Mutation analysis of Wilson disease in the Spanish population -- identification of a prevalent substitution and eight novel mutations in the ATP7B gene.

Wilson disease (WD) is a copper metabolism disorder characterized by hepatic and/or neurological damage. More than 200 mutations in the ATP7B gene causing this autosomal recessive defect have been reported. In certain populations, a high prevalence of particular mutations allows rapid screening and diagnosis of the disease. We identified the ATP7B alterations in Spanish patients with WD. Mutations in the ATP7B gene were analysed in a total of 64 individuals from 40 different WD families by PCR amplification, single-strand conformation polymorphism (SSCP) analysis and sequencing. Twenty-one different ATP7B gene mutations were identified, eight of which were novel. 74% of the disease alleles were characterized among the 40 unrelated probands. We identified a prevalent mutation in our population (Met645Arg), present in 55% of this 40 patients. The frequency of the remaining ATP7B alterations was low. In addition, 17 different polymorphic variants were found. There is remarkable allele heterogeneity in WD in the Spanish population. Nevertheless, SSCP screening for the most frequent mutations in our population is feasible and leads to the detection of about 74% of the mutated chromosomes. Molecular diagnosis of WD is very useful in clinical practice to confirm or support clinical suspicion.

[Transient cerebral oedema associated to hypoglycaemia]. / Edema cerebral transitorio asociado a hipoglucemia.

INTRODUCTION: In the adult, hypoglycaemia is documented as a consequence of overdose of insulin or oral hypoglycaemic agents. Neonatal hypoglycaemia is common but rarely symptomatic due to protective mechanisms. Very few reports on hypoglycaemic injury are available in adults and most of them in patients with poor outcome. CASE REPORT: Woman, 45 years old. She is referred to emergency room due to insulin overdose and coma of unknown duration. Glucose level was 15 mg% without other metabolic anomalies. Computerized tomography revealed brain oedema most obvious in the parieto-occipital lobes. Therapy with manitol and glucose was started with total functional recovery. CONCLUSIONS: Brain imaging in neonatal hypoglycaemia shows similar findings. In the acute phase oedema in the parieto-occipital cortex and underlying white matter and atrophy or malacic cysts in the chronic phase have been described. Chronic changes are related to long-term sequelae that vary from development delay and epilepsy to persistent vegetative state. In adults lesions involve hippocampus and basal ganglia. The reasons of different sensitivity of these regions are not fully clear. Differences in regional blood flow autoregulation or in excitotoxins receptors of aspartate have been postulated. Hypoglycaemia may induce blood-brain-barrier permeability and subsequently brain oedema.

Pulmonary mass and multiple lung nodules mimicking a lung neoplasm as amiodarone-induced pulmonary toxicity.

Amiodarone is an effective anti-arrhythmic agent. However, during long-term therapy, patients can develop severe adverse pulmonary reactions that are potentially life-threatening. A case of amiodarone-induced pulmonary toxicity is presented in a 78-year-old woman. She developed dyspnea and a pulmonary mass with associated multiple lung nodules mimicking a lung cancer following 5 years of treatment with amiodarone for atrial fibrillation. After drug withdrawal, and without any additional treatment, clinical and radiological improvement was observed, and radiological findings resolved completely within 6 months.

Chromosomal abnormalities and polymorphisms in infertile men.

The aim of this study was to determine the prevalence of alterations and normal variable chromosome features in males from infertile couples. Karyotyping was performed to 84 men attending the infertility clinic at the Hospital Clinic i Provincial of Barcelona (Spain). Sex chromosome abnormalities were detected in 19 patients (26.62%): 14 (16.67%) aneuploidies 47,XXY and 47,XYY, 3 (3.57%) Y-chromosome long arm deletions; 1 (1.19%) mosaic 45,x/46,XY and 1 (1.19%) Robertsonian translocation (45.X-15-Y+t(15p: Yq). Chromosomal polymorphisms were observed in 29 patients. Yqh+ was the most frequent variant in sex chromosomes and increased length in heterochromatin and satellites were present in autosomal chromosomes. The high prevalence of chromosomal abnormalities observed in infertile men justify the use of karyotyping to evaluate males enrolled in new assisted reproductive technologies programs.

