A method for the detection of carriers of duchenne muscular dystrophy—A preliminary report

This report of a method to detect carriers of Duchenne muscular dystrophy describes a technique of tissue autoradiography that identifies increased uptake of [³H]leucine into some individual muscle fibers in muscle biopsies. Three obligate and seven of 11 sisters of affected boys are identified as carriers in this series. This method of identification may be of value in identifying those carriers undetected by other means, but remains to be further evaluated.     

Reliability of the use of fructose 1-phosphate to detect Hunter cells in fibroblast-cultures of obligate carriers of the Hunter syndrome

Pulse-chase experiments measuring 35S-sulphate incorporation into acid mucopolysaccharides were performed in the presence and absence of fructose 1-phosphate on fibroblasts obtained from one skin-biopsy of 25 obligate Hunter carriers. The presence of fructose 1-phosphate significantly increased the accumulation of 35S-labelled acid mucopolysaccharides in fibroblast cultures of 23 obligate Hunter carriers. In one carrier, the accumulation of labelled acid mucopolysaccharides was significantly increased prior to the addition of fructose 1-phosphate, and in one of the 25 obligate carriers the 35S-sulphate incorporation was normal in the presence as well as in the absence of fructose 1-phosphate. Similar experiments performed on mixtures of Hunter cells and normal cells revealed that 20% Hunter cells should be present to obtain a significantly increased difference in between the incorporation in the presence and in the absence of fructose 1-phosphate. Fructose 1-phosphate had no effect on the accumulation of labelled mucopolysaccharides in fibroblast-cultures of seven women with no family history of mucopolysaccharidosis. The present results show that pulse-chase experiments measuring 35S-sulphate incorporation into fibroblasts, cultured in the presence of fructose 1-phosphate, can identify Hunter carriership, provided that the accumulation is normal prior to the addition of fructose 1-phosphate. Furthermore, 35S-sulphate incorporation in the absence of fructose 1-phosphate, higher than mean +4SD of normal control-fibroblasts indicates carriership.     

Biochemical,psychometric, and neuropsychological studies in heterozygotes for various lipidoses

Summary A sample of 38 clinically unaffected carriers for various lipidoses and their noncarrier relatives was studied with biochemical, psychological, and neuropsychological tests under blind conditions. The largest group of carriers was that for metachromatic leucodystrophy (MLD). The mean activity of arylsulphatase A or cerebroside sulphatase in the obligate carriers was 25%–30% of the control values, some heterozygotes showing little more activity than MLD patients. It was found that compared with the controls all heterozygotes (both obligate and facultative) differ unfavourably in some personality traits and in WISA subtests, including capacity for spatial cognition. These differences are especially obvious in a group of seven MLD carriers from the same family.With respect to reaction times, performance was significantly slower in MLD carriers, and particularly in those with enzyme activity lower than 30% of the control values.     

Identification and rapid detection of three Tay-Sachs mutations in the Moroccan Jewish population.

Infantile Tay-Sachs disease (TSD) is caused by mutations in the HEXA gene that result in the complete absence of beta-hexosaminidase A activity. It is well known that an elevated frequency of TSD mutations exists among Ashkenazi Jews. More recently it has become apparent that elevated carrier frequencies for TSD also occur in several other ethnic groups, including Moroccan Jews, a subgroup of Sephardic Jews. Elsewhere we reported an in-frame deletion of one of the two adjacent phenylalanine codons at position 304 or 305 (delta F304/305) in one HEXA allele of a Moroccan Jewish TSD patient and in three obligate carriers from six unrelated Moroccan Jewish families. We have now identified two additional mutations within exon 5 of the HEXA gene that account for the remaining TSD alleles in the patient and carriers. One of the mutations is a novel C-to-G transversion, resulting in a replacement of Tyr180 by a stop codon. The other mutation is a G-to-A transition resulting in an Arg170-to-Gln substitution. This mutation is at a CpG site in a Japanese infant with Tay-Sachs disease and was described elsewhere. Analysis of nine obligate carriers from seven unrelated families showed that four harbor the delta F304/305 mutation, two the Arg170----Gln mutation, and one the Tyr180----Stop mutation. We also have developed rapid, nonradioactive assays for the detection of each mutation, which should be helpful for carrier screening.     

Gyrate atrophy of the choroid and retina with hyperornithinemia: biochemical and histologic studies and response to vitamin B6.

