Genetic predictors of cardiovascular morbidity in Bardet-Biedl syndrome.

Bardet-Biedl syndrome is a rare ciliopathy characterized by retinal dystrophy, obesity, intellectual disability, polydactyly, hypogonadism and renal impairment. Patients are at high risk of cardiovascular disease. Mutations in BBS1 and BBS10 account for more than half of those with molecular confirmation of the diagnosis. To elucidate genotype-phenotype correlations with respect to cardiovascular risk indicators 50 patients with mutations in BBS1 were compared with 19 patients harbouring BBS10 mutations. All patients had truncating, missense or compound missense/truncating mutations. The effect of genotype and mutation type was analysed. C-reactive protein was higher in those with mutations in BBS10 and homozygous truncating mutations (p = 0.013 and p = 0.002, respectively). Patients with mutations in BBS10 had higher levels of C peptide than those with mutations in BBS1 (p = 0.043). Triglyceride levels were significantly elevated in patients with homozygous truncating mutations (p = 0.048). Gamma glutamyl transferase was higher in patients with homozygous truncating mutations (p = 0.007) and heterozygous missense and truncating mutations (p = 0.002) than those with homozygous missense mutations. The results are compared with clinical cardiovascular risk factors. Patients with missense mutations in BBS1 have lower biochemical cardiovascular disease markers compared with patients with BBS10 and other BBS1 mutations. This could contribute to stratification of the clinical service.

Epigenotype-phenotype correlations in Silver-Russell syndrome.

BACKGROUND: Silver-Russell syndrome (SRS) is characterised by intrauterine growth restriction, poor postnatal growth, relative macrocephaly, triangular face and asymmetry. Maternal uniparental disomy (mUPD) of chromosome 7 and hypomethylation of the imprinting control region (ICR) 1 on chromosome 11p15 are found in 5-10% and up to 60% of patients with SRS, respectively. As many features are non-specific, diagnosis of SRS remains difficult. Studies of patients in whom the molecular diagnosis is confirmed therefore provide valuable clinical information on the condition. METHODS: A detailed, prospective study of 64 patients with mUPD7 (n=20) or ICR1 hypomethylation (n=44) was undertaken. RESULTS AND CONCLUSIONS: The considerable overlap in clinical phenotype makes it difficult to distinguish these two molecular subgroups reliably. ICR1 hypomethylation was more likely to be scored as 'classical' SRS. Asymmetry, fifth finger clinodactyly and congenital anomalies were more commonly seen with ICR1 hypomethylation, whereas learning difficulties and referral for speech therapy were more likely with mUPD7. Myoclonus-dystonia has been reported previously in one mUPD7 patient. The authors report mild movement disorders in three further cases. No correlation was found between clinical severity and level of ICR1 hypomethylation. Use of assisted reproductive technology in association with ICR1 hypomethylation seems increased compared with the general population. ICR1 hypomethylation was also observed in affected siblings, although recurrence risk remains low in the majority of cases. Overall, a wide range of severity was observed, particularly with ICR1 hypomethylation. A low threshold for investigation of patients with features suggestive, but not typical, of SRS is therefore recommended.

Sexual rehabilitation of women with a spinal cord injury.

STUDY DESIGN: Literature review and a qualitative study. OBJECTIVE: This article reviews literature examining the sexual rehabilitation of women following spinal cord injury (SCI). It includes recommendations for improvements in initial clinical rehabilitation efforts and counselling services. SETTING: United Kingdom, Denmark and Sweden. METHODS: Articles concerning sexual rehabilitation following SCI from the last two decades have been reviewed and critiqued. Qualitative results from discussions with women with SCI in Denmark and Sweden are presented. RESULTS: The literature focuses on the effect of neurological change on women's ability to achieve sexual arousal and orgasm. Urinary and bowel incontinence, spasticity, vaginal lubrication and autonomic dysreflexia are the physical consequences of SCI that appear to have most impact on sexual activity. More recent studies have acknowledged that psychosocial factors such as age and partnership status may also affect the successful sexual rehabilitation. Discussions with women with SCI in Denmark and Sweden on their reactions to information and counselling offered during rehabilitation revealed an overwhelming need for the exchange of information and experience with other women with SCI, and a desire for opportunities for counselling after initial rehabilitation. CONCLUSION: Successful sexual rehabilitation of women with SCI demands a holistic approach that considers individual neurological, physical and psychosocial circumstances. Peer-counselling could make a significant contribution to the sexual rehabilitation of women with SCI.

Growth of (101) faces of tetragonal lysozyme crystals: measured growth-rate trends.

Previous extensive measurements of the growth rates of the (110) face of tetragonal lysozyme crystals have shown unexpected dependencies on the supersaturation. In this study, similar growth-rate measurements were performed for the (101) faces of the crystals. The data show a similar dependence on the supersaturation, becoming appreciable only at high supersaturations, reaching a maximum value and then decreasing. The (101) growth rates are larger at low supersaturations than the (110) growth rates under the same conditions and are smaller at high supersaturations. These trends suggest that the growth mechanism of the (101) face is similar to that of the (110) face: both processes involve the addition of multimeric growth units formed in solution, but the average size of the units for the (101) face is likely to be smaller than for the (110) face.

Locations of bromide ions in tetragonal lysozyme crystals.