Paternal isodisomy 13 in a normal newborn infant after trisomy rescue evidenced by prenatal diagnosis.

Maternal and paternal uniparental disomy of chromosome 13 have been associated with normal phenotypes. We report on a new case of paternal isodisomy 13 in a phenotypically normal girl. Prenatal diagnosis had shown a 46,XX,-13,der(13;13) karyotype in chorionic villi and a 45,XX,der(13;13) karyotype in amniocytes and fetal blood. Molecular studies demonstrated that the de novo der(13;13) was an isochromosome 13 of paternal origin. This observation supports the lack of imprinting effects on chromosome 13 and trisomy rescue as a mechanism leading to uniparental disomy in cases involving isochromosomes.

Analysis of the polymorphic (GT)(n) repeat at the dopamine beta-hydroxylase gene in Spanish patients affected by schizophrenia.

The presence of a polymorphic (GT)(n) repeat, a microsatellite repeat, at the human dopamine beta-hydroxylase (DBH) gene had been previously investigated in healthy people and in schizophrenic patients. The different DBH genotypes had been found to be associated to different DBH biochemical function, but no differences were found in the allelic and genotype frequencies between schizophrenic and control groups. To further clarify the potential involvement of the variation at the DBH gene in schizophrenia we have studied the DBH (GT)(n) repeat in a sample of 47 Spanish schizophrenic patients, in their healthy relatives (n = 72), and in a control population (n = 74). We have been able to identify five different variants of the DBH gene (A1, A2, A3, A4, A5) in the different groups. Subsequent statistical analysis revealed that the genotypes as well as the allele frequencies did not differ significantly among schizophrenic patients and the control population. Interestingly, the allelic variant A2 and the genotype A4/A2 were significantly more frequent in schizophrenic patients as compared with their healthy relatives. However, the association of the A2 allele with schizophrenia was not supported by the haplotype relative risk analysis of transmitted versus nontransmitted alleles. Therefore, although it will be important to extend the present analysis in a larger sample of schizophrenic patients and controls, our results suggest that the (GT)(n) does not seem to play a major role in the genetics of schizophrenia at least in this group of Spanish schizophrenic patients. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 96:88-92, 2000.

SRY gene transferred to the long arm of the X chromosome in a Y-positive XX true hermaphrodite.

Yp-specific sequences, including the testicular determinant gene SRY, have been detected and located in a 46,XX true hermaphrodite individual, using PCR amplification and fluorescent in situ hybridization (FISH). Among different Y chromosome loci tested, it was only possible to detect Yp sequences. The Y-centromere and Yq sequences were absent. Unexpectedly, the Y fragment was translocated to the long arm of one of the X chromosomes, at the Xq28 level, and the derivative (X) chromosome of the patient lacked q-telomeric sequences. To our knowledge, this is the first Yp/Xq translocation reported. The coexistence of testicular and ovarian tissue in the patient may have arisen by differential inactivation of the Y-bearing X chromosome, in which Xq telomeric sequences are missing. The possible origin of the Yp/Xq translocation, during paternal meiosis or in somatic paternal cells, is discussed.

Dentatorubropallidoluysian atrophy in a spanish family: a clinical, radiological, pathological, and genetic study.

