A variable myopathy associated with heterozygosity for the R503C mutation in the carnitine palmitoyltransferase II gene.

Adult-onset carnitine palmitoyltransferase II (CPT II) deficiency is an autosomal recessive disease characterized by muscle pain and stiffness with rhabdomyolysis and myoglobinuria in severe cases. Exercise, fasting, viral infection, anesthesia, or extremes in temperature may trigger symptoms. A 54-year-old woman exhibited a 35-year history of progressive weakness and myopathic symptoms. CPT II activity in the patient's lymphoblasts, cultured skin fibroblasts, and skeletal muscle was reduced to 47, 43, and 13% of normal, respectively. Respiratory chain enzymes were also reduced in muscle ranging from 22 to 49% of their respective normal reference means. beta-oxidation enzymes in fibroblasts ranged from 29 to 63% of normal. The patient, her father, and her 26-year-old son were all heterozygous for the R503C mutation. The patient's son has a lifelong history of myopathic symptoms while his grandfather only had mild weakness during childhood. Analysis of the V368I and M647V polymorphisms in the CPT2 gene showed that the mutant allele is linked to 368I and 647M in this family and that the normal allele is linked to 647V in the affected patient and her son, and to 647M in the patient's father. While the variability in CPT2 gene haplotypes may contribute to the phenotypic complexities in this family, it is also possible that an additional gene defect in the transport of mitochondrial proteins contributes to the complex phenotype in the patient. We present biochemical and molecular evidence for vertical transmission of a variable myopathy caused by heterozygosity for a single mutation, R503C, in the CPT2 gene.

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.

Novel mutations associated with carnitine palmitoyltransferase II deficiency.

The most common form of carnitine palmitoyltransferase II (CPT II) deficiency occurs in adults and is characterized by muscle pain, stiffness, and myoglobinuria, triggered by exercise, fasting, or other metabolic stress. This study reports the molecular heterogeneity of CPT2 mutations and their biochemical consequences among a series of 59 individuals who were suspected of having CPT II deficiency based on the decreased CPT activity observed in muscle or leukocytes samples, clinical findings, or referral for mutation analysis from other laboratories. Only 19 subjects were considered to be at particularly high risk of CPT II deficiency based on review of their clinical symptoms and residual CPT activity. The samples were initially screened for 11 mutations with allele-specific oligonucleotides (ASO). Extensive sequence analysis was subsequently performed on 14 samples which either had a CPT2 mutation detected by ASO screening or the residual CPT activity was below that observed in ASO positive samples. Three known (P50H, S113L, and F448L) and three novel mutations were identified among 13 individuals in this study. A single nucleotide polymorphism was also identified 11 bp distal to the CPT2 polyadenylation site that will be useful for linkage analysis. Two of the new mutations were single nucleotide missense mutations, R503C and G549D, that occurred in highly conserved regions of the CPT isoforms, and the third was a frameshift mutation, 413 delAG, caused by a 2-bp deletion upstream of a previously identified missense mutation, F448L. The 413 delAG mutation was the second most common mutation identified in our study (20% of mutant alleles) and all individuals with the mutation were of Ashkenazi Jewish ancestry suggesting a defined ethnic origin for the mutation. Despite rigorous mutation analysis, six of 13 individuals identified with CPT2 mutations remained as heterozygotes. We propose that heterozygosity for certain CPT2 mutations, S113L and R503C, is sufficient to render individuals at risk of clinical symptoms.

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.

Expression of cathepsin E in pancreas: a possible tumor marker for pancreas, a preliminary report.