Four patients with hyperomithinemia and gyrate atrophy of the choroid and retina age described. In vivo response to vitamin B6 is documented in three of the four patients by significant reduction of fasting serum ornithine and increase of lysine after oral B6 supplementation. Oral glucose tolerance testing in one patient resulted in marked changes in serum ornithine and lysine concentrations, in addition to mild glucose intolerance. Histochemical staining of punch muscle biopsies showed intracellular inclusions in type 2 muscle fibers. Tubular aggregates, approximately 60 nm in diameter and adjacent to the sarcoplasmic membrane, were seen on electron microscopy. Obligate heterozygotes had a mean serum ornithine slightly higher than normal, but there was considerable overlap with the normal range. Oral ornithine tolerance tests distinguished carriers from controls in only one of five cases. Deficient activity of ornithine ketoacid aminotransferase (OKT) in cultured skin fibroblasts was documented in all four patients. Approximately half-normal levels were found in obligate heterozygotes. In vitro response to B6 was manifest by increased OKT activity at increased concentrations of pyridoxal phosphate in fibroblasts from the patients.     

Improved identification of heterozygotes for homocystinuria due to cystathionine synthase deficiency by the combination of methionine loading and enzyme determination in cultured fibroblasts

Summary Previous data on tentative identification of the carrier state for homocystinuria due to cystathionine synthase deficiency using methionine loading or measurement of cystathionine synthase activity in tissue extracts are conflicting. We studied the results of standardized oral methionine loading in 20 obligate heterozygotes and compared them with those of determination of cystathionine synthase activity in cultured fibroblasts. Special attention was devoted to our recently reported observation on the small but striking differences in methionine metabolism between healthy pre- and postmenopausal women and men. Fasting and after load peak levels of methionine in serum did not discriminate the carriers from the control subjects. The mean fasting level of total homocysteine was only significantly higher in the group of premenopausal heterozygotes than in the corresponding control group. Nevertheless, the individual values overlapped with the normal range in 4 of 12 premenopausal heterozygotes. After loading peak levels of total homocysteine in 18 out of the 20 obligate heterozygotes exceeded the upper limit of the ranges in the three control groups. Thus, this parameter discriminated 90% of the obligate carriers. Measurement of cystathionine synthase activity in cultured fibroblasts from a skin biopsy identified the obligate heterozygotes to a similar degree (85%). No significant correlation between the measurements of cystathionine synthase activity and the after load peak levels of total homocysteine in the individual heterozygotes was established. Combination of both methionine loading and determination of cystathionine synthase activity in cultured fibroblasts identified all of these carriers.     

Skeletal muscle damage: a study of isotope uptake, enzyme efflux and pain after stepping

We have studied the occurrence of skeletal muscle uptake of 99mtechnetium pyrophosphate (Tc-PYP), creatine kinase (CK) release and muscle pain in normal subjects after exercise. Five subjects stepped on and off a high bench in such a way that one leg stepped up and the other down. Pain only developed in the muscles used for descending: quadriceps, adductors and gluteal muscles of one leg and the calf muscle of the other. A large rise in plasma CK occurred in four subjects but no increased Tc-PYP muscle uptake was seen in the quadriceps. In the four subjects with high CK effluxes, increased isotope uptake was seen in the thigh adductors used when stepping down; in the two subjects with the largest CK effluxes there was extensive uptake into the gluteal muscles. Muscle pain preceded and was not well correlated with either the magnitude of the enzyme release or the amount and distribution of increased muscle isotope uptake. We conclude that delayed onset muscle pain, the cause of which remains unknown, is a poor indicator of muscle damage as indicated by circulating muscle enzymes and muscle isotope uptake. Tc-PYP uptake by skeletal muscle can provide useful information about the localisation and time course of muscle damage.     

Muscle provocation test. A sensitive method for discrimination between carriers and noncarriers of Duchenne muscular dystrophy

Summary After 40 min of strenuous exercise on a bicycle ergometer (muscle provocation test, MPT), normal women and obligate carriers showed a progressive elevation of serum creatine kinase, with a peak 8 h after exercise. The diagnostic applicability of MPT for carrier detection is demonstrated.     