UNLABELLED: The object was to describe the clinical, radiological, pathological, and genetic findings in a Spanish family with dentatorubropallidoluysian atrophy (DRPLA). This is an inherited neurodegenerative disease, well recognised in Japan, but with few cases reported from Europe and America and no cases published from Spain. The clinical misdiagnosis of Huntington's disease is not infrequent. Pedigree analysis and clinical data of a family were collected. A genetic study was performed in two patients. Pathological information was obtained from the necropsy of one patient. RESULTS: Pedigree analysis showed an autosomal dominant pattern of inheritance. Age at onset varied from 5 to 55 years. Ataxia and chorea were present in most of the members. Some of these had a long course disease with late dementia. Four patients had seizures and early mental impairment. In one patient, cranial MRI showed cortical, brain stem and cerebellar atrophy, and white matter changes. In another patient, necropsy showed atrophy of the globus pallidus and lipofuscin deposits in dentate and pallidal neuronal cells. Genetic study showed an abnormal CAG triplet expansion in the B37 gene on chromosome 12. As in other cases previously reported, Spanish cases of DRPLA show intrafamilial phenotypic heterogeneity. Clinical and MRI data could differentiate DRPLA from Huntington's disease but definitive diagnosis requires molecular studies. Pathological studies are still necessary to correlate DRPLA brain involvement with the clinical and molecular findings.

[Clinical characterization, molecular and FISH studies in 80 patients with clinical suspicion of Williams-Beuren syndrome]. / Caracterización clínica y genética de 80 pacientes con sospecha clínica de síndrome de Williams-Beuren.

BACKGROUND: Williams-Beuren syndrome is a developmental disorder affecting vascular and connective tissues and central nervous system. The syndrome is caused by a submicroscopic deletion in the chromosome 7 implicating the 7q11.23 region. Fluorescence in situ hybridization (FISH) and molecular studies allow us to confirm the clinical suspicion of this syndrome. PATIENTS AND METHODS: We report clinical evaluation, FISH using Elastin Williams/D7S427 probe and molecular study with markers: D7S672, D7S653, D7S489B, D7S2476, D7S1870 and D7S489A, in 80 patients referred to test for Williams-Beuren syndrome. RESULTS: We found hemizygosity for the critical region in 36 patients. From 69 cases studied by FISH, 28 showed the deletion. Molecular studies in 78 cases showed loss of heterozygosity (LOH) in 26 patients. The patients presented the deletion from the paternal or maternal chromosome at equal frequency. Clinical evaluation of mental retardation, facial features, esotopia dental, malocclusion, hoarse voice, supravalvular aortic stenosis (SVAS), hernias, join limitation, WBS personality and mental retardation from positive and negative patients showed estatistical significant differences for all items except mental retardation and joint limitation. The most significant item was the presence of SVAS. CONCLUSION: This study confirms the usefulness of genetic studies as a diagnostic tool for William-Beuren Syndrome.

Trisomy/tetrasomy 21 mosaicism in CVS: interpretation of cytogenetic discrepancies between placental and fetal chromosome complements.

Trisomy/tetrasomy 21 mosaicism was found in chorionic villi (semidirect preparation) obtained from a 40 year old pregnant woman. Since both cell lines were abnormal, the couple elected for pregnancy termination. Placenta and fetal tissue samples were obtained for cytogenetic study. Long term cultured villi showed a non-mosaic trisomy 21 karyotype, while other tissues showed either a normal karyotype or normal/trisomy21 mosaicism. These discrepancies could be explained by a modified "bottle neck" embryogenic model with a trisomic zygote and a non-disjunction event taking place in one of the first divisions. Our case emphasises the need for confirmatory studies in other tissues when mosaicism is encountered in chorionic villi, even if all cell lines are abnormal.

Parental origin and meiotic stage of non-disjunction in 139 cases of trisomy 21.

The parental origin and the meiotic stage of non-disjunction have been determined in 139 Down syndrome patients with regular trisomy 21 and in their parents through the analysis of DNA polymorphism. The meiotic error is maternal in 91.60% cases and paternal in 8.39% of cases. Of the maternal cases, 72.41% were due to meiosis I errors (MMI) and 27.58% were due to meiosis II errors (MMII). Of the paternal cases, 45.45% were due to meiosis I (PMI) and 54.54% were due to meiosis II (PMII). The mean maternal ages were 31.6 +/- 5.3 (+/- SD) years in errors from MMI, 32.3 +/- 6.4 years in errors from MMII, 31.4 +/- 4.6 years in errors from PMI and 29.5 +/- 2.7 years in errors from PMII. No significant statistical differences were observed between maternal and paternal errors, further supporting the presence of a constant chromosome 21 non-disjunction error type.

