Somatic intragenic recombination within the mutated locus BLM can correct the high sister-chromatid exchange phenotype of Bloom syndrome cells.

Cells from persons with Bloom syndrome feature an elevated rate of sister-chromatid exchange (SCE). However, in some affected persons a minority of blood lymphocytes have a normal SCE rate. Persons who inherit the Bloom syndrome gene BLM identical by descent from a common ancestor very rarely exhibit this high-SCE/low-SCE mosaicism; conversely, mosaicism arises predominantly in persons who do not share a common ancestor. These population data suggested that most persons with Bloom syndrome in whom the exceptional low-SCE cells arise are not homozygous for a mutation at BLM but instead are compound heterozygotes. Following this clue, we carried out a genotype analysis of loci syntenic with BLM in 11 persons who exhibited mosaicism. In five of them, polymorphic loci distal to BLM that were heterozygous in their high-SCE cells had become homozygous in their low-SCE cells, whereas heterozygous loci proximal to BLM remained heterozygous. These observations are interpreted to mean that intragenic recombination between paternally derived and maternally derived mutated sites within BLM can generate a functionally wild-type gene and that low-SCE lymphocytes are progeny of a somatic cell in which such intragenic recombination had occurred.     

Unequal Exchange and Meiotic Instability of Disease-Resistance Genes in the Rp1 Region of Maize

The Rp1 region of maize was originally characterized as a complex locus which conditions resistance to the fungus Puccinia sorghi, the causal organism in the common rust disease. Some alleles of Rp1 are meiotically unstable, but the mechanism of instability is not known. We have studied the role of recombination in meiotic instability in maize lines homozygous for either Rp1-J or Rp1-G. Test cross progenies derived from a line that was homozygous for Rp1-J, but heterozygous at flanking markers, were screened for susceptible individuals. Five susceptible individuals were derived from 9772 progeny. All five had nonparental combinations of flanking markers; three had one combination of recombinant flanking markers while the other two had the opposite pair. In an identical study with Rp1-G, 20 susceptible seedlings were detected out of 5874 test cross progeny. Nineteen of these were associated with flanking marker exchange, 11 and 8 of each recombinant marker combination. Our results indicate that unequal exchange is the primary mechanism of meiotic instability of Rp1-J and Rp1-G.     

ENPP1 variants in patients with GACI and PXE expand the clinical and genetic heterogeneity of heritable disorders of ectopic calcification

Pseudoxanthoma elasticum (PXE) and generalized arterial calcification of infancy (GACI) are clinically distinct genetic entities of ectopic calcification associated with differentially reduced circulating levels of inorganic pyrophosphate (PPi), a potent endogenous inhibitor of calcification. Variants in ENPP1, the gene mutated in GACI, have not been associated with classic PXE. Here we report the clinical, laboratory, and molecular evaluations of ten GACI and two PXE patients from five and two unrelated families registered in GACI Global and PXE International databases, respectively. All patients were found to carry biallelic variants in ENPP1. Among ten ENPP1 variants, one homozygous variant demonstrated uniparental disomy inheritance. Functional assessment of five previously unreported ENPP1 variants suggested pathogenicity. The two PXE patients, currently 57 and 27 years of age, had diagnostic features of PXE and had not manifested the GACI phenotype. The similarly reduced PPi plasma concentrations in the PXE and GACI patients in our study correlate poorly with their disease severity. This study demonstrates that in addition to GACI, ENPP1 variants can cause classic PXE, expanding the clinical and genetic heterogeneity of heritable ectopic calcification disorders. Furthermore, the results challenge the current prevailing concept that plasma PPi is the only factor governing the severity of ectopic calcification.     

Continuous opportunistic and systematic screening for hypertension with computer help: analysis of non-responders.