One-carbon compounds transport studies in the obligate methylotroph

Cells of obligate methylotrophic Gram-negative bacterium Methylobacillus flagellatum KT which can only grow on methanol and methylamine media possess three different carriers mediating uptake of methylamin depending on growth conditions. All three uptake systems are energy-dependent, the methylamine uptake was inhibited by oxidative phosphorylation uncoupler and respiratory inhibitors. The first active transport system for methanol in the cells of obligate methylotroph was also demonstrated. The parameters of this system were measured, their dependence on energy, presence of respiratory inhibitors and uncoupler was shown.Abbreviations CCCP Carbonyl cyanide p-(trichloromethoxy)-phenylhydrazone - DCCD N,N-dicyclohexyl-carbodiimide     

Sandhoff disease heterozygote detection: a component of population screening for Tay-Sachs disease carriers. I. Statistical methods

Serum and leukocyte hexosaminidase profiles (total activity and percent heat-labile activity levels) in obligate Sandhoff disease (SHD) heterozygotes differ from those of obligate Tay-Sachs disease (TSD) heterozygotes and noncarrier individuals. We have developed a procedure to identify, with 95% sensitivity, carriers of the allele(s) for SHD among individuals screened in a TSD heterozygote identification program. Using multivariate statistical methods of cluster analysis and discriminant analysis on serum and leukocyte hexosaminidase profiles from 102 potential SHD carriers, a linear discriminant function to classify individuals as SHD carriers or SHD noncarriers was constructed. This function classifies the serum and leukocyte profiles from all 15 obligate SHD heterozygotes studied, as those of SHD carriers. A 95% isodensity ellipse derived from only the serum hexosaminidase profiles of the 15 SHD obligate carriers has been applied to a TSD screened sample of 37,843 Jewish and non-Jewish individuals. A potential recall rate of screened individuals for serum retests and leukocyte assays of 2.01% has been estimated. These statistical methods enhance the TSD heterozygote screening program by permitting one to detect SHD heterozygotes within the screened population.     

X-chromosome inactivation in the Wiskott-Aldrich syndrome: a marker for detection of the carrier state and identification of cell lineages expressing the gene defect

Recently developed techniques for the direct analysis of DNA have made possible the determination of patterns of cellular X-chromosome inactivation. These techniques provide a potential method for carrier detection for several X-linked human disorders in which obligate carriers show nonrandom X inactivation. By using restriction fragment length polymorphic (RFLP) gene-specific probes in conjunction with methylation-sensitive enzymes, we have characterized the patterns of X-chromosome inactivation in cell subsets from females belonging to 10 kindreds segregating for the X-linked immune deficiency disorder Wiskott-Aldrich syndrome (WAS). We show that selective inactivation of the X chromosome distinguishes obligate WAS carriers from noncarrier females and constitutes a valuable marker of the WAS carrier state. Selective inactivation phenomena were observed in the monocytes and T and B lymphocytes of obligate carriers, implying that the WAS gene defect is expressed in each of these cellular lineages. In conjunction with the use of linked DNA markers, RFLP-methylation analysis should render carrier detection feasible for the majority of females from WAS families. The results of such analyses also provide an initial step toward identifying the cellular level and molecular basis for WAS.     

Glucose uptake and glucose transporter proteins in skeletal muscle from undernourished rats

Undernutrition in rats impairs secretion of insulin but maintains glucose normotolerance, because muscle tissue presents an increased insulin-induced glucose uptake. We studied glucose transporters in gastrocnemius muscles from food-restricted and control anesthetized rats under basal and euglycemic hyperinsulinemic conditions. Muscle membranes were prepared by subcellular fractionation in sucrose gradients. Insulin-induced glucose uptake, estimated by a 2-deoxyglucose technique, was increased 4- and 12-fold in control and food-restricted rats, respectively. Muscle insulin receptor was increased, but phosphotyrosine-associated phosphatidylinositol 3-kinase activity stimulated by insulin was lower in undernourished rats, whereas insulin receptor substrate-1 content remained unaltered. The main glucose transporter in the muscle, GLUT-4, was severely reduced albeit more efficiently translocated in response to insulin in food-deprived rats. GLUT-1, GLUT-3, and GLUT-5, minor isoforms in skeletal muscle, were found increased in food-deprived rats. The rise in these minor glucose carriers, as well as the improvement in GLUT-4 recruitment, is probably insufficient to account for the insulin-induced increase in the uptake of glucose in undernourished rats, thereby suggesting possible changes in other steps required for glucose metabolism.     

