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A study of a family with a propositus suffering from classical thrombasthenia type I has shown that the new immunochemical methods detect heterozygotes with high reliability. There was no overlapping between heterozygotes and normals, and the concentration of the glycoproteins IIb-IIIa-complex is remarkable constant around 50-60% in the heterozygotes. Furthermore, heterozygotes as a group show an increased bleeding tendency.
Recent research have demonstrated that homozygotic and heterozygotic patients with ataxia telangiectasia (AT) show chromosomal rupture. Taking in consideration this characteristic, a study was designed, inducing chromosomal breaks in granulocytes from patients with AT, and heterozygotic carriers of AT compared with a group of healthy individuals. The number of chromosomic ruptures induced by 14 doses of radiation at 125 KV and 125 mA, was measured in all of them. Results suggest significant differences in the number of chromosomal structural alterations induced by radiation in granulocytes from heterozygotic carriers of AT, similar to structural alterations in lymphocytes from patients with AT. It is demonstrated that these alterations occur preferentially in one group C chromosome, both in homozygotic and heterozygotic patients with AT.
A screening program for cystic fibrosis (CF) heterozygotes was conducted in a large HMO prenatal population, to evaluate the level of interest among eligible patients, the effectiveness of prescreening education, attitudes toward the screening process, psychological effects, and utilization of prenatal diagnosis and its outcomes. The heterozygote identification rate and frequency of specific CFTR mutations were also assessed. Identified carriers were offered genetic counseling and testing of male partners. Prenatal diagnosis was offered if both parents were identified as carriers. A total of 5,161 women underwent carrier testing; 947 others completed survey instruments only. The acceptance rate of screening was high (78%), and pretest education by videotape was generally effective. Adverse psychological effects were not reported. Participants generally found screening to be desirable and useful. Screening identified 142 female heterozygotes, 109 couples in which the male partner was not a carrier, and 7 high-risk couples. The incidence of R117H mutations was much higher than expected. The number of identified carriers was much lower in Hispanics than in Caucasians. We conclude that large-scale prenatal screening for CF heterozygotes in the absence of a family history of CF is an acceptable method for identifying couples at risk for affected fetuses. Sufficient pretest education can be accomplished efficiently, test insensitivity is well accepted, adverse psychological events are not observed, and general patient satisfaction is high.
BACKGROUND: The high mortality associated with ovarian carcinoma is largely a reflection of the inability to diagnose the disease at an early stage; the identification of a histologic lesion or molecular marker associated early stages of transformation would represent an important advance in understanding the natural history of this cancer. The existence of individuals with germline mutations in the ovarian and breast carcinoma susceptibility gene BRCA1 represents a unique opportunity to search for such premalignant alterations in ovarian tissues that are at unusually high risk for tumorigenesis. In this study, the authors addressed the hypothesis that pathologically normal ovaries removed from BRCA1 heterozygotes are likely to display premalignant histologic, molecular, and/or cell biologic alterations that may provide insight into early stages of ovarian tumorigenesis. METHODS: Ovarian tissues from 18 BRCA1 heterozygotes and from 20 age-matched controls were examined in a blinded fashion for histologic evidence of surface epithelial pseudostratification, epithelial inclusion cysts, deep cortical invaginations of surface epithelium, increased stromal cell activity, and surface papillomatosis. Immunohistochemical analyses for expression of BRCA1, p53, and ERBB-2 and quantitation of cell proliferation (Ki-67 expression) and apoptosis (TUNEL assay), were also performed on all specimens. RESULTS: Although histologic alterations were observed, there was no difference in frequency between cases and controls. Analysis of BRCA1 expression revealed ubiquitous nuclear immunoreactivity in the surface epithelial cells of all ovaries. Similarly, no evidence was found of p53 overexpression in any ovarian tissue or of a difference in ERBB-2 expression between cases and controls. Finally, no differences were observed in epithelial cell proliferation or apoptosis. CONCLUSIONS: Clinically, normal ovaries from BRCA1 heterozygotes do not show evidence of premalignant alterations in histology, molecular markers, cell proliferation, or apoptosis, indicating that such changes are likely rare.
We present an inventive method that generates intramolecular controls for SNP analysis, termed Mirror SNPs. Using the ovine diseases of callipyge and scrapie as examples, we describe the PCR-driven production of balanced heterozygote copies of the various SNPs implicated in these diseases. In the absence of a callipyge-positive control DNA, we generated a balanced heterozygote Mirror SNP that represents both the wild-type and mutant forms of the causal polymorphism. Simultaneous analysis of this artificial Mirror SNP and the Real (target) SNP was used to prove the absence of the mutant form of the nucleotide at the Real SNP position in tested samples. Scrapie susceptibility was assessed using a PCR-driven system which generated four separate Mirror SNPs, and these enabled the confirmation of an apparent departure from 'balanced' heterozygote appearance at Real SNPs tested. Mirror SNP technology is generic and will enable the accurate assessment of rare and medically important SNP variants, more accurate frequency determinations, and the potential assessment of SNPs in 'mixed template' samples common in forensic analyses.