Assaying the granulocyte-macrophage colony-stimulating factor (GM-CSF) as a mitogen of immature cells in fetal blood cultures.

Based on the presence of immature cells in fetal blood, and in an attempt to shorten the cytogenetic reporting time, three simultaneous one-day culture regimes were established in 23 fetal blood samples: (a) the standard phytohemagglutinin (PHA)-stimulated lymphocytes culture, (b) a culture using the granulocyte-macrophage colony-stimulating factor (GM-CSF) as an alternative mitogen, and (c) an unstimulated culture. Diagnostic success rates achieved by these three methods were as follows: 43 per cent (95 per cent CI: 23-64) (GM-CSF), 30 per cent (95 per cent CI: 12-49) (PHA) and 9 per cent (unstimulated). These three regimes were also assayed in three-day cultures giving 100 per cent diagnostic success rate for the PHA and GM-CSF, and 62 per cent (95 per cent CI: 41-83) for the unstimulated. A moderate correlation was found between the initial concentration of cultured erythroblasts and the metaphase count in one-day GM-CSF-stimulated (r=0.43, p=0.01) and unstimulated (r=0.35, p=0.05) cultures, suggesting that erythroblasts may be in part responsible for the mitotic index observed in these two regime cultures. In conclusion, our experience suggests that immature cells in fetal blood may be successfully cultured for diagnostic purposes.

Alpha1-antichymotrypsin gene polymorphism and susceptibility to Parkinson's disease.

OBJECTIVE: To determine whether the alpha1-antichymotrypsin AA genotype (ACT-AA) confers susceptibility for developing Parkinson's disease (PD) in the Spanish population. BACKGROUND: A correlation between the ACT-AA genotype and the risk of developing PD has been recently reported in the Japanese population. METHODS: The ACT genotypes of 71 patients diagnosed with clinically definite PD were compared with those of 109 age-matched healthy control subjects. RESULTS: The authors found that the ACT-AA polymorphism frequency was not increased significantly in the PD group (31%) compared with the control group (28.4%). The ACT allelic distribution was also similar for familial and sporadic PD, for female and male patients, and for the different clinical subtypes of PD. The age at onset of PD was significantly lower in the ACT-AA patients compared with non-ACT-AA patients. When the actual age was considered, the ACT-AA frequency was higher in PD patients < or =50 years old (50%) compared with that present in patients >50 years old (26.8%), but the same effect was found in control subjects. CONCLUSIONS: The ACT-AA polymorphism is not related to an increased risk of developing PD in the Spanish population. The ACT-AA overrepresentation in PD and control subjects < or =50 years old suggests that this polymorphism could be associated with life-threatening conditions other than PD.

A presenilin 1 mutation (Ser169Pro) associated with early-onset AD and myoclonic seizures.

OBJECTIVE: To characterize the mutation responsible for early-onset AD in a large Spanish kindred. BACKGROUND: Mutations in the presenilin 1 (PS1) gene have been identified and are known to be responsible for 18 to 50% of familial early-onset AD cases. METHODS: Patients were characterized clinically. The proband was further studied with EEG, CSF analysis, CT, brain biopsy, and histology. Other members were studied using EEG, CT, MRI, and SPECT. Genetic analysis of PS1 was performed using PCR amplification of PS1 exons and direct sequencing followed by PS1 modeling of the normal and mutant PS1 proteins. RESULTS: A novel mutation (Ser169Pro) in exon 6 of the PS1 gene was identified in different affected members. The Ser169Pro mutation is located at a site of the PS1 protein that is not a cluster of mutations. The mutation was not present in 100 general population controls and in 50 unrelated sporadic AD cases. The Ser169Pro mutation is associated with generalized myoclonic seizures several years after the initial symptoms of AD, a very early AD onset (< or =35 years), and a rapidly progressive cognitive decline. CONCLUSIONS: The absence of the PS1 Ser169Pro mutation in the general population and in sporadic AD cases together with its detection in the affected members of this kindred suggests that it is a pathogenic mutation. The serine to proline change predicts a kink in the alpha-helix of the transmembrane domain of the PS1 protein that could radically disrupt its normal structure. Further characterization of the effect of this mutation could help identify the function of the PS1 protein and the pathogenic mechanisms of AD.