For two years an office computer was used to identify patients to prompt for opportunistic screening and call for systematic screening. After the two years 92% of patients on the list had had blood pressure readings recorded within the previous five years, of which 34% resulted from special prompts and 22% from screening letters. Those who failed to respond to letters were sent questionnaires, and their records were compared with those of screened patients. With the help of a microcomputer it is practicable to sustain a continuous screening rate of between 90% and 95%.     

Nonsense mutations of the von Willebrand factor gene in patients with von Willebrand disease type III and type I.

von Willebrand disease (vWD) is the most common inherited bleeding disorder in humans. The disease is caused by qualitative and quantitative abnormalities of the von Willebrand factor (vWF). Genomic DNA from 25 patients with vWD type III, the most severe form of the disease, was studied using PCR followed by restriction-enzyme analysis and direct sequencing of the products. Nonsense mutations (CGA----TGA) were detected in exons 28, 32, and 45 by screening of all the 11 CGA arginine codons of the vWF gene. Two patients were found to be homozygous and five heterozygous for the mutation. Both parents and some of the relatives of the homozygous patients carry the mutation. These are the first reported examples of homozygous point mutations associated with the severe form of vWD. In the three heterozygous probands, one of the parents carried the mutation and had vWD type I. Family studies including parents and family members with or without vWD type I indicated that these three heterozygous patients are likely to be compound heterozygous. Twenty-one individuals from these seven families with vWD type I were found to be heterozygous for the mutation.     

Gall stones in sickle cell disease in the United Kingdom.

The prevalence of gall stones was studied prospectively by abdominal ultrasound examination in 131 patients with sickle cell disease aged 10-65 years. Of 95 patients with homozygous sickle cell disease, 55 (58%) had gall stones or had had a cholecystectomy. Gall stones were present in four out of 24 (17%) patients with haemoglobin S + C disease and two out of 12 (17%) with haemoglobin S beta thalassaemia. The presence of gall stones was not related to sex, geographical origin, or haematological variables and was not associated with abnormal results of liver function tests. Symptoms typical of biliary colic were reported by 32 out of 47 adult patients with gall stones, and cholecystitis or cholestasis was diagnosed in 18. Cholecystectomy was performed in 29 patients with good relief of symptoms in most cases. Postoperative complications were common, occurring in 10 of the 28 patients who could be evaluated, but not generally serious; they were considerably lessened by a preoperative exchange transfusion that reduced the haemoglobin S concentration to below 40%. It is suggested that all patients with sickle cell disease should be screened for gall stones and that elective cholecystectomy should be performed in those with symptoms or complications.     

Prevention of pneumococcal infection in children with homozygous sickle cell disease.

The efficacy of prophylactic penicillin and of 14 valent pneumococcal vaccine in preventing pneumococcal infection in homozygous sickle cell (SS) disease was investigated in 242 children aged 6 months to 3 years at entry. In the first five years of the trial there were 11 pneumococcal infections in the pneumococcal vaccine treated group, 10 by serotypes present in the vaccine. Type 23 accounted for five of these, and there was evidence of higher infection rates in those given the vaccine before age 1. No pneumococcal isolations occurred in the penicillin group while receiving penicillin, although four isolations occurred within one year of stopping penicillin. Probably the most effective prophylaxis against pneumococcal infection requires penicillin beyond the age of 3. The age at which pneumococcal vaccine should be given must await further data on antibody response and clinical efficacy in these patients.     

