Linkage analysis using multiple DNA polymorphic markers in normal families and in families with fragile X syndrome

Summary Linkage data, using the polymorphic markers 52A (DXS51), F9, 4D-8(DXS98), and St14(DXS52), are presented from 14 fragile X pedigrees and from 7 normal pedigrees derived from the collection of the Centre d'Étude du Polymorphisme Humaine. A multipoint linkage analysis indicates that the most probable order of these four loci in normal families is DXS51-F9-DXS98-DXS52. Recombination frequencies ( ) corresponding to maximum LOD scores ( ) were obtained by two-point linkage analysis for a nuber of linkage groups, including: DXS51-F9 ( =5.94, =0.03), F9-DXS98 ( =0.51, =0.26), F9-DXS52 ( =0.84, =0.27), and DXS98-DXS52 ( =0.32, =0.20). A multipoint linkage analysis of these loci, including the fragile X locus, was also performed for the fragile X population and the data support the relative order (DSX51, F9, DXS98)-FRAXA-DXS52. Recombination frequencies and maximum LOD scores, which again were derived from two-point linkage analyses, were obtained for the linkage groups DXS51-F9 ( =9.96, =0) and F9-DXS52 ( =0.07, =0.45), as well as for the groups DXS51-FRAXA ( =2.42, =0.15), F9-FRAXA ( =1.30, =0.18), DXS98-FRAXA ( =0.05 =0.36), and DXS52-FRAXA ( =2.42 =0.15). The linkage data was further tested for the presence of genetic heterogeneity both within and between the fragile X and normal families for the intervals DXS51-F9, F9-DXS52, F9-FRAXA, and DXS52-FRAXA using a modification of the A test. Except for the interval F9-FRAXA (P<0.10) there was no evidence of genetic heterogeneity for each of the various linkage groups examined. The heterogeneity detected for the interval F9-FRAXA, however, was most likely due to one family (Fx-28) that displayed very tight linkage between these two loci.     

Close linkage between Norrie disease,a cloned DNA sequence from the proximal short arm,and the centromere of the X chromosome

Summary Norrie disease (ND) is an X-linked recessive disorder with congenital blindness (atrophia bulborum hereditaria, pseudoglioma). Six kindreds segregating for ND were studied for linkage with polymorphic markers of the human X chromosome. No recombination was observed between the ND-locus (NDP) and the DXS7 locus, the latter followed as a DNA-restriction fragment length polymorphism, detected by the recombinant DNA probe L1.28, and assigned to the region Xp11.2–Xp11.3. The maximum lod scores are at . Linkage data between NDP and the other genetic markers used in the present study are in keeping with this assignment of the mutation to the proximal Xp.     

Evidence for linkage of the gene causing familial Mediterranean fever to chromosome 17q in non-Ashkenazi Jewish families: second locus or type I error?

Familial Mediterranean fever (FMF) is an autosomal recessive disorder of unknown pathogenesis, characterized by recurrent, selflimited attacks of fever with synovitis, peritonitis, or pleurisy. Using DNAs from affected Israeli families, we have recently mapped the gene causing FMF (designated MEF) to the short arm of chromosome 16, with two-point lod scores in excess of 20. In this report we consider the possibility of a second FMF susceptibility locus. Before discovering linkage to markers on chromosome 16, we had found suggestive evidence for linkage to chromosome 17q, with the following maximal two-point lod scores: D17S74 (pCMM86), = 2.47, ( = 0.20); D17S40 (pLEW101), = 2.15( = 0.15); D17S35 (CRI-pP3-1), = 1.78 ( = 0.15); D17S46 (pLEW108), = 1.69 ( = 0.18), D17S254, = 2.30 ( = 0.20). Moreover, multipoint linkage analysis using D17S74 and D17S40 as fixed loci gave = 3.27 approximately 10 centimorgans (cM) telomeric to D17S40. Data with the chromosome 17 markers alone in our families suggested locus heterogeneity. Nevertheless, our families were not separable into complementary subsets showing linkage either to chromosome 16 or to chromosome 17. We also examined the possibility that the positive lod scores for chromosome 17 might reflect a secondary, modifying locus. By several measures of disease severity, families with positive lod scores for chromosome 17 loci had no worse disease than those with negative lod scores for these loci. We conclude that chromosome 17 does not encode a major FMF susceptibility gene for some of the families, nor does it encode a disease-modifying gene. Rather, it would appear that linkage to chromosome 17 is a false positive (type I) error. These results reemphasize the fact that a lod score of 3.0 corresponds to a posterior probability of linkage of 95%, with an attendant 1 in 20 chance of observing a false positive.     