Prevalence of the Cys282Tyr and His63Asp HFE gene mutations in Spanish patients with hereditary hemochromatosis and in controls.

BACKGROUND/AIMS: A mutation (Cys282Tyr) of the HFE gene has recently been reported to be present in most of the patients with hereditary hemochromatosis of Northern European ancestry, but in a lower frequency in Italy. No data are so far available on the prevalence of these mutations in Spain. Therefore, we initiated the present study to determine if the reported Cys282Tyr HFE mutation is also the main cause of hereditary hemochromatosis in Spain. In addition, we investigated the presence of the His63Asp HFE mutation in patients and in controls. METHODS: Thirty-one hereditary hemochromatosis patients and 485 controls were screened for the Cys282Tyr and the His63Asp mutations, using polymerase chain reaction amplification of genomic DNA, followed by digestion with the restriction enzymes Rsa I or Dpn II, respectively, and the separation of the products by electrophoresis. RESULTS: Twenty-seven out of 31 (87.1%) hereditary hemochromatosis patients were homozygous for the Cys282Tyr mutation. None of the patients was homozygous for the His63Asp mutation, and two patients (6.5%) were compound heterozygous (Cys282Tyr/His63Asp). Only one of 512 (0.2%) controls was homozygous for the Cys282Tyr mutation, and 29 (5.7%) were heterozygous. The Cys282Tyr mutation is present with an allelic frequency of 90.3+/-7.5% in patients with hereditary hemochromatosis and 3.0+/-1.1% in controls. Twenty out of 487 (4.1%) controls were His63Asp homozygous, while 171 (35.1%) were heterozygous. The His63Asp mutation is present with an allelic frequency of 21.7+/-2.7% in controls. CONCLUSIONS: The high frequency of the Cys282Tyr mutation in hereditary hemochromatosis patients indicates that this mutation is the most common defect associated with hereditary hemochromatosis in Spain. The finding of some patients with the wild genotype at position 282 suggests the existence of other changes in the HFE gene or in other loci involved in the disease. We have found one of the highest allelic frequencies reported for the His63Asp mutation in our controls (21.7+/-2.7%).

Prevalence of Y chromosome microdeletions in oligospermic and azoospermic candidates for intracytoplasmic sperm injection.

OBJECTIVE: To determine the prevalence and type of Y chromosome microdeletions in 136 consecutively seen intracytoplasmic sperm injection (ICSI) candidates and in 50 consecutively seen azoospermic men attending an infertility clinic. DESIGN: Controlled clinical study. SETTING: Genetics laboratory and infertility clinic at a University hospital. PATIENT(S): One hundred eighty-six men who were seen at an infertility clinic and who were referred to a genetics counseling service for genetic assessment before ICSI. INTERVENTION(S): Collection of semen and blood samples. MAIN OUTCOME MEASURE(S): Semen analysis; serum FSH, LH, and T levels; karyotype analysis; and presence or absence of several single tagged site markers along the Y chromosome (sY274, sY238, sY276, sY84, sY102, sY143, sY153, sY254, sY269, sY202, sY158, sY160). RESULT(S): Yq chromosome microdeletions were detected in 10 (5.4%) of 186 consecutively seen ICSI candidates. The number of microdeletions was much higher in azoospermic patients (16%; 8 of 50) than in oligospermic patients (1.5%; 2 of 136). Two of the azoospermic patients with a Yq microdeletion also had sex chromosome aneuploidy mosaicism. No microdeletions were detected in 100 consecutively seen fathers who were included as controls. CONCLUSION(S): The prevalence of Yq microdeletions in the azoospermic group was much higher than in the oligospermic group and was consistent with the prevalence of Yq microdeletions detected in other series of azoospermic men in different geographic areas. All Yq microdeletions found in our patients belong to the AZFc region, indicating that microdeletions of the AZFa and AZFb regions are infrequent among oligospermic ICSI candidates or azoospermic males in our population.