Abnormal anti-Epstein Barr virus antibodies in carriers of the X-linked lymphoproliferative syndrome and in females at risk

The asymptomatic hemizygous female carriers of the X-linked lymphoproliferative syndrome (XLP) have abnormal antibody responses to EBV. This suggests partial expression of the defect that leads to EBV-provoked life-threatening diseases in their affected sons. EBV specific antibodies were measured in 65 serum samples of 12 obligate carrier females and seven of their daughters (females at risk) during periods ranging from 1 to 5 yr. Abnormal qualitative antiviral capsid antigen (VCA) IgG titers were nearly fourfold higher than normal controls, two carriers had persistent IgM anti-VCA antibody, two-thirds had persistent IgA anti-VCA antibody, and half of the women had titers to early antigen (EA). Five of seven females exhibited a similar persistent pattern. In contrast, none of the unaffected family members nor 23 normal controls expressed IgA or IgM titers to VCA even with high exposure to the virus, and anti-EA was detected in only one control. Therefore, these findings may prove useful for detecting carriers of the syndrome. Abnormal anti-EBV titers similar to the carrier pattern have been reported in patients and other immunosuppressed individuals, and are indicative of active viral infection.     

Distribution of three alpha-chain beta-hexosaminidase A mutations among Tay-Sachs carriers.

DNA from 176 carriers of the Tay-Sachs gene was tested for the presence of the three mutations most commonly found among Ashkenazi Jews: the so-called insertion, splice junction, and adult mutations. Among 148 Ashkenazi Jews tested, 108 had the insertion mutation, 26 had the splice junction mutation, five had the adult mutation, and nine had none of the three. Among 28 non-Jewish carriers tested, most of whom were obligate carriers, four had the insertion mutation, one had the adult mutation, and the remaining 23 had none of the three.     

Calcium uptake in mitochondria from different skeletal muscle types

The kinetics of calcium (Ca2+) uptake have been studied in mitochondria isolated from the different types of skeletal muscle. These studies demonstrate that the Ca2+ uptake properties of skeletal mitochondria are similar to those from liver and cardiac mitochondria. The Ca2+ carriers apparently have a high affinity for Ca2+ (Michaelis constants in the microM range). The relationship between Ca2+ uptake and initial Ca2+ concentration (10(-5) to 10(-7) M) is sigmoid in all mitochondria from the different skeletal muscle types suggesting that the uptake process is cooperative. Hill plots reveal coefficients of approximately 2 for mitochondria from fast-twitch muscle and 3.5 for slow-twitch muscle, adding further evidence to the concept that the uptake process is cooperative. An analysis of the potential role of mitochondria in the sequestration of Ca2+ during muscular contraction demonstrated that mitochondria from slow-twitch muscle of both rats and rabbits can potentially account for 100% of the relaxation rate at a low frequency of stimulation (5 Hz). In fast-twitch muscle, the mitochondria appear unable to play a significant role in muscle relaxation, particularly at stimulation frequencies that are considered in the normal physiological range. In summary, it appears that Ca2+ uptake by mitochondria from slow-twitch skeletal muscle has kinetic characteristics which make it important as a potential regulator of Ca2+ within the muscle cell under normal physiological conditions.     

Influence of active muscle mass on glucose homeostasis during exercise in humans

To study the effect of increasing amounts of exercising muscle mass on the relationship between glucose mobilization and peripheral glucose uptake, seven young men (23-28 yr) bicycled for 70 min at a work load of 55-60% VO2max. From minute 30 to 50, arm cranking was added and total work load increased to 82 +/- 4% VO2max. During leg exercise, hepatic glucose production (Ra) increased in parallel with peripheral glucose uptake (Rd) and euglycemia was maintained. During arm + leg exercise, Ra increased more than Rd and accordingly plasma glucose increased from 5.11 +/- 0.22 to 8.00 +/- 0.66 mmol/l (P less than 0.05). Plasma catecholamines increased three- to four-fold more during arm + leg exercise than during leg exercise. Leg glucose uptake increased with time regardless of arm cranking. Net leg lactate release during leg exercise was reverted to a net leg lactate uptake during arm + leg exercise. The rate of glycogen breakdown in exercising leg muscle was not altered by addition of arm cranking. In conclusion, when large amounts of muscle mass are active, plasma catecholamines increase sharply and mobilization of glucose exceeds peripheral glucose uptake. This indicates that mechanisms other than feedback regulation to maintain euglycemia are involved in hormonal and substrate mobilization during intense exercise in humans.     

