Genotyping microarray as a novel approach for the detection of ATP7B gene mutations in patients with Wilson disease.

Wilson disease (WD) is an autosomal recessive inherited disorder of copper metabolism that is caused by mutations in the ATP7B gene. To date, more than 300 mutations have been described in this gene. Molecular diagnostics of WD utilizes restriction enzyme digestion, multiplex ligation-dependent probe amplification or a direct sequencing of the whole gene. To simplify and speed up the screening of ATP7B mutations, we have developed a genotyping microarray for the simultaneous detection of 87 mutations and 17 polymorphisms in the ATP7B gene based on the arrayed primer extension reaction. The patient's DNA is amplified in four multiplex polymerase chain reactions, fragmented products are annealed to arrayed primers spotted on a chip, which enables DNA polymerase extension reactions with fluorescently labeled dideoxynucleotides. The Wilson microarray was validated by screening 97 previously genetically confirmed WD patients. In total, we detected 43 mutations and 15 polymorphisms that represent a majority of the common mutations occurring in the Czech and Slovak populations. All screened sequence variants were detected with 100% accuracy. The Wilson chip appears to be a rapid, sensitive and cost-effective tool, representing the prototype of a disease chip that facilitates and speeds up the screening of potential WD patients.

Molecular screening of Smith-Lemli-Opitz syndrome in pregnant women from the Czech Republic.

Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive metabolic disorder. SLOS is caused by the mutations in the gene for 3beta-hydroxysterol Delta(7) reductase (DHCR7; EC 1.3.1.21), which maps to chromosome 11q12-13. DHCR7 catalyses the final step in cholesterol biosynthesis-the reduction of 7-dehydrocholesterol to cholesterol. Clinical severity ranges from mild dysmorphism to severe congenital malformation and intrauterine lethality. Pregnant women are offered a biochemical screening test for Down syndrome in the second trimester, where the suspicion for SLOS could be registered, when the unconjugated estriol (uE3) level appears low. A group of 456 fetuses with a high risk for SLOS were examined by DNA analysis. We confirmed SLOS in 5 fetuses and 11 fetuses were carriers. One novel mutation (p.G30A) was detected. The most frequently found mutations, c.964-1G > C and p.W151X, are also the most severe ones. At least one of these mutations was detected in each fetus with SLOS. This suggests that the biochemical screening of pregnant women probably uncovers mainly more severely affected fetuses. We confirmed SLOS also in two patients whose prenatal screening was negative. Both of them had nonsense mutation on one allele. It stands to reason that some modifying factors may play a role in the reduction of the uE3 level in the mother's serum.

[Effect of amaranth oil and intermittent hypoxic training on ultrastructural and metabolic changes in the liver induced by fluorine and low doses of radiation].

Ultrastructural and metabolic changes were studied during chronic fluorine intoxication and low doses of radiation (total dose is 1 Gr) in liver cells and tissues in rats fed with amaranth oil and treated with intermittent hypoxic training (IHT). The obtained data detected the ordered and compact position of mitochondria, peroxisomes, lipoprotein droplets with light electronic density, glycogen granules and also agranular endoplasmatic retuculum channels, which may be linked with reorganization of metabolic pathway of energy supply from fat acids via gluconeogenesis. Simultaneously the decrease of TBA-reactive substances accumulation with the considerable increase in activity of the antioxidant enzymes (catalase, glutathione peroxidase) and index of general antioxidant activity have been established. Therefore, the combined effect of IHT and amaranth oil on adequate occurence of free radical reactions provides the effective adaptation of organism to fluorine intoxication and ionizing radiation via the restoration of homeostasis on metabolic and ultrastructural levels. The obtained results allow to recommend IHT and amaranth oil for complex correction of changes, induced by fluorine intoxication and ionizing radiation.

Maternal hyperphenylalaninemias in healthy Czech population of pregnant women: 30 years experience with screening, prevention and treatment.