Exome Sequencing Identifies a Founder Frameshift Mutation in an Alternative Exon of USH1C as the Cause of Autosomal Recessive Retinitis Pigmentosa with Late-Onset Hearing Loss

We used a combined approach of homozygosity mapping and whole exome sequencing (WES) to search for the genetic cause of autosomal recessive retinitis pigmentosa (arRP) in families of Yemenite Jewish origin. Homozygosity mapping of two arRP Yemenite Jewish families revealed a few homozygous regions. A subsequent WES analysis of the two index cases revealed a shared homozygous novel nucleotide deletion (c.1220delG) leading to a frameshift (p.Gly407Glufs*56) in an alternative exon (#15) of USH1C. Screening of additional Yemenite Jewish patients revealed a total of 16 homozygous RP patients (with a carrier frequency of 0.008 in controls). Funduscopic and electroretinography findings were within the spectrum of typical RP. While other USH1C mutations usually cause Usher type I (including RP, vestibular dysfunction and congenital deafness), audiometric screening of 10 patients who are homozygous for c.1220delG revealed that patients under 40 years of age had normal hearing while older patients showed mild to severe high tone sensorineural hearing loss. This is the first report of a mutation in a known USH1 gene that causes late onset rather than congenital sensorineural hearing loss. The c.1220delG mutation of USH1C accounts for 23% of RP among Yemenite Jewish patients in our cohort.     

n of 1 trials comparing a non-steroidal anti-inflammatory drug with paracetamol in osteoarthritis.

OBJECTIVE--To evaluate the efficacy of paracetamol and a non-steroidal anti-inflammatory drug for symptom relief in osteoarthritis. DESIGN--Double blind, randomised, controlled trials in individual patients (n of 1 trials). Three treatment cycles with two weeks'' each of paracetamol (1 g twice daily) and diclofenac (50 mg twice daily) prepared in identical gelatin capsules. SETTING--General practices in metropolitan Sydney, Australia. SUBJECTS--25 patients (median age 64 years) with pain of osteoarthritis (median duration of disease eight years) considered by their general practitioners to require regular treatment. 20 were already taking non-steroidal anti-inflammatory drugs. MAIN OUTCOME MEASURES--Diary of pain and stiffness, function, and side effects. RESULTS--15 patients completed the study, five withdrew early but had made a therapeutic decision, and five dropped out very early. Results from 20 patients were analysed. Several patterns of response evolved. Eight of the 20 patients found no clear difference, symptoms being adequately controlled by paracetamol; five indicated a clear preference for the non-steroidal anti-inflammatory drug; two showed control of symptoms after their initial two weeks of the non-steroidal anti-inflammatory drug which continued throughout subsequent treatment changes; in five the non-steroidal anti-inflammatory drug may have been better but neither agent gave satisfactory control. After three months nine of the 20 patients had adequate symptom control with paracetamol alone. CONCLUSIONS--Of 1 studies--that is, randomised trials in individual patients--are clinically useful in deciding treatment in heterogeneous conditions which require long term symptomatic relief. In osteoarthritis many patients currently receiving or being considered for non-steroidal anti-inflammatory drugs may achieve adequate control with paracetamol.     

Aluminium bone disease in patients receiving plasma exchange with contaminated albumin.

Aluminium balance studies were carried out on eight patients with various immunological disorders who were receiving plasma exchange with albumin solutions known to be contaminated with aluminium. Four patients with impaired renal function (creatinine clearance less than 50 ml/min) retained between 60% and 74% of the aluminium infused during a single plasma exchange. Transiliac bone biopsy specimens from three patients in this group had a high content of aluminium and showed histological evidence of current or previous bone disease related to aluminium. Two of these patients suffered intermittent bone pain. The main route of excretion of injected aluminium was in urine, only a small proportion of the total input being removed in the "plasma bag" during plasma exchange. The extent of aluminium retention and bone deposition was not reflected by the plasma aluminium concentration before or after plasma exchange. Treatment of five patients with intravenous desferrioxamine increased the plasma aluminium concentration and urinary output of aluminium in those with evidence of aluminium retention. These studies show that patients with poor renal function receiving treatment with albumin contaminated with aluminium retain the metal and deposit it in bone, where it may eventually cause aluminium bone disease. Plasma exchange should be used with caution in patients with renal impairment.     