Thymidylate synthase (TS) gene polymorphisms are important as prognostic factors in cancer chemotherapy, but recent results describe that the TS enhancer region (TSER) polymorphic genotypes may also modulate risk for malignancies. Two functionally important and ethnically diverse polymorphisms are present on the TS transcript, TSER, a repeat polymorphism (2 or 3 repeats; 2R, 3R) affecting TS expression, and a 6 bp ins/del polymorphism on the 3' UTR (position TS1494, del6 or ins6), which may influence mRNA stability. Hungarian population has one of the highest colorectal cancer (CRC) mortality rates in Europe, and several elevated dietary risk factors affect a large part of the population. In our study (99 primary CRC cases), population analysis of the patient group genotype frequencies revealed a departure from the Hardy-Weinberg equilibrium and significant heterozygote deficiency (p < 0.05) at the TSER locus. Despite the strong linkage between the 2 polymorphic loci, case TS1494del genotype frequencies were normally distributed, as well as the genotype frequencies of the healthy control population (n = 102), at both loci. Case-control comparison demonstrated a lower relative risk of TSER heterozygotes (OR = 0.47; CI = 0.27-0.83; p = 0.008) and a possible higher prevalence of the 3R3R&ins6/del6 in the CRC group. The observation that heterozygotes are those less susceptible for CRC in the Hungarian population may support the possibility of 2 different pathways in which TS may play a role in colorectal carcinogenesis, probably nutrient (or folate)-dependently. The lack of similar genotype effect seen with TS1494del polymorphism and the increased presence of one genotype combination (3R3R&ins6/del6) in the patient group suggest a possible TS haplotype effect influencing CRC risk.
Neuromuscular junctions (NMJs) were identified by revealing the presence of cholinergic receptors (AChR) with alpha-bungarotoxin coupled to the fluorescent dye cascade blue in 9- and 60-day-old normal and heterozygote mdx mice. Dystrophin was detected by an immunoperoxidase technique. All the muscle fibers of the normal animals observed in cross sections were immunoreactive for dystrophin and an accumulation of dystrophin was observed at all NMJs identified by alpha-bungarotoxin. In the 9-day-old mdx heterozygote animals, dystrophin positive, negative, and partially positive muscle cross sections were observed. Four different observations were made in these heterozygote animals on the coexistence of AChR and dystrophin. First, alpha-bungarotoxin sites (i.e., NMJs) were observed on dystrophin positive muscle fiber cross sections with an accumulation of dystrophin at these sites. Second, alpha-bungarotoxin sites were observed on dystrophin positive fibers without a dystrophin accumulation at NMJs. Third, there was a coexistence of alpha-bungarotoxin and dystrophin labelling at NMJs of muscle fibers with perimeters labelling negative for dystrophin. Fourth, NMJs, identified by alpha-bungarotoxin, were observed on muscle fibers negative for dystrophin even at the NMJ. These observations suggest that dystrophin is present not only in the muscle membrane but also in the presynaptic nerve terminals.
A 64-year-old Japanese male suffering from very slowly progressive amyloidosis was studied by immunohistopathologic, mass spectrometric, and molecular genetic methods. After confirming the immunoreactivity of transthyretin (TTR) in the amyloid deposits using an anti-TTR polyclonal antibody, matrix-assisted laser desorption ionization/time-of-flight-mass spectrometry (MALDI/TOF-MS) was employed to look for the presence of variant TTR(s) in the serum. Two variant forms of TTR, one with a molecular weight 32 Da greater and another with a molecular weight 19 Da less than that of normal TTR encoded by the two respective alleles, were detected in this patient. Direct sequence analysis confirmed the presence of a double substitution: one at codon 30 from GTG (Val) to ATG (Met) and the other at codon 104 from CGC (Arg) to CAC (His) in the two alleles. MALDI/TOF-MS of the parents of the proband revealed that his father was a heterozygote of ATTR Arg104His and his mother was a heterozygote of ATTR Val30Met. The total TTR and retinol binding protein (RBP) concentrations in the serum samples of the proband were very high compared with those of FAP ATTR Val30Met patients and control subjects. We report here a new compound heterozygote in the TTR gene with familial amyloidotic polyneuropathy (FAP).