Ductal cancers of the pancreas frequently express markers of gastrointestinal epithelial cells. Cathepsin E (CTSE) is a non-secretory, intracellular, but non-lysosomal proteinase found in the highest concentration in the superficial epithelial cells of the stomach. The aims of our study were to examine the expression of CTSE in the pancreas, to establish an assay system of CTSE and to evaluate the diagnostic usefulness of CTSE in the pancreatic juice. Eleven patients with pancreatic ductal adenocarcinoma, 10 with mucin-producing adenoma, 3 with intraductal papillary hyperplasia and 43 with chronic pancreatitis were examined. Surgically resected pancreatic tissues were subjected to immunohistochemistry for CTSE. Pancreatic juice was collected from the patients and subjected to sandwich ELISA and Western analysis for detecting CTSE. Positive staining for CTSE was observed in pancreatic ductal adenocarcinoma by immunohistochemistry. CTSE was also expressed in mucin-producing adenoma, intraductal papillary hyperplasia and mucinous hyperplasia. CTSE in the pancreatic juice was present in 8 of 11 patients with pancreatic ductal adenocarcinoma, 5 of 10 patients with mucin-producing tumor, 1 of 3 patients with intraductal papillary hyperplasia and 4 of 43 patients with chronic pancreatitis. The detection frequency of CTSE in the pancreatic juice was significantly higher in the patients with pancreatic ductal adenocarcinoma than in the patients with chronic pancreatitis. Our findings suggest that the expression of CTSE is associated with the pathogenesis of pancreatic ductal adenocarcinoma, that CTSE in the pancreatic juice seems to be a useful marker for a definitive diagnosis and that CTSE may be expressed at a relatively early stage of multistep carcinogenesis in pancreatic lesions.

Genetic mapping studies of 40 loci and 23 cosmids in chromosome 11p13-11q13, and exclusion of mu-calpain as the multiple endocrine neoplasia type 1 gene.

Forty loci (16 polymorphic and 24 non-polymorphic) together with 23 cosmids isolated from a chromosome 11-specific library were used to construct a detailed genetic map of 11p13-11q13. The map was constructed by using a panel of 13 somatic cell hybrids that sub-divided this region into 19 intervals, a meiotic mapping panel of 33 multiple endocrine neoplasia type 1 (MEN1) families (134 affected and 269 unaffected members) and a mitotic mapping panel that was used to identify loss of heterozygosity in 38 MEN1-associated tumours. The results defined the most likely order of the 16 loci as being: 11pter-D11S871-(D11S288, D11S149)-11cen-CNTF-PGA-ROM1-D11S480-PYGM- SEA-D11S913-D11S970-D11S97- D11S146-INT2-D11S971-D11S533-11qter. The meiotic mapping studies indicated that the most likely location of the MEN1 gene was in the interval flanked by PYGM and D11S97, and the results of mitotic mapping suggested a possible location of the MEN1 gene telomeric to SEA. Mapping studies of the gene encoding mu-calpain (CAPN1) located CAPN1 to 11q13 and in the vicinity of the MEN1 locus. However, mutational analysis studies did not detect any germ-line CAPN1 DNA sequence abnormalities in 47 unrelated MEN1 patients and the results therefore exclude CAPN1 as the MEN1 gene. The detailed genetic map that has been constructed of the 11p13-11q13 region should facilitate the construction of a physical map and the identification of candidate genes for disease loci mapped to this region.

Evidence of genetic heterogeneity in Romano-Ward long QT syndrome. Analysis of 23 families.

BACKGROUND: The Romano-Ward long-QT Syndrome (LQTS) is an autosomal dominant inherited trait characterized by prolonged QT interval on ECG, life-threatening arrhythmias, syncope, and sudden death in affected individuals. A gene responsible for this disorder has been shown to be linked to the Harvey ras-1 locus (H-ras-1) DNA marker on the short arm of chromosome 11 (11p) in 7 families. The purpose of this study was to determine, by analyzing 23 families with LQTS for linkage to chromosome 11p, whether evidence exists for more than one gene causing LQTS (ie, locus heterogeneity). METHODS AND RESULTS: Twenty-three families (262 family members) were clinically evaluated using medical histories, ECGs, and Holter recordings. Each corrected QT interval (QTc) were determined using Bazett's formula. Blood for DNA extraction and cell line immortalization was obtained after informed consent. Southern blotting and polymerase chain reaction were performed, and linkage analysis carried out using the LINKAGE computer program (v 5.03). Genetic heterogeneity was determined using the HOMOG 2 (v 2.51) computer program. Twenty-three families were studied for evidence of linkage to chromosome 11p using the H-ras-1 locus probe pTBB-2 and multiple flanking markers, including tyrosine hydroxylase (TH). Two-point linkage analysis using pTBB-2 and TH markers was consistent with linkage in 15 of 23 families, with the maximum single-family LOD score of +3.038 occurring at theta = 0. However, 8 of 23 families had negative LOD scores, with the values in 4 families being less than -2 at theta = 0, consistent with exclusion of linkage. Analysis with the HOMOG program was consistent with genetic heterogeneity (P < .0001). Multipoint linkage data using pTBB-2 and TH were also examined for evidence of heterogeneity. HOMOG analysis of multipoint LOD scores from 100 cM surrounding the H-ras-1 locus also supported heterogeneity (P < .001). CONCLUSIONS: In the 23 families with LQTS analyzed for linkage to the H-ras-1 locus on chromosome 11p15.5, 15 of 23 families had LOD scores consistent with linkage. The remaining 8 of 23 families had negative LOD scores, 4 of which were definitively excluded from linkage. Thus, genetic heterogeneity is definitively (P < .001) demonstrated for this disorder.