Mutations in glucokinase and other genes detected in neonatal and type 1B diabetes patient using whole exome sequencing may lead to disease-causing changes in protein activity

Monogenic diabetes is caused by mutations that reduce β-cell function. While Sanger sequencing is the standard method used to detect mutated genes. Next-generation sequencing techniques, such as whole exome sequencing (WES), can be used to find multiple gene mutations in one assay. We used WES to detect genetic mutations in both permanent neonatal (PND) and type 1B diabetes (T1BD).A total of five PND and nine T1BD patients were enrolled in this study. WES variants were assessed using VarioWatch, excluding those identified previously. Sanger sequencing was used to confirm the mutations, and their pathogenicity was established via the literature or bioinformatic/functional analysis. The PND and T1BD patients were diagnosed at 0.1–0.5 and 0.8–2.7?years of age, respectively. Diabetic ketoacidosis was present at diagnosis in 60% of PND patients and 44.4% of T1BD patients. We found five novel mutations in five different genes. Notably, patient 602 had a novel homozygous missense mutation c.1295C?>?A (T432?K) in the glucokinase (GCK) gene. Compared to the wild-type recombinant protein, the mutant protein had significantly lower enzymatic activity (2.5%, p?=?0.0002) and V_max (1.23?±?0.019 vs. 0.33?±?0.016, respectively; p?=?0.005). WES is a robust technique that can be used to unravel the etiologies of genetically heterogeneous forms of diabetes. Homozygous inactivating mutations of the GCK gene may have a significant role in PND pathogenesis.     

Statistical and mutational analysis of chronic granulomatous disease in Japan with special reference to gp91-phox and p22-phox deficiency

Chronic granulomatous disease (CGD) is a group of inherited disorders of host defense caused by a mutation in any of the four components of phagocyte NADPH oxidase, namely gp91-, p22-, p47-, and p67-phox. We have made a precise statistical analysis of 229 registered patients from 195 families in Japan and mutation analysis of 28 and 5 independent patients, respectively, with gp91- and p22-phox deficiency. The gp91- and p22-phox proteins form the membrane cytochrome b558, which plays important roles in the assembly of the active oxidase and electron-transfer reaction, and the lesions in either subunit account for more than 80% of cases. The ratio of male to female patients was 6.6/1, the incidence was calculated to be about 1 out of 220,000 birth, and the life expectancy of the patients born in the 1970s was estimated to be 25-30 years old. For the X-linked gp91-phox deficiency, we found five missense and nine nonsense mutations, seven deletions, three insertions, and four splice site mutations, which included the following novel mutations: four missense, five nonsense, six deletions, one insertion, and two splice site abnormalities. With regard to p22-phox deficiency, two homozygous nonsense mutations and one homozygous deletion, a missense mutation together with a splice site mutation, and two different missense mutations were found. These mutations have not been reported before. Based on the present and reported data from Japan, we discuss the molecular defects of the disease and the difference in statistics between western countries and Japan.     

Analysis of steroid 21-hydroxylase gene in five unrelated Japanese patients with 21-hydroxylase deficiency

DNA samples from five unrelated Japanese patients with 21-hydroxylase (21-OHase) deficiency were studied by Southern analysis using human 21-OHase cDNA. Patterns seen after digestion with not only TaqI but also KpnI showed that two out of the five patients were homozygous for a deletion of the 21-OHase B gene. This result supports the report that the 21-OHase B gene is functional. In the other three, smaller mutations might be responsible for the disorder. The parents of one of the two patients with the deletion had a common ancestor. Hybridization patterns of DNA from members of the family of the patient were consistent with an autosomal recessive mode of inheritance of the deletion that correlates with the clinical phenotype. The deletion segregated with HLA-Aw 24; Bw 61; Cw 3. Heterozygous carriers of 21-OHase deficiency could be detected by comparing the patterns as well as the HLA haplotypes in this family. The application of the family study to the prenatal diagnosis is also discussed.     

Sister-chromatid exchanges in beta-thalassaemic patients under conditions of in vivo and in vitro depletion of folic acid.