Detection of duchenne muscular dystrophy carriers: quantitative echography and creatine kinasemia

Summary Data obtained from simultaneous determinations of serum creatine-kinase levels and estimation of ultrasound attenuation values in muscles greatly improved the detection of obligate carriers of Duchenne muscular dystrophy than when only one of these methods was employed alone. Eleven carriers out of 19 had a high creatine-kinasemia level and nine carriers out of 19 had a high (abnormal) attenuation value. Because of the limited overlapping between the two parameters studied, we were able to recognize 17 obligate carriers out of the 19. This indicates that the parameters studied concern different features of the disease, and the practical and theoretical considerations are discussed. The techniques are discussed together with molecular genetic investigations.     

Allan-Herndon syndrome. II. Linkage to DNA markers in Xq21.

The original family with the Allan-Herndon type of X-linked mental retardation has been investigated for linkage by using DNA probes spanning the length of the X chromosome. Available for study, over 3 generations, were 13 affected males, three obligate carriers, and three normal sons of the obligate carriers. Initial disease-to-marker analysis suggested linkage to three markers (DXYS2 [7b], DXS250 [GMGX22], and DXS3 [p19-2]) located in Xq21. All three exhibited the same maximum lod score of 2.3 at a maximum theta of .05. Multipoint analysis using LINKMAP and a set of four DNA markers (DXYS1-DXYS2-DXS3-DXS94) gave a multipoint lod score of 3.58 for a location of the Allan-Herndon syndrome near locus DXYS1 (pDP34). Therefore, our data indicate that the gene for the Allan-Herndon syndrome is likely located in Xq21.     

X chromosome inactivation in carriers of Barth syndrome.

Barth syndrome (BTHS) is a rare X-linked recessive disorder characterized by cardiac and skeletal myopathy, neutropenia, and short stature. A gene for BTHS, G4.5, was recently cloned and encodes several novel proteins, named "tafazzins." Unique mutations have been found. No correlation between the location or type of mutation and the phenotype of BTHS has been found. Female carriers of BTHS seem to be healthy. This could be due to a selection against cells that have the mutant allele on the active X chromosome. We therefore analyzed X chromosome inactivation in 16 obligate carriers of BTHS, from six families, using PCR in the androgen-receptor locus. An extremely skewed X-inactivation pattern (>=95:5), not found in 148 female controls, was found in six carriers. The skewed pattern in two carriers from one family was confirmed in DNA from cultured fibroblasts. Five carriers from two families had a skewed pattern (80:20-<95:5), a pattern that was found in only 11 of 148 female controls. Of the 11 carriers with a skewed pattern, the parental origin of the inactive X chromosome was maternal in all seven cases for which this could be determined. In two families, carriers with an extremely skewed pattern and carriers with a random pattern were found. The skewed X inactivation in 11 of 16 carriers is probably the result of a selection against cells with the mutated gene on the active X chromosome. Since BTHS also shows great clinical variation within families, additional factors are likely to influence the expression of the phenotype. Such factors may also influence the selection mechanism in carriers.     

Identification of functioning sweat pores and visualization of skin temperature patterns in X-linked hypohidrotic ectodermal dysplasia by whole body thermography

Summary In this preliminary study, non-invasive infrared thermography has been used to visualize individual sweat pores and whole body skin temperature patterns in subjects with X-linked hypohidrotic ectodermal dysplasia (XHED) and normal controls. The findings in eight obligate heterozygotes and four affected males were compared to six normal female controls and to six non-manifesting females at risk for carrier status. Sweat secretion from individual pores in circumscribed areas was imaged using a high spatial resolution SPRITE infrared detector system working in the 8–14 m band. In seven out of eight obligate heterozygotes, skin areas devoid of active sweat glands were found on the face, the hands or the trunk. Tear front movement over the cornea was also visualized and abnormal patterns were identified in obligate heterozygotes. Whole body skin temperature patterns, obtained with an Agema 780 Medical Thermovision system, identified abnormal skin temperature distributions, including characteristic aberrant cas-cade back patterns, in obligate carriers. Two out of six at risk females had skin temperature patterns comparable with obligate heterozygotes and we have tentatively concluded that they are carriers. Thermal imaging may be used for the examination of at risk non-manifesting females in families with a single affected male. The results of this study suggest that the random X-inactivation in females with XHED, as well as producing relatively large skin areas with sweat pore aplasia, is also associated with abnormal temperature patterns that are consistent with altered peripheral vascular perfusion.