INTRODUCTION: The increased level of phenylalanine (Phe) in maternal blood--hyperphenylalaninemia (mHPA) has a detrimental effect on the early development of healthy foetus (1965). The toxic effect causes spontaneous abortion or retards intrauterine growth, skeletal malformation, cardiac anomalies can appear. However the most frequent are microcephaly, mental retardation and hypotrophy. PATIENTS AND METHODS: Simultaneously with the introduction of obligatory "Newborn Screening Program" in CR also the facultative screening for mHPA was introduced ("Maternal Hyperphenylalaninemia Preventive Screening Program"). Since 1975 till now 222,990 healthy pregnant women (16-47 yrs) from city Prague and its area (cca 2 mil. inh.) have been screened for increased Phe in blood by Efron's chromatographic screening test (1964); Phe cut off value: 240 micromol/l. Nonfasting venous blood has been taken in 2nd-3rd month of pregnancy during the first antenatal visit. All positive cases have been verified with quantitative Phe estimation on amino acid analyzer incl. pterines analysis in urine. For differentiation of detected mHPAs the Güttler's scheme (1980) has been used. Mutations for Phe-hydroxylase gene analyzed by restriction enzyme digestion after Guldberg (1994). RESULTS: The average incidence of mHPA detected at the beginning of pregnancy was found 1:8675. The major part (65.3%) of all detected mHPA belongs to mild or moderate form of phenylketonuria (PKU) with most frequent PAH gene mutations R408W, Y414C, IVS11 nt8g-a, R158Q, IVS12ntlg-a and R261Q. 19.2% corresponds to atypical or classical PKU with prevailing mutation R408W. Only in 15.3% were detected non-PKU (persistent HPA) with mutations R408W, Y414C, IVS12ntlg-a, IV11nt8g-a and A403V. 28 offsprings born from pregnancies on low-phenylalanine diet (LPD) introduced at least 2 months before the conception and during the whole pregnancy show normal psychomotoric development. In 7 offsprings without LPD or after delayed introducing or on PLD or badly monitored showed malformations (microcephaly, hypotrophy, skeletal malformations) or died. DISCUSSION: Relatively high incidence of mHPA detected in healthy population of pregnant women of Prague area differs from findings of Buist (1989) or Levy (1994) from American pregnant women screened for mHPA from umbilical blood. We consider that screening performed at the beginning of pregnancy from nonfasting venous blood is more effective compared to umbilical blood from two reasons: the Phe level in maternal blood is increased during first trimester of pregnancy due to succing effect of placenta in comparison to decreased Phe level at the end of labour. Umbilical blood for screening of mHPA is not quite suitable to detect the atypical or mild forms of Phe disturbances which prevailed in our Slavonic population of pregnant women. (Tab. 5, Fig. 7, Ref. 16.)

Splicing mutations, mainly IVS6-1(G>T), account for 70% of fumarylacetoacetate hydrolase (FAH) gene alterations, including 7 novel mutations, in a survey of 29 tyrosinemia type I patients.

Hereditary tyrosinemia type I (HTI) is an autosomal recessive disease characterized by a deficiency in fumarylacetoacetate hydrolase (FAH) activity. In this work, the FAH genotype was established in a group of 29 HTI patients, most of them from the Mediterranean area. We identified seven novel mutations-IVS8-1(G>A, IVS10-2(A>T), 938delC, E6/I6del26, W78X, Q328X, and G343W-and two previously described mutations-IVS6-1(G>T) and IVS12+5(G>A). Fully 92.8% of the patients were carriers of at least one splice site mutation, with IVS6-1(G>T) accounting for 58.9% of the total number of alleles. The splice mutation group of patients showed heterogeneous phenotypic patterns ranging from acute forms with severe liver malfunction to chronic forms with renal manifestations and slow progressive hepatic alterations. Qualitative FAH cDNA expression was the same in all IVS6-1(G>T) homozygous patients regardless of their clinical picture. One patient with a heterozygous combination of a nonsense (Q328X) and a frameshift (938delC) mutation showed an atypical clinical picture of hypotonia and repeated infections. Despite the high prevalence of IVS12+5(G>A) in the northwestern European population, we found only two patients with this mutation in our group.