Anions have been shown to play a dominant role in the crystallization of chicken egg-white lysozyme from salt solutions. Previous studies employing X-ray crystallography have found one chloride ion binding site in the tetragonal crystal form of the protein and four nitrate ion binding sites in the monoclinic form. In this study the anion positions in the tetragonal form were determined from the difference Fourier map obtained from lysozyme crystals grown in bromide and chloride solutions. Five possible anion-binding sites were found in this manner. Some of these sites were in pockets containing basic residues while others were near neutral, but polar, residues. The sole chloride ion binding site found in previous studies was confirmed, while four further sites were found which corresponded to the four binding sites found for nitrate ions in monoclinic crystals. The study suggests that most of the anion-binding sites in lysozyme remain unchanged even when different anions and different crystal forms of lysozyme are employed.

The effect of protein impurities on lysozyme crystal growth.

While bulk crystallization from impure solutions is used industrially as a purification step for a wide variety of materials, it is a technique that has rarely been used for proteins. Proteins have a reputation for being difficult to crystallize and high purity of the initial crystallization solution is considered paramount for success in the crystallization. Although little is written on the purifying capability of protein crystallization or of the effect of impurities on the various aspects of the crystallization process, recent published reports show that crystallization shows promise and feasibility as a purification technique for proteins. To further examine the issue of purity in macromolecule crystallization, this study investigates the effect of the protein impurities, avidin, ovalbumin, and conalbumin at concentrations up to 50%, on the solubility, crystal face growth rates, and crystal purity of the protein lysozyme. Solubility was measured in batch experiments while a computer controlled video microscope system was used to measure the ¿110¿ and ¿101¿ lysozyme crystal face growth rates. While little effect was observed on solubility and high crystal purity was obtained (>99.99%), the effect of the impurities on the face growth rates varied from no effect to a significant face specific effect leading to growth cessation, a phenomenon that is frequently observed in protein crystal growth. The results shed interesting light on the effect of protein impurities on protein crystal growth and strengthen the feasibility of using crystallization as a unit operation for protein purification.

Crystallization of chicken egg-white lysozyme from ammonium sulfate.

Chicken egg-white lysozyme was crystallized from ammonium sulfate over the pH range 4.0-7.8, with protein concentrations from 100 to 150 mg ml(-1). Crystals were obtained by vapor-diffusion or batch-crystallization methods. The protein crystallized in two morphologies with an apparent morphology dependence on temperature and protein concentration. In general, tetragonal crystals could be grown by lowering the protein concentration or temperature. Increasing the temperature or protein concentration resulted in the growth of orthorhombic crystals. Representative crystals of each morphology were selected for X-ray analysis. The tetragonal crystals belonged to the P4(3)2(1)2 space group with crystals grown at pH 4.4 having unit-cell dimensions of a = b = 78.71, c = 38.6 A and diffracting to beyond 2.0 A. The orthorhombic crystals, grown at pH 4.8, were of space group P2(1)2(1)2 and had unit-cell dimensions of a = 30.51, b = 56.51 and c = 73.62 A.

Orthorhombic lysozyme solubility.

The orthorhombic, or high-temperature, form of chicken egg-white lysozyme typically appears at temperatures >/=298 K. Solubility diagrams have been determined for this form of lysozyme from pH 4.0 to 5.4 in 0.2 pH increments using the micro-column technique. Data were collected in the 297-317 K temperature range which resulted in phase diagrams similar in overall shape to those obtained for the lower temperature tetragonal form. Specifically, the solubility increased with increasing temperature and decreased with increasing precipitant concentration. However, the solubility of the orthorhombic form is considerably less sensitive to temperature than the tetragonal form, resulting in a more flattened slope. On the other hand, pH effects on the high-temperature form were opposite to those on the low-temperature form. When holding the precipitant concentration constant, the solubility decreased with increasing pH for the orthorhombic form. Previous tetragonal data were incorporated with these orthorhombic data to produce intercept values. These values varied with both pH and precipitant concentration, but the general tendency of the slope was to decrease with increasing pH.

Studies on tetragonal lysozyme crystal growth rates.

A computer-controlled apparatus able to simultaneously follow the face growth rate of up to 40 crystals was developed. This apparatus was used to investigate the effects of solution pH on the (110) and (101) face growth rates of tetragonal lysozyme. Growth rates were measured at pH 4.0, 4.4, 4.8 and 5.2, in 0.1 M sodium acetate buffer with 5% NaCl, 295 K. Initial crystal sizes ranged from 10 to 40 microm. Plots of log supersaturation ratio (either C/C(sat) or C/C(sat) - 1) versus log(growth rate) are not linear, typically having a slope of approximately 8 at the lowest growth rates determined (10(-6) microm s(-1)), which falls off to a slope of approximately 2 at the highest growth rates (10(-2) microm s(-1)) measured. Ratios of C/C(sat) ranged from 4 to >20. The data show that lower solubility solutions require higher supersaturation ratios for equivalent growth rates. Data for the growth rate of the (101) face at pH 4.0 were widely scattered, especially at lower supersaturation ratios. Time-lapse video of crystals at low supersaturations shows that initially only the (110) faces grow, leaving 'notches' at the (110)-(110) corners. These corners then fill in and macro-steps appear on the (101) faces which rapidly move inward in the form of an octagon, restoring the crystal to a 'normal' appearance. This phenomenon has been observed for tetragonal crystals grown in either still or flowing solutions. Flowing solutions at lower supersaturations also gave cases where the corners did not fill in, with the (110) faces continuing to grow out until growth ceased.