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.

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.

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)

Mapping the gene for hereditary hyperparathyroidism and prolactinoma (MEN1Burin) to chromosome 11q: evidence for a founder effect in patients from Newfoundland.

An autosomal dominant syndrome of prolactinomas, carcinoids, and hyperparathyroidism was described in four Newfoundland kindreds in 1980 and in one kindred from the Pacific Northwest in 1983. Because this syndrome shares many features with multiple endocrine neoplasia type 1, the gene for which maps to proximal chromosome 11q, we performed linkage studies with chromosome 11 markers in prolactinoma families to determine whether the two genes map to the same location. All proximal chromosome 11q markers gave positive LOD scores, and no recombinants were seen with PYGM (LOD score 15.25, recombination fraction .0). All affected individuals from Newfoundland shared the same PYGM allele, providing evidence for a founder effect. The disease in the Pacific Northwest kindred cosegregated with a different PYGM allele.

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)

Isolation and characterization of recombinant human cathepsin E expressed in Chinese hamster ovary cells.

The cDNA sequence encoding precursor forms of human cathepsin E (CE), an intracellular aspartic proteinase, was expressed in Chinese hamster ovary cells using an SV40 promotor-driven expression vector. By immunoelectron microscopic studies using an anti-human CE antibody and by Percoll density gradient fractionation, the expressed CE was found to be in two different intracellular fractions; the cytosolic compartment and the vacuolar system. The CEs in both the cytosolic and the vacuolar fractions were highly purified by a simple method involving Percoll density gradient fractionation, chromatography on concanavalin A-Sepharose, Mono Q, and TSK-GelG2000SW, and termed s-CE and v-CE, respectively. The v-CE was further separated into a major (v-CE1) and a minor (v-CE2) form by Mono Q chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting revealed that the s-CE and v-CE1 consists of two polypeptides of 90 and 84 kDa, whereas v-CE2 is composed of 84- and 82-kDa polypeptides. The NH2-terminal amino acid sequence analyses showed that the 90- and 84-kDa proteins from both s-CE and v-CE started with Ser3 and Lys30 of the sequence of human gastric CE predicted from its cDNA sequence, respectively, and that the NH2 terminus of the 82-kDa protein of v-CE2 is the Ile37. Upon acid treatment at pH 3.5 and 37 degrees C for 5 min, the 90- and 84-kDa forms are rapidly converted to the 82-kDa form, indicating that the 90-, 84- and 82-kDa proteins are the pro-CE, the intermediate form, and the mature CE, respectively. All the forms of CE are N-glycosylated with high-mannose-type oligosaccharides. The catalytic properties of s-CE and v-CE are comparable to those of natural human CE. These results suggest that the recombinant CE is initially synthesized on membrane-bound ribosomes as a N-glycosylated preproenzyme and that, after cleavage of the signal segment, the 90-kDa proenzyme is proteolytically processed to the intermediate (84 kDa) and mature (82 kDa) forms by the transport system.

Pepsinogen C gene polymorphisms associated with gastric body ulcer.

