Variant maple syrupurine disease in mother and daughter

Intermittent MSUD in a mother and her daughter is reported. Fibroblast cultures were studied for branched-chain keto acid decarboxylase and results show that the mother has approximately 12% while the daughter has 5% of the normal enzyme activity. Other key members in the family were also studied for enzyme activity. It appears that the child has inherited an abnormal gene from her homozygous mother and another abnormal gene from her heterozygous father.A classification based on the degree of residual enzyme activity and protein tolerance places the mother in grade III and the daughter in grade II category. Classical MSUD, where the enzyme activity is less than 2% of normal, belongs to grade I.     

G6PD lozere and trinacria-like

Summary Two new G6PD variants have been found in red blood cells of the members of a French family originating from Lozere. The father is hemizygous for an electrophoretically fast variant with mild enzyme deficiency (50–60% of normal). The abnormal paternal G6PD gene is segregating in his daughter who is double heterozygous for maternal and paternal variants. This mutant enzyme, different from previously described variants is designated as Gd Lozère. The mother is heterozygous for another G6PD variant. Two sons are hemizygous for this latter mutant enzyme characterized by a moderate deficiency (25–30% of normal) and slower electrophoretic mobility with some slightly altered kinetic properties. This G6PD has been identified as Gd Trinacria like.These two abnormal enzymes are not associated with any hemolytic problem. Case reported is the first showing the segregation of two new mutant enzymes, distinct from common G6PD variants, among the members of the same family.     

Adenosine deaminase deficiency due to heterozygous abnormality consisting of a deletion of exon 7 and the absence of enzyme mRNA

An adenosine deaminase (ADA;EC 3.5.4.4)-deficient B lymphoblastoid cell line BADO5 derived from a Japanese patient with severe combined immunodeficiency disease and two B lymphoblastoid cell lines, BAMO5 from his mother and BAFO5 from his father, were characterized. To identify mutations affecting ADA activity, we prepared cDNAs to ADA mRNAs of the BADO5 cell line for nucleotide sequencing. Sequence analysis of one of the BADO5 ADA cDNA clones revealed deletion of exon 7, and one point mutation of base 629 from G to A that did not affect the amino acid sequence. All clones of the BADO5 cell line so far examined showed the absence of exon 7 by Southern blotting analysis. Ribonuclease protection assay with an RNA probe spanning from exon 5 to exon 11 showed that the BADO5 ADA mRNA had a deletion of exon 7, the BAMO5 mRNA had normal length, and the BAFO5 mRNA had two species with a deletion of exon 7 and with normal length. Consequently, the patient's ADA genes resulted from one allele of the BAMO5 ADA gene that did not produce a detectable mRNA, and the other allele of the BAFO5 ADA gene producing an aberrant mRNA without exon 7.     

An unusual case of blood group ABO inheritance: O from AB X O

An unusual blood group inheritance, that is, a phenotype O child from AB X O parents, was found in a Japanese family. Since two other children from the parents are blood type B, this is not a case of Cis-AB inheritance. The mother is not blood A/B chimera, and normal levels of blood group N-acetylgalactosaminyltransferase (A-enzyme) and galactosyltransferase (B-enzyme) were detected in her plasma. Therefore, the mother is genetically true AB heterozygous. The two sons with phenotype B had normal levels of plasma B-enzyme, but had no A-enzyme, and the father and the daughter with phenotype O had neither A- nor B-enzyme in their plasma. The analyses of 24 genetic marker systems indicated that the O daughter was a true child of the parents. The affirmative probability of parentage on the O daughter was calculated to be .9999999917 by Bayes' theorem. We concluded that the genotype of the O daughter was not the usual 00, and that this rare O expression might be due to a new structural mutation or a deletion in either maternal A or B gene during oogenesis.     

