concordance with the polymorphic markers. No disease-associated mutations were identified in twenty-five families screened for myosin VI mutations by SSCP analysis. Three coding single nucleotide polymorphisms (cSNPs) were identified in myosin VI that did not alter the amino acid sequence. Myosin VI mutations may be rare in the human deaf population or alternatively, may be found in a population not yet examined. The determination of the MYO6 genomic structure will enable screening of individuals with non-syndromic deafness, Usher's syndrome, or retinopathies associated with human chromosome 6q for mutations in this unconventional myosin.

Human gastric adenocarcinoma cathepsin B: isolation and sequencing of full-length cDNAs and polymorphisms of the gene.

Four full-length cDNA clones coding for preprocathepsin B were isolated from a human gastric adenocarcinoma cDNA library (AGS 1-6-30-1) and analyzed for possible sequence modifications that might be linked to altered intracellular trafficking and secretion of cathepsin B (CTSB) in malignant tumors. Comparison of AGS 1-6-30-1 cDNAs with human kidney/hepatoma cDNAs revealed: (1) three potential N-glycosylation sites instead of two, (2) a nucleotide (nt) substitution in the coding region for the propeptide from GTG to CTG which would result in a Val26-->Leu change, (3) three silent nt replacements in the coding region for the mature protein, (4) five single-nt differences in the 5'- and 3'-UTR (untranslated regions), (5) heterogeneity in the 5'-UTR, and (6) a 10-bp insertion in the 3'-UTR. The 10-bp insertion in the 3'-UTR may alter the stability of CTSB mRNA transcripts and thereby the expression of CTSB. These clones should be useful for expressing human tumor CTSB and analyzing the function of this enzyme in malignant progression. Two restriction-fragment length polymorphisms (RFLPs), EcoRI and TaqI, were detected by Southern blot analysis of genomic DNA from 36 unrelated Caucasians. Inheritance and distribution of the EcoRI alleles (13.0 and 11.0 kb) and the TaqI alleles (5.7 and 4.6 kb) indicated they were independent polymorphisms. In contrast to the EcoRI alleles of 13.0 and 11.0 kb observed in the population survey, genomic DNA from two AGS gastric adenocarcinoma subclones revealed two EcoRI alleles of 13.0 and 7.8 kb.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular and cytogenetic studies of an X;autosome translocation in a patient with premature ovarian failure and review of the literature.

We have identified a patient with premature ovarian failure (POF) and a balanced X;autosome translocation: 46,X,t(X;6)(q13.3 or q21;p12) using high-resolution cytogenetic analysis and FISH. BrdU analysis showed that her normal X was late-replicating and translocated X earlier-replicating which is typical of balanced X;autosome rearrangements. Molecular studies were done to characterize the breakpoint on Xq and to determine the parental origin. PCR probes of tetranucleotide and dinucleotide repeat polymorphisms, and genomic probes were used to study DNA from the patient, her chromosomally normal parents and brother, and somatic cell hybrids containing each translocation chromosome. The translocation is paternally derived and is localized to Xq13.3-proximal Xq21.1, between PGK1 and DXS447 loci, a distance of 0.1 centimorgans. A "critical region" for normal ovarian function has been proposed for Xq13-q26 [Sarto et al., Am J Hum Genet 25:262-270, 1973; Phelan et al., Am J Obstet Gynecol 129:607-613, 1977; Summitt et al., BD:OAS XIV(6C):219-247, 1978] based on cytogenetic and clinical studies of patients with X;autosome translocations. Few cases have had molecular characterization of the breakpoints to further define the region. While translocations in the region may lead to ovarian dysfunction by disrupting normal meiosis or by a position effect, two recent reports of patients with premature ovarian failure and Xq deletions suggest that there is a gene (POF1) localized to Xq21.3-q27 [Krauss et al., N Engl J Med 317:125-131, 1987; Davies et al., Cytogenet Cell Genet 58:853-966, 1991] or within Xq26.1-q27 [Tharapel et al., Am J Hum Genet 52:463-471, 1993] responsible for POF.(ABSTRACT TRUNCATED AT 250 WORDS)

Hereditary hyperparathyroidism and multiple ossifying jaw fibromas: a clinically and genetically distinct syndrome.