Linkage relationships and allelic associations of the cystic fibrosis locus and four marker loci

Summary The linkage relationships between the cystic fibrosis (CF) locus and four marker loci (MET-H, MET-D, D7S8 and D7S16), allelic associations between these loci and the extent of informativity at these marker loci were investigated in a sample of 206 families with at least one child affected by CF. The data were contributed by 11 laboratories from Europe and Israel. The maximum lod scores and recombination frequency estimates ( ) (and confidence limits of ) were: 18.3 at =0.007(0.001–0.038) for CF vs. MET, 11.0 at (0.001–0.068) for CF vs. D7S8, and 5.7 at =0.0(0.0–0.064) for CF vs. D7S16. A gene order of CF-MET-D7S8 was best supported by the data, but its preference to the order D7S8-CF-MET is mainly based on one single family. There are significant allelic associations between CF, MET, D7S8 and D7S16; these allelic associations affect the risk of random individuals to be carriers of CF.     

Confirmation and refinement of the localisation of the c-MEL locus on chromosome 19 by physical and genetic mapping

Summary The human gene locus c-MEL was identified following transfection of genomic DNA from the human melanoma cell line NK14; it has previously been assigned to chromosome 19 (p13.2–q13.2) by analysis of somatic cell hybrids. We have further refined the position of this gene to the proximal region of 19p (cen-p13.2), using cell hybrids containing only fragments of human chromosome 19. We have confirmed this physical localisation by linkage analysis with a recently described restriction fragment length polymorphism for the c-MEL gene, and mapped the locus within the region of the low density lipoprotein receptor gene (LDLR) (Lod 4.43, ) and the anonymous marker D19S11 (13.1.25) (Lod 9.33, ). This gene thus maps to a region of chromosome 19 involved in karyotypic abnormalities in a variety of malignancies including melanomas and leukaemias.     

Likely linkage: Inv with Jk

Summary In a data base consisting of 1665 pairs of loci linkage between Inv and Jk is significant . Recombination is nearly the same in the two sexes . The reason why this linkage was not noticed earlier is discussed.     

Spondyloepiphyseal dysplasia tarda: linkage with genetic markers from the distal short arm of the X chromosome

Summary A linkage study of six families with spondyloepiphyseal dysplasia tarda (SEDL) has been performed. A linkage to site DXS41 ( =0.08; =3.07) and DXS92 ( =0.05; =2.95) has been established. We propose, that the SEDL locus lies on the distal part of the short arm of the X chromosome.     

Estimating the Recombination frequency for the PTC-Kell linkage

Summary Two data sets are analyzed for linkage between the PTC and Kell blood group loci. The original report of close linkage for these loci was that of Conneally et al. (1976), where the maximum likelihood estimate of was 0.05. These two new data sets give a combined maximum likelihood estimate of _m=f =0.28. Estimating the recombination frequency for the sexes separately gave _m =0.29, _f =0.23. The combined maximum likelihood estimate over all published data sets including this report is _m=f =0.14, _max=8.94. There is statistically significant evidence of heterogeneity among the published studies.     

Genetic mapping of four DNA markers (DXS16, DXS43, DXS85, and DXS143) from the p22 region of the human X chromosome

Summary Linkage analysis of four polymorphic anonymous DNA markers from the Xp22 region was performed using families from the Centre d'Etude du Polymorphisme Humain. The loci DXS43 (pD2) and DXS16 (pXUT23) were found to be tightly linked ( = 0.02 at = 14.96) and proximal to both DXS85 (782) and DXS143 (dic56). Multipoint linkage analysis suggests the order:     

Linkage relationships of X-linked choroideremia to DXYS1 and DXS3

Summary Choroideremia is a distinct blinding condtion with an X-linked pattern of inheritance. We have analyzed two RFLPs, DXS3 and DXYS1, for linkage with the choroideremia locus (TCD) within three kindreds. A maximum LOD score of 3.98 was obtained at . Contrary to previous reports, the present data demonstrate that these two RFLPs are not tightly linked to the choroideremia gene locus.     