[Molecular analysis of Wilson disease]. / Molekulární analýza Wilsonovy choroby.

BACKGROUND: Wilson disease is an autosomal recessive disorder, characterized by cooper accumulation and intoxication of the organism. Molecular basis of the disease represent mutations in the gene for the copper-transporting ATPase (ATP7B). METHODS AND RESULTS: The submitted paper deals with results of molecular-genetic examination in 130 unrelated families in which Wilson disease was diagnosed. By denaturing gradient gel electrophoresis (DGGE), the exons with abnormal sequences were detected. Followed by sequencing, 17 causal mutations and 9 silent polymorphism were found. Five novel mutations were detected. After analysis of 260 mutant alleles, 214 (82.3%) were identified. The most frequent mutation, H1069Q, occurred in our population with the frequency of 65.8%. Incidence of other mutations, however, did not exceed 5%. CONCLUSIONS: DNA analysis of the Wilson disease offers prompt and reliable results in affected families. It can help to identify asymptomatic and heterozygote siblings at genetic counselling.

Spectrum of low density lipoprotein receptor mutations in Czech hypercholesterolemic patients.

The aim of our study was to define mutations causing familial hypercholesterolemia (FH) phenotype in Czech hypercholesterolemic individuals. A combination of heteroduplex analysis, SSCP, DGGE, DNA sequencing and PCR/restriction analysis was used for this purpose. Molecular searching in the promoter region and coding sequence of the low density lipoprotein receptor (LDLR) gene in 130 patients from 68 unrelated families resulted in the identification of 37 sequence variations. Thirty of them are most likely disease causing mutations. Nineteen mutations were novel (two nonsense, five missense, six nucleotide(s) insertions and six nucleotide(s) deletions). Their pathological effect can be predicted on the basis of their position with respect to previously reported mutations with an estimated reduction of the receptor activity and/or premature termination of translation. These results expand our knowledge of mutations responsible for FH. Seven nucleotide variations were characterized as silent polymorphisms. Hum Mutat 18:253, 2001.

[The hepatic form of Wilson's disease in young patients]. / Jaterní forma Wilsonovy nemoci u mladých pacientu.

UNLABELLED: Wilson's disease (WD) is a hereditary disorder of the copper metabolism with very varied clinical and biochemical symptoms. Hepatic and neurological forms are the most frequent manifestations of this rare disease. In schoolchildren and adolescents symptoms of liver damage predominate. In a retrospective study 19 patients were evaluated with biochemical signs of hepatopathy manifested before the age of 18 years. The diagnosis of WD was established at the age of 7 to 27 years. One female patient was admitted with fulminant hepatic failure which was treated by acute transplantation of the liver in the Institute of Clinical and Experimental Medicine in Prague. Only 9 of 18 patients with chronic hepatic affection at the time of diagnosis met the Sternlieb diagnostic criteria. These patients had reduced ceruloplasmin levels (0.08-0.18 g/l) and a high copper content in the hepatic dry matter (783 ug/g +/- 323 [SD]). In the remaining 9 patients the ceruloplasmin level was normal, however, in 8 a high copper content of the hepatic dry matter was found (696 ug/g (+)- 352[SD]. The last patient from this group had Kayser-Fleischer's (K-F) ring. It was possible to confirm the high copper content in the hepatic dry matter only after one year's penicillinamine treatment because at the time of the diagnosis poor coagulation did not permit to perform a liver biopsy. There was a statistically significant difference in the copper content of the hepatic dry matter in patients meeting and not meeting Sternlieb's criteria. Statistically significant differences between both groups were found in the plasma copper levels and in the 24-hour urinary copper excretion. Histological examination of the liver under a light microscope revealed findings from minimal changes associated with the presence of glycogen nuclei in hepatocytes to the picture of active chronic hepatitis. In all 19 patients the gene mutation H1069Q was examined and the results were positive in 39.8%. In 3 asymptomatic patients it was present in the homozygous form. CONCLUSION: Early detection of the atypical form of WD remains very difficult. The gold standard is still in all cases assessment of copper in the dry liver tissue. In the near future an important place will be held also by direct DNA analysis although its use is limited not only by the large number of known mutations but also by the financial costs of the method.