A large previously reported family with hyperparathyroidism has been reinvestigated recently because of the occurrence of multiple ossifying jaw fibromas in two affected members of the third generation similar to the jaw tumors of four of five affected members of the first generation. These maxillary and mandibular tumors can be differentiated from the "brown tumors" of hyperparathyroidism because they can appear and enlarge even though the hypercalcemia is surgically corrected. These tumors are histologically distinct fibroosseous lesions without the giant cells seen in "brown tumors." The parathyroid enlargement was mostly uniglandular, with multiple tumors found occasionally. Studies in DNA linkage were performed within this large family and a similar family in Houston to determine if the gene for this syndrome, termed HRPT2, is linked to DNA markers on chromosome 11, to which the gene for multiple endocrine neoplasia (MEN) type 1 has been linked. (This linkage is supported by our findings in one family with MEN 1 reported here.) Linkage studies were also performed with markers on chromosome 10, to which the genes for MEN 2A and MEN 2B have been linked. Evidence against close linkage with chromosome 10 and chromosome 11 markers suggests that this clinically distinct syndrome is also genetically distinct.

Genetic variation of human aspartic proteinases.

The human aspartic proteinases include pepsinogen A, pepsinogen C, cathepsin D, cathepsin E and renin. Comparative analysis of the proteinase genes reveals a high degree of similarity with regard to their respective coding sequences and the location of exon-intron junctions. Despite strong conservation of the regions containing the active site aspartyl groups, genetic polymorphisms have been identified for each of the proteinase genes with the exception of cathepsin D. These genetic polymorphisms are useful for localization of genes on linkage maps as well as determination of gene copy number. The chromosomal location of each aspartyl proteinase has been determined by a variety of gene mapping methods employing recombinant DNA probes including; analysis of somatic cell hybrid mapping panels, in situ hybridization to metaphase chromosome preparations and family linkage analysis with polymorphic markers. Pepsinogen A exhibits the most extensive polymorphism among aspartic proteinases which can be detected by either by protein electrophoresis or by DNA analysis. Southern blot hybridization with respective DNA probes and polymerase chain reaction (PCR) amplification have revealed nucleotide differences located within the coding and noncoding portions of the aspartic proteinase genes. These polymorphisms can be used to investigate potential roles of each proteinase in genetically influenced clinical conditions. The development of additional highly polymorphic markers detected by PCR amplification of divergent nucleotide sequence repeats will greatly assist with documentation of the effect of genetic variation of the aspartic proteinases may have in specific clinical diseases such as ulcer and hypertension.

Monoclonal antibodies to pig angiotensinogen recognize minor idiotypes.

We have produced monoclonal antibodies to a highly purified pig (P) angiotensinogen preparation and characterized their ability to bind [125]I-P- angiotensinogen. Lymphocytes of RBF/Dn mice immunized with P-angiotensinogen were fused with FOX-NY myeloma cells and clones were isolated by binding to [125]I-P-angiotensinogen and by an immunodot blot assay. Three of 16 clones which recognized P-angiotensinogen were characterized. Isolated monoclonal antibodies bound only 10-15% of the total [125]I-P-angiotensinogen; however, the bound counts could be displaced with unlabelled P-angiotensinogen. None of the monoclonals inhibited the cleavage of P-angiotensinogen by homologous renin, nor did they bind to the NH-terminal angiotensin I (ANG I) peptide. Little or no binding was detected to angiotensinogens in human, monkey, rat, rabbit, sheep or bovine serum. Mixtures of the clones and analysis of the immune complexes by PAGE indicated that different binding sites on different P-angiotensinogen were detected by some of the monoclonals, while the same or competing sites were recognized by others. No combination of clones tested significantly increased the amount of P-angiotensinogen bound. We interpret these findings to indicate that monoclonal antibodies to 'purified' pig P-angiotensinogen recognize species-specific minor epitope subsets of the protein, but not antigenic determinants common to all.

Pepsinogens, pepsins, and peptic ulcer.

The role of pepsin in the pathogenesis of peptic ulcer has been the subject of intense study and debate for many years. Two difficulties inherent in distinguishing between the role of acid alone vs acid and pepsin are that a) acid-containing gastric juice always contains pepsin, and, b) that hydrogen ion concentration (pH) is a major determinant of the activity of pepsin. However, studies in animal models of peptic ulcer indicate clearly that pepsin, in combination with acid, produces much more severe and more extensive mucosal damage than acid alone. Recent interest in pepsin and its precursor, pepsinogen, has stemmed from the finding that each is remarkably heterogeneous, and that the heterogeneity has a genetic basis. Results of studies using radioimmunoassays specific for the 2 major forms of pepsinogen, pepsinogen I and pepsinogen II, have shown that serum levels of pepsinogen I and pepsinogen II, and the ratio of pepsinogen I to pepsinogen II, can be used as noninvasive probes of gastric mucosal structure and function, indicators of the genetics and heterogeneity of duodenal ulcer, and as markers of increased risk for duodenal ulcer and gastric ulcer.