We evaluated Alabama hemochromatosis probands (n = 74) and normal control subjects (n = 142) for expression of the hemochromatosis-associated mutations nt 845G-->A (845A; Cys282Tyr) and nt 187C-->G (His63Asp) in a gene linked to the major histocompatibility complex (MHC). We also tabulated parameters of iron metabolism and iron overload in probands and in obligate heterozygote family members of homozygous Cys282Tyr probands. Among probands, 59.4% were Cys282Tyr homozygotes and 20.3% were heterozygotes; 20.3% did not express this mutation. In normal control subjects, 14.7% were heterozygous for the Cys282Tyr mutation; one normal control subject was homozygous for the Cys282Tyr mutation. None (0 of 44) of our Cys282Tyr-homozygous hemochromatosis probands had the His63Asp mutation. Of the Cys282Tyr-heterozygous and -negative probands, the His63Asp mutation occurred in 26.7% (4/15) and 53.3% (8/15), respectively. In normal control subjects, 23.2% were heterozygous for the His63Asp mutation; 2.8% were homozygous. Induction phlebotomy requirements and other manifestations of iron overload were significantly greater in Cys282Tyr homozygotes than among other probands. Cys282Tyr-heterozygous probands had significantly higher values of serum iron parameters than did obligate Cys282Tyr heterozygotes whose values were, on the average, normal. Co-expression of HLA-A3, HLA-B7, and D6S105(8) was significantly more frequent in all subgroups of probands stratified by Cys282Tyr expression than in normal control subjects. These results demonstrate that the severity of iron overload in hemochromatosis is affected significantly by genetic factors. Further, our findings support the hypothesis that one or more MHC-linked genes other than that corresponding to the Cys282Tyr and His63Asp mutations contributes to increased iron absorption and iron overload in hemochromatosis probands.
The failure to find an increased frequency of ATM mutations in large cancer cohorts, especially breast cancer, is contrary to what was anticipated based on the increased cancer susceptibility of obligate ATM heterozygotes from families with ataxia-telangiectasia (A-T). We hypothesize that this paradox might be resolved if two types of ATM heterozygotes exist and the phenotypes differ, i.e., those with truncating types of mutations (ATM(trunc)), that make no protein, and those with missense types of mutations (ATM(mis)), that make reduced amounts of defective protein. The phenotype of ATM(trunc/trunc) mutations is the A-T syndrome; the phenotype of ATM(mis/mis) mutations, judging from the few homozygous patients that have been documented, appears to include some neurological features and cancer susceptibility but not the A-T syndrome. Evidence is reviewed which suggests that ATM(mis/wt) mutations are technically more difficult to detect than ATM(trunc/wt) mutations. Despite this, most large cancer cohort studies have identified mainly missense mutations and few truncating mutations. This model would require a paradigm shift for cancer risk analyses, to recognize the existence of different allele frequencies for the two types of A-T heterozygotes.
We have identified deficient biopterin synthesis in four probands and one sib with persistent postnatal hyperphenylalaninemia. The metabolic findings were associated with a benign clinical presentation and normal biopterin level in cerebrospinal fluid in the newborn period, indicating the peripheral (hepatic) form of this autosomal recessive phenotype. Impaired development was apparent at 3 months in one proband not treated early. Treatment with oral tetrahydropterin restored adequate phenylalanine hydroxylase activity; it also maintained or improved CNS function. The deficient enzyme in these subjects is 6-pyruvoyl tetrahydropterin synthase (PTS). Erythrocyte activity of PTS in homozygotes (or compound heterozygotes) is less than 10% of normal. Heterozygotes have 20%-50% of normal PTS activity (enzyme phenotype), a finding compatible with a range of gene dosage effects, some abnormal. The metabolic phenotype in heterozygotes (urine biopterin excretion) did not correlate with erythrocyte PTS activity. The complex relationship between erythrocyte PTS activity, and biopterin synthesis in these families indicates genetic heterogeneity at the PTS locus.
The discriminatory power of a quantitative heterozygote test for Maple Syrup Urine Disease (MSUD) which we have been using is analyzed on the presumption that a single rare mutant allele is involved in MSUD. Bayes theorem then predicts that only a small portion of persons from the general population with activities in the heterozygous range really are heterozygotes. In addition, overlap of heterozygous and normal homozygous activity distributions requires rather high activities in first-cousin matings of a patient's sib in order to obviate the necessity for prenatal diagnosis. Thus, the principle emerges that quantitative heterozygote tests for rare autosomal recessive diseases cannot fulfill the task they were designed for.
Peripheral lymphocytes of three Fanconi's anemia (FA) patients and of five heterozygotes have been tested for their susceptibility to chromosomal breakage and sister chromatid exchange (SCE) induction by diepoxybutane (DEB). As previously shown, DEB dramatically increases the chromosomal breakage level in both homozygotes and heterozygotes. Slightly, but significantly, reduced spontaneous levels of SCE were found in cultures from FA patients and FA gene carriers as compared with controls. SCE rates were not enhanced in lymphocytes grown in the presence of DEB, irrespective of the time at which it had been added to the culture, and were comparable in cells of FA patients and FA heterozygotes. This was in contrast with SCE rates in control cells, which showed increases positively related to the length of DEB treatment.