In order to investigate the effect of folate depletion, lymphocyte sister-chromatid exchange (SCE) rates were compared among homozygous beta-thalassaemic patients with low folic acid levels, heterozygous beta-thalassaemic patients with normal folate levels and healthy persons with normal haemoglobin, in cultures with both normal and depleted folate conditions. Significantly higher SCE rates were found in homozygous patients in all assays, but the in vitro folate depletion did not induce an increase in SCE frequency in any group.     

Early onset of severe familial amyotrophic lateral sclerosis with a SOD-1 mutation: potential impact of CNTF as a candidate modifier gene

Mutations in the copper/zinc superoxide dismutase 1 (SOD-1) gene are found in approximately 20% of patients with familial amyotrophic lateral sclerosis (FALS), or amyotrophic lateral sclerosis 1. Here we describe a 25-year-old male patient who died from FALS after a rapid disease course of 11 mo. Sequencing of the SOD-1 gene revealed a heterozygous T-->G exchange at position 1513 within exon 5, coding for a V-->G substitution at position 148 of the mature protein. Genetic analysis of this family revealed the same mutation in both his healthy 35-year-old sister and his mother, who did not develop the disease before age 54 years. Screening for candidate modifier genes that might be responsible for the early onset and severe course of the disease in the 25-year-old patient revealed an additional homozygous mutation of the CNTF gene not found in his yet unaffected sister. hSOD-1G93A mice were crossbred with CNTF(-/-) mice and were investigated with respect to disease onset and duration, to test the hypothesis that CNTF acts as a candidate modifier gene in FALS with mutations in the SOD-1 gene. Such hSOD-1G93A/CNTF-deficient mice develop motoneuron disease at a significantly earlier stage than hSOD-1G93A/CNTF-wild-type mice. Linkage analysis revealed that the SOD-1 gene was solely responsible for the disease. However, disease onset as a quantitative trait was regulated by the allelic constitution at the CNTF locus. In addition, patients with sporadic amyotrophic lateral sclerosis who had a homozygous CNTF gene defect showed significantly earlier disease onset but did not show a significant difference in disease duration. Thus, we conclude that CNTF acts as a modifier gene that leads to early onset of disease in patients with FALS who have SOD-1 mutations, in patients with sporadic amyotrophic lateral sclerosis, and in the hSOD-1G93A mouse model.     

Survival and desferrioxamine in thalassaemia major.

A small randomised trial and observation of all patients homozygous for beta-thalassaemia in Britain born in or before 1963 indicated that those patients who had received average weekly doses of more than 4 g of desferrioxamine over the previous few years were less likely to die in the near future than were patients of similar ages who had received less, or no, desferrioxamine.     

Respiratory system response to local cooling in subjects with single nucleotide polymorphism rs11562975 of the TRPM8 temperature-sensitive ion channel gene

Genome studies in ethnic Russians revealed subjects having a heterozygous genotype with allele C at the single-nucleotide polymorphism rs11562975 (G → C), which is in exon 6 of the TRPM8 temperature-sensitive ion channel gene. The subjects had increased sensitivity to cold and produced a hypometabolic response to local skin cooling and to temperature-independent activation of the TRPM8 ion channel by menthol, including a decrease in gas exchange, pulmonary ventilation, and oxygen extraction coefficient. Subjects with the homozygous genotype GG had a lower sensitivity to cold, and their response to local skin cooling was adequate in terms of thermoregulation, including a decrease in respiratory heat loss and an increase in lipid metabolism.     

Plasma exchange and human factor VIII concentrate in managing haemophilia A with factor VIII inhibitors.