Mapping of polypeptide genes by two-dimensional gel electrophoresis of hybrid cell extracts.

A series of 18 Chinese hamster X human somatic cell hybrid subclones segregating human chromosomes were analyzed by two-dimensional gel electrophoresis to determine the chromosomal location of the human genes responsible for the expression of specific proteins. Two independent methods, side by side comparison and double label autoradiography-fluorography, were used to identify the human proteins that were unambiguously resolved from those of the Chinese hamster (CH). Of the 83 human spots resolved, 22 were determined to be associated with nine specific chromosomes or chromosome regions by correlation with quantitative cytogenetic analysis of the hybrid clones. An additional five spots were associated with three different chromosomes that had identical patterns of segregation. A total of 12 resolvable polypeptides of apparently human origin were present in all 18 hybrid subclones and may be coded for by one of the nine human autosomes that were also consistently present. Future investigations using a variety of available methods to determine the identity of the polypeptide spots will permit the mapping of genes for specific enzymes and for other classes of proteins that are not detectable by the commonly used zymogram methods.

Mapping of polypeptide genes by two-dimensional gel electrophoresis of hybrid cell extracts.

A series of 18 Chinese hamster X human somatic cell hybrid subclones segregating human chromosomes were analyzed by two-dimensional gel electrophoresis to determine the chromosomal location of the human genes responsible for the expression of specific proteins. Two independent methods, side by side comparison and double label autoradiography-fluorography, were used to identify the human proteins that were unambiguously resolved from those of the Chinese hamster (CH). Of the 83 human spots resolved, 22 were determined to be associated with nine specific chromosomes or chromosome regions by correlation with quantitative cytogenetic analysis of the hybrid clones. An additional five spots were associated with three different chromosomes that had identical patterns of segregation. A total of 12 resolvable polypeptides of apparently human origin were present in all 18 hybrid subclones and may be coded for by one of the nine human autosomes that were also consistently present. Future investigations using a variety of available methods to determine the identity of the polypeptide spots will permit the mapping of genes for specific enzymes and for other classes of proteins that are not detectable by the commonly used zymogram methods.

Assignment of the gene for cytoplasmic superoxide dismutase (Sod-1) to a region of chromosome 16 and of Hprt to a region of the X chromosome in the mouse.

In the search for homologous chromosome regions in man and mouse, the locus for cytoplasmic superoxide dismutase (SOD-1; superoxide:superoxide oxidoreductase, EC 1.15.1.1) is of particular interest. In man, the SOD-1 gene occupies the same subregion of chromosome 21 that causes Down syndrome when present in triplicate. Although not obviously implicated in the pathogenesis, SOD-1 is considered to be a biochemical marker for this aneuploid condition. Using a set of 29 mouse-Chinese hamster somatic cell hybrids, we assign Sod-1 to mouse chromosome 16. Isoelectric focusing permits distinction between mouse and Chinese hamster isozymes, and trypsin/Giemsa banding distinguishes mouse from Chinese hamster chromosomes. The mouse fibroblasts used were derived from a male mouse carrying Searle's T(X;16)16H reciprocal translocation in which chromosomes X and 16 have exchanged parts. Analysis of informative hybrids leads to regional assignment of Sod-1 to the distal half of mouse chromosome 16 (16B4 --> ter). Because the Chinese hamster cell line (380) used for cell hybridization is deficient in hypoxanthine phosphoribosyltransferase (HPRT; IMP: pyrophosphate phosphoribosyltransferase, EC 2.4.2.8), that part of the mouse X chromosome carrying the complementing Hprt gene can be identified by selection in hypoxanthine/aminopterin/thymidine medium and counterselection in 8-azaguanine. Mouse Hprt is on the X(T) translocation product containing the proximal region X cen --> XD.

Immunochemical, electrophoretic, and genetic heterogeneity of pepsinogen I. Characterization with monoclonal antibodies.

Two immunologic subclasses of human pepsinogen I alpha-PG I and beta-PG I, have been identified based on their reactivity toward a murine monoclonal antibody that recognizes an epitope on the alpha-PG I isozymogens. The antibody was used to purify the major alpha- and beta-isozymogens from gastric mucosa and to determine their contributions to the previously described genetic polymorphism of PG I. The alpha-epitope was localized to the pepsin region of the molecules. The two major alpha-PG I isozymogens (Pg 3 alpha and Pg 5 alpha) and the major beta-PG I isozymogen (Pg 4 beta) were demonstrated to contain net charge differences located in the respective pepsin and activation peptide regions. We propose that the alpha- and beta-subclasses contain net charge amino acid substitutions encoded by the corresponding pepsinogen genes: PGA3, PGA4, and PGA5.