Two groups of heterozygotes, one for metachromatic leukodystrophy (MLD) and the other for Tay-Sachs disease, were given a battery of neuropsychological tests, a standard neurological examination, and an EEG. Neurological and EEG findings were unremarkable for both groups. The MLD heterozygotes showed deficits in the neuropsychological tests involving spatial or constructional components, but not in tests involving language skills. The Tay-Sachs heterozygotes showed no consistent deficit on any component of the neuropsychological tests.
A three day loading with sodium bromide was performed in 19 healthy controls, 16 definite cystic fibrosis heterozygotes, and 14 homozygote patients with cystic fibrosis. After three days sodium, potassium, chloride, and bromide were determined in serum and sweat. Using a multivariate discriminant analysis two nonelementary functions with the features sodium index, bromide index, potassium index, and chloride in sweat allowed us to identify the three groups. The reclassification of the probands, however, was correct in only 69.3%. A second analysis with the groups of controls and heterozygotes only resulted in a unidimensional nonelementary function with the features potassium index, bromide/sodium in sweat, and sodium index. The reclassification of the probands was correct in 91.4%. The discriminant values were 8.28 +/- 0.96 in the controls and 11.67 +/- 1.03 in the heterozygotes with an overlap of twice the standard deviations between 9.61 and 10.20.
One of the mechanisms by which chronic valproic acid (VPA) therapy induces serum and tissue depletion of carnitine in normal controls is through inhibition of plasmalemmal carnitine uptake (Tein et al 1993). To determine the effect of VPA on proven heterozygotes for the plasmalemmal carnitine transporter defect, we studied this system in cultured human skin fibroblasts with reduced Vmax for the carnitine transporter using L-[3H]carnitine. There was en exponential dose-dependent decrease in carnitine uptake with increasing VPA concentrations and the relative inhibitory effect was the same for all three carnitine concentrations for a given cell line. Importantly, the lower the maximal velocity of carnitine uptake of the heterozygote, the lower the number of carnitine transporters and the lower the carnitine uptake per given concentration of VPA. The degree of inhibition was also directly proportional to the time of VPA preincubation up to a specific maximal saturation time. The maximal effect of VPA exposure time was reached by 10 days in the control cell line and by 3 days in the two heterozygote lines, probably reflecting earlier saturation. We conclude that patients who are heterozygous for the plasmalemmal carnitine transporter defect are at increased risk for VPA-associated serum and tissue depletion of carnitine through inhibition of plasmalemmal carnitine uptake.
We have determined the nucleotide sequence surrounding a BclI restriction fragment length variation near the aprt gene of CHO cells. By BclI digestion of the PCR-amplified DNA from a variety of APRT-deficient mutants derived from CHO cells, we were able to infer the following. First, all three heterozygotes of class II, which are known to undergo the second mutational step via a large deletion event occurring at high frequency, are mutant at the chromosome Z4-linked allele, and wild type at the Z7 allele. Second, both class-III heterozygotes, which mutate to the APRT- phenotype at low frequency, are mutant at the Z7 allele, wild type at the Z4 allele. A total of 12 class-I lines, defined as having already undergone a deletion event and yielding fully APRT- mutants at low frequency were all found to have lost the Z7-linked allele. We conclude that the Z7-linked allele is substantially more susceptible to mutation by the large deletion event than is the Z4-linked allele. This supports a hypothesis we advanced earlier to explain the existence of the class-III heterozygotes but does not support previous work suggesting that a chromosomal inversion break-point junction near the Z4-linked aprt allele is responsible for the high frequency deletion event.
Niemann-Pick disease type C (NPC) is an autosomal recessive neurovisceral lysosomal storage disorder in which cholesterol lipidosis results from defective intracellular transport of unesterified cholesterol. The primary molecular defect of NPC is unknown; regulatory mechanisms of cholesterol metabolism are impaired, resulting in retarded esterification of exogenous cholesterol with accumulation of unesterified cholesterol in lysosomes and secondary storage of glycolipids and sphingomyelin. In obligate heterozygotes from a feline NPC model, cultured skin fibroblasts challenged with exogenously derived cholesterol exhibited intermediate rates of cholesterol esterification and accumulation of unesterified cholesterol. Liver lipid analyses of obligate heterozygote cats demonstrated intermediate cholesterol and sphingomyelin concentrations. Vacuolated skin fibroblasts were found in 2 of 3 heterozygote cats, and occasional cortical neurons exhibited intracellular inclusions immunoreactive for GM2-ganglioside. Ultrastructural studies provided evidence of storage in liver and brain. We believe these morphological and biochemical findings are the first example of manifestations of CNS abnormalities in a genetic carrier for a neuronal storage disease.