National hospital discharge survey: annual summary, 1997.

OBJECTIVES: This report presents national estimates of the use of non-Federal short-stay hospitals in the United States during 1997 and selected trend data. Estimates are provided by demographic characteristics of patients discharged, geographic region of hospitals, conditions diagnosed, and surgical and nonsurgical procedures performed. Measurements of hospital use include number and rate of discharges and days of care, and the average length of stay. METHODS: The estimates are based on data collected through the National Hospital Discharge Survey. The survey has been conducted annually by the National Center for Health Statistics since 1965. In 1997, data were collected for approximately 300,000 discharges. Of the 501 eligible non-Federal short-stay hospitals in the sample, 474 (95 percent) responded to the survey. Diagnoses and procedures were coded according to the International Classification of Diseases, 9th Revision, Clinical Modification, or ICD-9-CM.

Ambulatory and inpatient surgery: national patterns for the elderly.

In 1996 the elderly made 13.6 million visits to ambulatory and inpatient settings for surgery. This was 401.0 visits per 1,000 population, which was approximately three and a half times the rate of 114.4 for persons under 65. A total of 25.3 million procedures were performed during these visits. More of the elderly's procedures were performed during inpatient visits (14.5 million procedures) than during ambulatory visits (10.7 million procedures). Three-quarters of the procedures performed on the elderly were in four categories: operations on the eye, cardiovascular operations, operations on the digestive system, and miscellaneous diagnostic and therapeutic procedures. The leading procedures for this age group were extraction of lens, insertion of lens, arteriography and angiocardiography, and endoscopies of small and large intestines. Women who comprised the majority of the elderly population had a larger number of procedures than men, but elderly men had a higher rate per 1,000 population. The 65-74 year-old group had the largest number of procedures, followed by 75-84 year-olds and then the 85 years and older group, reflecting the decreasing population size of these age groups. But the 75-84 year and 85 years and over groups had higher rates of procedures than those aged 65 to 74.

Changing patterns of surgical care in the United States, 1980-1995.

National inpatient and ambulatory surgery data were combined to examine changes over time in the location and amount of surgical care. The main pattern was a decline in the rate of inpatient operations that was outweighed by growth in ambulatory operations. However, the rate of inpatient operations did not decrease for patients age 65 years or over, despite the growth in ambulatory surgery. Other patterns seen for specific types of operations were the substitution of ambulatory for inpatient operations, increases primarily in the rate of inpatient operations, and decreases in total operations. These patterns have implications for control of health care costs.

Ambulatory and inpatient procedures in the United States, 1996.

OBJECTIVES: This report presents estimates of surgical and nonsurgical procedures performed in the United States during 1996. Data are presented by characteristics of patients, region of the country, and procedure categories for ambulatory and inpatient procedures separately and combined. METHODS: Estimates in this report are based on data collected from the National Hospital Discharge Survey (NHDS) and the National Survey of Ambulatory Surgery-(NSAS). NHDS provides data on hospital inpatient care, and NSAS provides data on ambulatory surgery in hospitals and in freestanding ambulatory surgery centers. For NHDS, data were collected for approximately 282,000 discharges from 480 non-Federal short-stay hospitals (95 percent response rate). For NSAS, data were collected for approximately 125,000 ambulatory surgery discharges from 488 hospitals and freestanding ambulatory surgery centers (81 percent response rate). RESULTS: An estimated 71.9 million procedures were performed on 39.9 million discharges from hospitals and freestanding ambulatory surgery centers during 1996: 40.4 million procedures were for inpatients, and 31.5 million were for ambulatory patients. Females had more procedures than males, and the rate of procedures increased with age in ambulatory and inpatient settings. The leading procedures for ambulatory surgery patients and inpatients combined were arteriography and angiocardiography, endoscopy of small intestine, endoscopy of large intestine, and extraction of lens.

Detailed diagnoses and procedures, National Hospital Discharge Survey, 1996.