Plasma exchanges were combined with human factor VIII concentrate therapy in the treatment of major bleeding episodes in five patients with haemophilia A and factor VIII inhibitors. All patients had a good clinical response to combined treatment. Inhibitor levels showed satisfactory falls before rapid secondary increases of inhibitor levels took place. A sixth patient with von Willebrand''s disease and a factor VIII clotting activity inhibitor was successfully prepared for operation using plasma exchange. Postoperative haemostasis and healing were normal. In two patients the plasma exchanges were relatively more effective than the administered human factor VIII in reducing the levels of factor VIII inhibitor. Combined plasma exchange and human factor VIII treatment may offer a rapidly effective means of reducing factor VIII inhibitor levels in this group of patients, together with significant saving of costs.     

Characterization of apoA- and apoB-containing lipoprotein particles in a variant of familial apoA-I deficiency with planar xanthoma: the metabolic significance of LP-A-II particles.

This study describes a variant of familial apoA-I deficiency associated with a moderate risk for premature coronary artery disease. The proband, a 25-year-old man of Philippine origin, and his 62-year-old maternal aunt had peripheral corneal opacification, xanthelasma, and planar xanthoma; the aunt had coronary artery bypass surgery at 61 years of age. Proband's parents and three brothers were asymptomatic and apparently healthy. The characteristic apolipoprotein features of affected patients were the immunochemically and chemically undetectable apoA-I, reduced levels of apoA-II, apoC-II, apoC-III, and apoD, and normal levels of apoB and apoE; except for negligible levels of high density lipoprotein (HDL)-cholesterol (2-3 mg/dl), their plasma lipid profile was normal. The apoA-I levels in all five unaffected relatives were more than one SD below the normal mean values for their age and sex; the HDL-cholesterol levels of proband's unaffected brothers were below the 10th percentile of normal control values. Patient's very low density lipoprotein (VLDL), low density lipoprotein (LDL), and HDL contained 1.4, 80.4, and 18.1%, whereas those of control subjects contained 2.7, 28.8, and 68.1% of the total apolipoprotein mass, respectively. In unaffected relatives, the levels of LP-A-I, but not LP-A-I:A-II, were significantly lower than in controls. Neither of the two patients had detectable concentrations of LP-A-I or LP-A-I:A-II. Their HDL only consisted of LP-A-II particles, the levels of which (7-13 mg/dl) were similar to those of unaffected relatives or controls. There was no difference in the lipid composition of LP-A-II between patients and their relatives. However, LP-A-II from patients contained substantial amounts of apoC-peptides and apoE (0.40-0.98 mg/mg apoA-II), whereas those from unaffected relatives were free of these minor apolipoproteins. In patients, among all four major apoB-containing lipoproteins, only the levels of LP-B and LP-B:C were slightly higher than those in controls. Results of this study suggest a genetic cause for this variant of apoA-I deficiency characterized most probably by autosomal recessive inheritance. It appears that patients are likely to be homozygous for a gene present in single dose in the parents and brothers of the affected proband.(ABSTRACT TRUNCATED AT 400 WORDS)     

Frequency of Ag-stained nucleolus organizer regions in the acrocentric chromosomes of man

Summary The Ag-stainability of the nucleolus organizer region (NOR) was studied in the acrocentric chromosomes identified by Q-banding of cultured lymphocytes in 51 karyotypically normal persons (31 males and 20 females). A consistent pattern of Ag-positive NORs was found in each individual. Ninety percent of the individuals have a modal number of 8–10 Ag-positive NORs per cell. The frequency of Ag-positive NORs is similar in all five acrocentrics. A statistically nonsignificant lower frequency is found in chromosome 22. Ag-negative NORs on both homologues were found in four cases. The observed frequency distribution of individuals with homozygous NOR-positive, heterozygous, and homozygous negative acrocentric chromosomes was in accordance with the Hardy-Weinberg law in all five pairs of the acrocentric chromosomes as well as in total. No sex difference was observed in our material.A.-V. Mikelsaar is visiting exchange scientist of the Österreichische Bundesministerium für Wissenschaft und Forschung