Multipoint linkage analysis of the short arm of the human X chromosome in families with X-linked muscular dystrophy

Summary Sixteen three generation families from the West of Scotland with Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD) have been studied using the Xg blood group and seven cloned DNA sequences which recognise DNA polymorphisms on the short arm of the X chromosome (Xp). Linkage has been established between DMD and probe 754 with a maximum lod score () of 4.47 at a recombination fraction ( ) of 0.04. DMD has also been linked to probe 99-6 (=3.75, =0.03). Combining the data in this study with that of previously published work has established linkage between DMD and L1.28 (=4.42, =0.17) and altered the linkage estimate between BMD and L1.28 (=3.50, =0.22).An approximate order for the loci has been deduced by the study of recombinant chromosomes in phase known families informative for three or more loci. The proposed order is centromere-L1.28-754-DMD/BMD-99-6-D2-782-Xg. These results conclusively map both DMD and BMD to the central region of Xp and add weight to the original suggestion that they may be allelic.     

Breathing pattern and growth: comparative aspects

Summary The resting oxygen consumption and breathing pattern of nine newborn and adult species (ranging in body size from mouse to human) have been compared on the basis of data collected from the literature. Minute ventilation is similarly linked to at both ages, the percent of extracted as O₂ about 2.2. Tidal volume/kg is an interspecies constant in newborns and adults, approximately 8 ml/kg. Breathing frequency decreases with the increase in size in a different way at the two ages: large species have newborns breathing at rates 2–3 times above the corresponding adults' values, while in the small species newborns and adults breathe at almost the same rate. Therefore the newborns of the smallest species have both and below the expected values, implying a greater inability to cope with the external demands than newborns of larger species. Several considerations indicate that in the smallest newborns the mechanical properties of the respiratory system could be a constraint to resting ventilations larger than observed. It is therefore possible that their low is the cause, and not the effect, of the relatively small .     

Use of the force-velocity test to determine the optimal braking force for a sprint exercise on a friction-loaded cycle ergometer

A group of 15 untrained male subjects pedalled on a friction-loaded cycle ergometer as fast as possible for 5–7 s to reach the maximal velocity (V{immax}) against different braking forces (F _B). Power was averaged during a complete crank rotation by adding the power dissipated againstF _B to the power necessary to accelerate the flywheel. For each sprint, determinations were made of peak power output ( ) power output attained atV _max ( ) calculated as the product ofV _max andF _B and the work performed to reachV _max expressed in mean power output ( ). The relationships between these parameters andF _B were examined. A biopsy taken from the vastus lateralis muscle and tomodensitometric radiographs of both thighs were taken at rest to identify muscle metabolic and morphometric properties. The value was similar for allF _B. Therefore, the average of values was defined as corrected maximal power ( ). This value was 11 higher than the maximal power output uncorrected for the acceleration. Whereas the determination did not require high loads, the highest value ( ) was produced when loading was heavy, as evidenced by the -F _B parabolic relationship. For each subject, the braking force ( ) giving was defined as optimal. The , equal to 0.844 (SD 0.108) N · kg⁻¹ bodymass, was related to thigh muscle area (r = 0.78,P < 0.05). The maximal velocity ( ) reached against this force seemed to be related more to intrinsic fibre properties (% fast twitch b fibre area and adenylate kinase activity). Thus, from the determination, it is suggested that it should be possible to predict the conditions for optimal exercise on a cycle ergometer.     

The Kidd (JK) blood group locus assigned to chromosome 18 by close linkage to a DNA-RFLP

Summary The close linkage between the PstI-restriction fragment length polymorphism (RFLP) disclosed by the L2.7 genomic DNA probe and the Kidd blood group locus is described. The maximum lod score is+8.53 at recombination fraction . The upper probability limit of the recombination fraction is θ =₁ 0.11. The L2.7 probe, previously assigned provisionally to chromosome 17, is by the present study assigned to chromosome 18. This also assigns the Kidd blood group locus (JK) to chromosome 18. Accepting previous deletion mapping, the shortest regions of overlap (SRO) for JK is 18q11-12, whereas one of our hybrids assigns L2.7 to 18q11-pter, suggesting centromeric localisation of the linkage group. JK has been assigned previously to chromosome 2 because of its provisional linkage to IGK which in turn has been mapped to 2p12. Our own JK-IGK linkage data do in fact support the previous positive lod scores at high recombination fractions (total lods+4.12 at θ₁ = 0.30). No obvious explanation for the conflicting gene mapping data is found.     