OBJECTIVES: This report presents national estimates of the use of non-Federal short-stay hospitals in the United States during 1996. Estimates of first-listed diagnoses, all-listed diagnoses, days of care for first-listed diagnoses, and all-listed procedures are shown by sex and age of patient and geographic region of hospital. METHODS: The estimates are based on data collected through the National Hospital Discharge Survey for 1996. The survey has been conducted annually by the National Center for Health Statistics since 1965. In 1996, data were collected for approximately 282,000 discharges from 480 non-Federal short-stay hospitals. Diagnoses and procedures are presented according to their code number in the International Classification of Diseases, 9th Revision, Clinical Modification (ICD-9-CM).

Ambulatory and inpatient procedures in the United States, 1995.

OBJECTIVES: This report presents estimates of surgical and nonsurgical procedures performed in the United States during 1995. Data are presented by characteristics of patients, region of the country, and procedure categories for ambulatory and inpatient procedures separately and combined. METHODS: Estimates in this report are based on data collected from the National Hospital Discharge Survey (NHDS) and the National Survey of Ambulatory Surgery (NSAS). NHDS provides data on hospital inpatient care, and NSAS provides data on ambulatory surgery in hospitals and in freestanding ambulatory surgery centers. For NHDS, data were collected for approximately 263,000 discharges from 466 non-Federal short-stay hospitals (92 percent response rate). For NSAS, data were collected for approximately 122,000 ambulatory surgery discharges from 489 hospitals and freestanding ambulatory surgery centers (80 percent response rate). RESULTS: An estimated 69.2 million procedures were performed on 38.7 million discharges from hospitals and freestanding ambulatory surgery centers during 1995: 39.8 million procedures were for inpatients, and 29.4 million were for ambulatory patients. Females had more procedures than males, and the rate of procedures increased with age in ambulatory and inpatient settings. The leading procedures for ambulatory surgery patients and inpatients combined were endoscopy of small intestine, arteriography and angiocardiography, extraction of lens, and endoscopy of large intestine.

[Direct detection of mutations in the LDL receptor gene in patients with familial hypercholesterolemia]. / Prímá detekce mutací v genu pro LDL receptor u pacientu s familiární hypercholesterolémií.

BACKGROUND: Familial hypercholesterolemia is one of the most frequent hereditary metabolic diseases. As a result of the functional disorder of the molecule of the LDL receptor LDL cholesterol is not sufficiently eliminated from the blood stream and exerts an atherogenic effect. The objective of the study was to introduce direct detection of mutations in the gene for the LDL receptor and characterize the spectrum of mutations in the Czech population. METHODS AND RESULTS: The authors analyzed a group of 84 unrelated patients where on the basis of clinical and biochemical criteria the diagnosis of FH was established. From the group 12 patients were eliminated (14.3%) where a mutation 3500 in the gene for apolipoprotein (apo) B-100 was detected. This mutation is most frequently the cause of a familial defect of apo B-100 (FDB), which cannot be differentiated clinically or biochemically from FH. In the LDL receptor gene a total of 11 mutations were found in 14 unrelated patients (16.7%), incl. 7 mutations not described hitherto. CONCLUSIONS: This is the first systematic characteristic of the spectrum of mutations in the LDL receptor gene in the Czech population. Molecular genetic analysis of the gene for the LDL receptor in affected families can contribute towards early assessment of the diagnosis of FH and thus to prevention of life threatening cardiovascular episodes in asymptomatic subjects.

National Survey of Ambulatory Surgery: 1994.