Molecular, cytogenetic, and clinical characterisation of six XX males including one prenatal diagnosis.

Cytogenetic analysis, fluorescent in situ hybridisation (FISH), and molecular amplification have been used to characterise the transfer of Yp fragments to Xp22.3 in six XX males. PCR amplification of the genes SRY, RPS4Y, ZFY, AMELY, KALY, and DAZ and of several other markers along the Y chromosome short and long arms indicated the presence of two different breakpoints in the Y fragment. However, the clinical features were very similar in five of the cases, showing a male phenotype with small testes, testicular atrophy, and azoospermia. All these patients have normal intelligence and a stature within the normal male range. In the remaining case, the diagnosis was made prenatally in a fetus with male genitalia detected by ultrasound and a 46,XX karyotype in amniocytes and fetal blood. Molecular analysis of fetal DNA showed the presence of the SRY gene. FISH techniques also showed Y chromosomal DNA on Xp22.3 in metaphases of placental cells. To our knowledge, this is the second molecular prenatal diagnosis reported of an XX male.

Significant changes in the tau A0 and A3 alleles in progressive supranuclear palsy and improved genotyping by silver detection.

BACKGROUND: Progressive supranuclear palsy (PSP) is characterized by intraneuronal inclusions of neurofibrillary tangles formed by aggregated tau protein. A significant association between the tau gene A0/A0 genotype and PSP recently has been reported. OBJECTIVES: To determine if a significant association between the tau gene A0/A0 genotype and PSP could be found in an independent population with a genetic background different from that in which the initial association was reported, and to standardize a nonradioactive method for tau gene genotyping. SETTING: Hospital and university research laboratories. SUBJECTS AND METHODS: To facilitate genotyping of the tau gene, we standardized the conditions for silver-based detection of the tau gene dinucleotide polymorphism. Thirty patients from Spain clinically diagnosed as having probable PSP were included in the study and compared with different control groups. RESULTS: A highly significant overrepresentation of the A0/A0 genotype (P<.001) and a decrease in the frequency of the A0/A3 genotype were found in the Spanish patients with PSP compared with the control group. A method based on silver detection was standardized for the genotyping of the tau gene. CONCLUSIONS: The detection of a significant association between the tau gene A0/A0 genotype and PSP in 2 independent populations rules out genetic stratification as an explanation for the association and indicates that the presence of the tau A0/A0 genotype is a risk factor for developing PSP independent of genetic background. Alternatively, the results could be interpreted as a protective effect of the A3 allele.

Cytogenetic studies in fetal blood.

In order to assess the effectiveness and reliability of cytogenetic diagnosis provided by fetal blood, we report the first 186 cases of fetal blood sampling performed for rapid karyotype between 19-37 weeks of pregnancy in our Prenatal Diagnosis Unit. The overall diagnostic success rate was 98%, achieving 100% in the last period of the study. Chromosomal anomalies were detected in 16% (29/182) of the fetuses. In malformed fetuses this rate increased from 8-9% in isolated malformation or markers of aneuploidy to 50% in multiple malformations. In pregnancies in which a previous cytogenetic study in amniotic fluid was inconclusive, fetal blood made it possible to obtain a definitive result, with no discrepancies found at phenotypic follow-up examination. Interestingly enough, one of the four previously defined as pseudomosaicisms was found to be a non-mosaic in fetal blood, and only 1 of 4 mosaicisms was confirmed in fetal blood. In conclusion, cytogenetic analysis of fetal blood samples appears to be effective, rapid and reliable to establish the fetal karyotype in selected cases.