(H)NCAHA and (H)CANNH experiments for the determination of vicinal coupling constants related to the ϕ-torsion angle

Summary A set of three-dimensional triple-resonance experiments is described which provide , , and coupling constants. The pulse sequences generate E.COSY-like multiplet patterns and comprise a magnetization transfer from the amide proton to the α-proton or vice versa via the directly bound heteronuclei. For residues with the ¹H^α spin resonating close to the H₂O signal, a modified HNCA experiment can be employed to measure the vicinal ¹H^N,¹H^α couplings. Ambiguities associated with the conversion of values into ϕ-angle constraints for protein structure determination can be resolved with the knowledge of the heteronuclear ³J-couplings. In favourable cases, stereospecific assignments of glycine α-protons can be obtained by employing the experiments described here in combination with NOE data. The methods are applied to flavodoxin from Desulfovibrio vulgaris.     

Genetische Untersuchungen bei funktionell-obstruktiver subvalvulärer Aortenstenose (irregulär hypertrophischer Kardiomyopathie)

Summary Family investigations have been carried out on 32 propositi with functional obstructive subvalvular aortic stenosis. In the families of 15 propositors members were further-more affected. According to our own observations as well as to the literature this heart disease shows an autosomal dominant mode of inheritance with a reduced penetrance and variable manifestations. Using the maximum-likelihood-method, the risk of the brothers or sisters to be affected is calculated as being between (k=0, r_min=1) and (k=1, r_min=1). The probability of parents in the “familial” observations to be affected is between 32.9±8.1 and 33.3±8.1. Only one half of the cases observed are “familial”, the others are “sporadic”. It is not impossible, that a part of the sporadic cases could be dominant mutants, because the mean age of the fathers at the birth is higher than expected.

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Direktor: Prof. Dr. med. P. E. Becker

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Activity and oxygen consumption during hypoxic exposure in high altitude and lowland sceloporine lizards

Summary Resting rates of O₂ consumption against , exercise endurance times and during recovery from vigorous exercise were measured inSceloporus occidentalis captured near sea level and inS. graciosus captured above 2850 m. Oxygen consumption against was also measured inS. occidentalis captured above 2850 m. When was recorded continuously, as ambient was slowly reduced from 155 Torr, it became directly dependent upon ambient between 110 and 120 Torr. The critical for the high altitude lizards was lower than that for the lowland lizards, which enabled the former to maintain relatively higher 's when ambient was reduced below 120 Torr. The high altitude lizards also had significantly greater endurance when stimulated to exercise at 1600 m ( 130 Torr). Both the higher under hypoxia and the greater endurance roughly parallel a significantly greater maximum in the high altitude lizards. At a simulated altitude of 3600 m ( 100 Torr), maximum and rate of recovery of the O₂ debt calculated from post active were significantly reduced in the lowland but not the high altitude lizards. The effects of simulated altitude conditions on the lowland but not the mountaine animals indicate adaptations to altitude in these sceloporine lizards. We did not find any consistent relationship between organ/body weight ratios or hematocrit and our measures of endurance or the altitude at which the lizards were captured.     

The dissociation and separation of bovine adenohypophysial cells

Bovine adenohypophysial tissue was dissociated by sequential enzymatic incubation in a continuous flow system. Dispersed cells separated into discrete fractions after centrifugation in isopycnic bovine serum albumin gradients. The dispersed and separated cells were prepared for microscopic identification and differential counts by centrifugal cytology. Radioimmunoassays for LH, FSH, TSH, and Prl were used to corroborate the differential counts and determine the homogeneity of the fractions. The thyrotrophs banded at an average density ( ) of 1.0417, the FSH-secretory cells at , the LH-secretory cells at , and the Prl-secretory cells at . A 7–16 fold enrichment of different cell populations was possible. In bovine hypophyses each hormone appears to be formed by specific cells: the average TSH concentrations of the thyrotrophs were 5.1 pg/cell and the average LH and FSH concentrations were 4.7 and 4.9 pg/cell for LH-and FSH-secreting cells, respectively. The average Prl concentration was 4.9 pg/cell for Prl-secreting cells.     