In 1994, 27.7 million surgical and nonsurgical procedures were performed during 18.4 million visits to ambulatory surgery settings. Eighty-five percent of the ambulatory procedures (23.4 million) were performed in hospital-based settings and 15 percent (4.3 million) were in freestanding settings. More than half of the ambulatory procedures were in three major categories: operations on the digestive system, the eye and the musculoskeletal system. The leading ambulatory procedures were extraction of lens (performed almost 2 million times), endoscopy of large intestine with or without biopsy (1.8 million) and insertion of prosthetic lens (1.6 million). Women had significantly more ambulatory surgery procedures than men. The ambulatory procedure performed most frequently on children under 15 was myringotomy. Also common for children were tonsillectomies and adenoidectomies. For those aged 15-44, frequently performed ambulatory procedures included endoscopies of large and small intestine; dilation and curettage of uterus; bilateral destruction or occlusion of fallopian tubes; and arthroscopy of knee. In the 45-64 year old group, endoscopies of large and small intestine were the most commonly performed ambulatory procedures. Extraction of lens and insertion of prosthetic lens were the leading ambulatory procedures for those 65 years and older, though endoscopies of large and small intestine were also performed frequently for this age group.

The mouse rostral cerebellar malformation gene encodes an UNC-5-like protein.

Migration of neurons from proliferative zones to their functional sites is fundamental to the normal development of the central nervous system. Mice homozygous for the spontaneous rostral cerebellar malformation mutation (rcm(s)) or a newly identified transgenic insertion allele (rcm(tg)) exhibit cerebellar and midbrain defects, apparently as a result of abnormal neuronal migration. Laminar structure abnormalities in lateral regions of the rostral cerebellar cortex have been described in homozygous rcm(s) mice. We now demonstrate that the cerebellum of both rcm(s) and rcm(tg) homozygotes is smaller and has fewer folia than in the wild-type, ectopic cerebellar cells are present in midbrain regions by three days after birth, and there are abnormalities in postnatal cerebellar neuronal migration. We have cloned the rcm complementary DNA, which encodes a transmembrane receptor of the immunoglobulin superfamily. The sequence of the rcm protein (Rcm) is highly similar to that of UNC-5, a Caenorhabditis elegans protein that is essential for dorsal guidance of pioneer axons and for the movement of cells away from the netrin ligand, which is encoded by the unc-6 gene. As Rcm is a member of a newly described family of vertebrate homologues of UNC-5 which are netrin-binding proteins, our results indicate that UNC-5-like proteins may have a conserved function in mediating netrin-guided migration.

Ambulatory surgery in the United States, 1994.

OBJECTIVES: This report presents estimates of surgical and nonsurgical procedures done on an ambulatory basis in hospitals and freestanding ambulatory surgery centers in the United States during 1994. Data are presented by types of facilities, age, sex, geographic region, and disposition for ambulatory surgery visits. Major categories of procedures and diagnoses are shown by age, sex, and region. METHODS: The estimates are based on data collected from the national Survey of Ambulatory Surgery, conducted for the first time in 1994 by the national Center for Health Statistics. The 1994 data were abstracted from 118,000 medical records in 494 hospitals and freestanding ambulatory surgery centers.

Ambulatory and inpatient procedures in the United States, 1994.

OBJECTIVES: This report presents estimates of surgical and nonsurgical procedures performed in the United States during 1994. Data are presented by characteristics of patients, region of the country, and procedure categories for ambulatory and inpatient procedures separately and combined. METHODS: Estimates in this report are based on data collected from the National Hospital Discharge Survey (NHDS) and the National Survey of Ambulatory Surgery (NSAS). The NHDS provides data on hospital inpatient care, and the NSAS provides data on ambulatory surgery in hospitals and in freestanding ambulatory surgery centers. For the NHDS, data were collected for approximately 277,000 discharges from 478 non-Federal short-stay hospitals (93 percent response rate). For the NSAS, data were collected for about 118,000 ambulatory surgery discharges from 494 hospitals and freestanding ambulatory surgery centers (80 percent response rate). RESULTS: An estimated 68.4 million procedures were performed on 38.0 million discharges from hospitals and freestanding ambulatory surgery centers during 1994: 40.7 million procedures were for inpatients, and 27.7 million were for ambulatory patients. Females had more procedures than males did and the rate of procedures increased with age in both ambulatory and inpatient settings. The leading procedures for ambulatory surgery patients and inpatients combined were endoscopy of large intestine, arteriography and angiocardiography, endoscopy of small intestine, and extraction of lens.