This study was aimed to investigate the association of restriction fragment length polymorphisms (RFLPs) for pepsinogen genes with peptic ulcer disease. Eighty unrelated controls, 61 patients with gastric ulcer, and 57 patients with duodenal ulcer were studied. No genetic polymorphisms for pepsinogen A were detected by EcoRI digestion in Japanese subjects but a 100 base pairs insertion-deletion RFLP for the pepsinogen C gene was observed. The allele frequencies of the large (3.6 kilobase EcoRI fragment) and the small fragment (3.5 kilobase EcoRI fragment) were 80.6% and 19.4% respectively in controls, 55.4% and 44.6% in patients with gastric body ulcer, 79.4% and 20.6% in patients with gastric angular ulcer, 71.4% and 28.6% in patients with gastric antral ulcer, and 75.4% and 24.6% in patients with duodenal ulcer. The allele frequency of the small fragment was significantly higher in patients with gastric body ulcer than in controls and in patients with gastric angular or antral ulcer. The genotypes which possessed the small fragment were significantly more frequent in patients with gastric body ulcer (78.4%) than in controls (33.8%) and in patients with gastric angular or antral ulcer (37.5%). These results suggest that there is a significant association between the genetic polymorphism at the pepsinogen C gene locus and gastric body ulcer, and that the pepsinogen C RFLP is a useful marker of the genetic predisposition to this disorder. These results also indicate genetic heterogeneity of gastric ulcer disease, and suggest that the pepsinogen C RFLP may be a useful subclinical marker to explain the differences in genetic aetiologies of gastric body ulcer and gastric angular or antral ulcer.

Detection of a polymorphism within the pepsinogen C gene with PCR: construction of a linkage map around PGC from 6p11-6p21.3.

An insertion/deletion polymorphism between exons 7 and 8 of the pepsinogen C gene (PGC), previously detectable with Southern analysis, was formatted for detection with PCR. Alleles were rapidly typed by UV irradiation of ethidium bromide-stained agarose gels. Whereas Southern analysis revealed two alleles, the smaller fragments generated with PCR allowed the resolution of three alleles that were previously scored as a single allele and increased the heterozygosity of the system from 0.20 to 0.53. After a set of reference families was genotyped with the PCR-based polymorphism, a linkage map around the PGC gene on chromosome 6 was constructed. This included the HLA cluster and the highly informative D6S223 locus. PGC lies 22 cM proximal to HLA-DPB and between D6S5 and D6S4 at distances of 4.5 and 13.1 cM, respectively.

The human loci DNF15S2 and D3S94 have a high degree of sequence similarity to acyl-peptide hydrolase and are located at 3p21.3.

The short arm of chromosome 3 undergoes genetic loss in most small-cell lung cancers and renal cell carcinomas. The most frequently deleted region includes the DNF15S2 locus (mapped to 3p21), suggesting that a putative recessive tumor-suppressor gene might be located nearby. A cosmid clone, cA476, contains the D3S94 locus and two HTF islands and detects a PstI RFLP. We have isolated cDNAs homologous to conserved fragments within cA476; and these cDNAs have 96% sequence similarity to a cDNA derived from the DNF15S2 locus. Sequence information from cDNAs derived from both the rat and pig acyl-peptide hydrolase (E.C.3.4.19.1) gene show that they have a high degree of sequence similarity to cDNAs derived from D3S94 and DNF15S2, suggesting that they are all the same locus. Cosmid cA476 (DNF15S2) has been mapped, by fluorescent in situ hybridization, to chromosome 3p21.3. D3S94 and DNF15S2 are quite distinct from aminoacylase 1 (ACY1), which has been physically linked to D3S2, D3S92, and D3S93, all localized within 3p21.1.

Human gastric cathepsin E gene. Multiple transcripts result from alternative polyadenylation of the primary transcripts of a single gene locus at 1q31-q32.

Genomic clones containing portions of the human cathepsin E (CTSE) gene were isolated from cosmid and lambda recombinant libraries. The regions corresponding to coding, the 5'- and 3'-untranslated, and the exon-intron boundaries of the CTSE gene were identified by sequence and hybridization analysis. The size and placement of the nine exons found in the 17.5-kilobase CTSE gene was highly conserved relative to other aspartic proteinases and provided additional evidence that these proteinases are derived from a common ancestral gene. Segregation and linkage analysis of two informative restriction fragment length polymorphisms (MspI and DraI) indicated that there is a single human CTSE locus located at chromosome 1q31-q32 which is closely linked to the renin gene. Three CTSE transcripts (3.6, 2.6, and 2.1 kilobases) were identified in gastric fundic and antral mucosa poly (A+) RNA, and these appeared identical in size and relative abundance to those contained in poly(A+) RNA from cultured gastric adenocarcinoma cell lines containing CTSE. Sequence analysis of cDNA clones and comparison with the 3'-flanking untranslated region in genomic clones provided evidence that alternative polyadenylation of the primary transcript resulted in the 2.6- and 2.1-kilobase transcripts which constituted greater than 95% of CTSE transcripts found in the stomach.

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.