Water uptake by barley roots as affected by the osmotic and matric potential in the rhizosphere

Summary The water uptake rates of roots in saline soils are depressed by the simultaneously decreasing matric and osmotic water potentials in the soil surrounding the roots (rhizospheric soil). Unfortunately there are no reliable tools available for direct measurements of the effect of decreasing water potentials in the rhizospheric soil on the uptake rate of soil water by roots. This paper presents some results of a vegetation technique for studying the effect of different combinations of osmotic and matric water potentials in the rhizospheric soil on the water uptake rates of barley roots. Water uptake rates were reduced to a greater extent by decreasing soil matric water potentials than by decreasing soil osmotic water potentials. According to the results of this experiment, there was no relationship between the total soil water potential of a sandy soil and the water uptake rates when the roots were exposed to different combinations of and .     

Two-substrates packed-bed bioreactors: inhibition of enzyme and competitive binding of substrate and product to a ligand

An analytical model is developed to describe the performance of a packed-bed immobilized enzyme reactor in which parallel processes take place. In particular, two-substrate reaction, inhibition of the enzyme by one of the reaction products, and binding of one substrate and/or one product to an added ligand are taken into account. In addition, substrates and product diffusion into the porous catalyst are also considered. Using this model, numerical simulations were performed. The results point to the fact that, when all the above processes occur concomitantly, a variety of performance characteristics can be obtained, depending on the particular values of the related parameters. Moreover, under certain conditions, the reactor performance can be improved by controlled addition of ligand.List of Symbols A total concentration of ligand - C _1,i concentration of Substrate-1 in the pores of stage i - C _2,i concentration of Substrate-2 in its free form in the pores of stage i - _2,i concentration of the Substrate-2-Ligand Complex in the pores of stage i - total concentration of Substrate-2 in the pores of stage i - _i concentration of the Product-Ligand Complex in the pores of stage i - concentration of the free Product in the pores of stage i - total concentration of the Product in the pores of stage i - internal (pore) diffusion coefficient for the Substrate-Ligand Complex - D ₁ internal (pore) diffusion coefficient of Substrate-1 - D ₂ internal (pore) diffusion coefficient of Substrate-2 - effective (pore) diffusion coefficient for Substrate-2 - internal (pore) diffusion coefficient for the Product - internal (pore) diffusion coefficient for the Product-Ligand Complex - effective (pore) diffusion coefficient for the Product - K thermodynamic equilibrium constant for binding Substrate-2 to Ligand - K _m,1,K _m,2 Michaelis constants for Substrates-1 and 2, respectively - effective Michaelis constant for Substrate-2 - K _p thermodynamic equilibrium constant for binding the reaction Product to Ligand - effective equilibrium constant for binding Substrate-2 to Ligand - effective equilibrium constant for binding the reaction Product to Ligand. - K _b inhibition constant - K _q inhibition constant - effective inhibition constant - effective inhibition constant - k _a, k _d association and dissociation rate constants for Substrate-2 — Ligand complex - association and dissociation constants for Product —Ligand complex - n total number of elementary stages in the reactor - Q volumetric flow rate throughout the reactor - R _j,i reaction rate of Substrate-j in stage i, in terms of volumetric units - S _1,0 concentration of Substrate-1 in the reactor feed - total concentration of Substrate-2 in the reactor feed - S _1,i–1,S _1,i concentration of Substrate-1 in the bulk phase leaving stages i–1 and i, respectively - S _2,i concentration of Substrate-2 in its free form, in the bulk phase leaving stage i - _2,i–1, _2,i concentration of Substrate-2 in the bulk phase leaving stage i–1 and i, respectively - total concentration of Substrate-2 in the bulk phase leaving stages i–1 and i, respectively - _i concentration of the Product-Ligand Complex in the bulk phase of stage i - concentration of free Product in the bulk phase of stage i - total concentration of Product in the bulk phase of stage i - V total volume of the reactor - V _m maximal reaction rate in terms of volumetric units - y axial coordinate of the pores - y ₀ depth of the pores Greek Symbols ₁ dimensionless parameter - dimensionless parameter - dimensionless parameter - ₁ dimensionless parameter - dimensionless parameter - _1,i dimensionless concentration of Substrate-1 in pores of stage i - dimensionless total concentration of Substrate-2 (in both free and bound form) in pores of stage i - dimensionless total concentration of the reaction product in the pores of stage i - ₁ dimensionless parameter - dimensionless parameter - dimensionless parameter - dimensionless parameter - dimensionless parameter - dimensionless position along the pore - volumetric packing density of catalytic particles (dimensionless) - porosity of the catalytic particles (dimensionless) - _1,i dimensionless concentration of Substrate-1 in the bulk phase of stage i - dimensionless total concentration of Substrate-2 (in both free and bound form) in the bulk phase of stage i