A large deletion causes apparent homozygosity for the D1152H mutation in the cystic fibrosis transmembrane regulator (CFTR) gene

We report the case of a patient with an apparent homozygosity for the D1152H mutation located in exon 18 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The parents had no personal history of cystic fibrosis (CF) and referred to our laboratory after the diagnosis of fetal bowel hyperechogenicity. The proband presented with meconium ileus and normal sweat chloride test. Sequencing of the CFTR exon 18 together with quantitative genomic assays, such as real-time PCR and the multiplex ligation probe amplification (MLPA) techniques, were performed and revealed that the father was heterozygous for the D1152H mutation and the mother carried a large deletion of the CFTR gene encompassing the genomic sequence including the same mutation. The child inherited D1152H from his father and the large deletion of the CFTR gene from his mother. We suggest that D1152H likely acts as a mild mutation with a dominant effect on the severe deletion of exon 18, considering that after 3 years of clinical examinations the child shows no classical signs and symptoms of CF. Not testing for large deletions in subjects with apparent homozygosity for a mutated CFTR allele could lead to the misidentification of CFTR mutation carrier status.     

Deletion of chromosome region 13q14 is transmissible and does not always predispose to retinoblastoma

Summary During routine screening of retinoblastoma patients for esterase D activity in red blood cell lysates a patient was identified with only 50% of normal enzyme activity. Chromosome analysis showed that this patient had a small deletion within chromosome region 13q14. Parental studies showed that, whereas the father had normal enzyme levels, the mother had esterase D levels which were also 50% of normal and a similar small 13q14 deletion. Ophthalmological examination failed to demonstrate any retinal abnormality in either parent. Thus wer present the first case not only of the direct transmission of a 13q14 deletion within a family but also of an individual in whom the deletion has not predisposed to tumour formation.     

FMR1 gene deletion/reversion: a pitfall of fragile X carrier testing

The Fragile X syndrome is, in the majority of cases, caused by CGG trinucleotide amplification within the FMR1 gene. The syndrome is rarely caused by point mutations or deletions. Here we describe a family with 2 sons and 1 daughter affected by Fragile X syndrome and 2 unaffected daughters whose carrier status was unknown prior to this study. Analysis of DNA from each of the 2 daughters revealed two alleles in the normal size range. However, 1 daughter carried one allele of 10 CGG repeats that was not present in either the mother or the father. No evidence for mosaicism could be detected. Haplotype analysis of flanking polymorphic markers revealed that the 10 CGG allele was derived from the mutated allele inherited from the mother. Thus, this case most likely represents an additional case of a reverse mutation from a premutation allele in a female to a normal-sized allele in the offspring. It remains unclear how frequently such reversion events occur. The observation has important consequences for genetic testing, because many laboratories prescreen for the Fragile X syndrome by determining the length of the CGG repeat using PCR. If this shows alleles in the normal size range, a diagnosis of Fragile X syndrome is considered to be excluded. Because the routine PCR and/or Southern blot analyses alone may yield false-negative results in cases of a regression of the number of CGG repeats, we strongly recommend the inclusion of fragment length or haplotype analysis when determining the carrier status within Fragile X syndrome families.     

The Finnish type of the LDL receptor gene mutation: molecular characterization of the deleted gene and the corresponding mRNA

In one third of Finnish patients with the heterozygous form of familial hypercholesterolemia the disease is due to a gross deletion at the 3'-end of the LDL receptor gene. The present study demonstrates that an 8-kb deletion completely eliminates exons 16 and 17 and a part of exon 18. Cloning and partial sequencing of a DNA fragment from the mutated allele indicated that the 5'-boundary of the deletion lies within intron 15 while the 3'-breakpoint is located at nucleotide 3390 in exon 18. RNA blot hybridization studies revealed that the mutated allele encodes a truncated 4.2 kb mRNA (normal, 5.3 kb). This type of mutation has not been reported in other ethnic groups.     

Glucose phosphate isomerase deficiency with hereditary hemolytic anemia in a Spanish family: clinical and familial studies.

A new case of glucose phosphate isomerase deficiency associated with cogenital nonspherocytic hemolytic anemia is described in a 12-year-old girl of Spanish origin. The parents exhibited erythrocyte glucose phosphate isomerase activity between 50 and 60% of normal. The enzyme of the propositus had normal Michaelis-Menten constants both for F-6-P and G-6-P, but abnormal pH optimum and decreased heat stability at 48 degrees C. On starch-gel electrophoresis the father's enzyme was normal but the mother's showed a cathodic migrating band in addition to the normal one. The enzyme from the propositus exhibited only one band with cathodal mobility of 116% of the main band found in normal subjects. It is postulated that the propositus is double heterozygous for two abnormal alleles, and the mother contributes a mutant allele with abnormal electrophoretic mobility and thermolability at 48 degrees C whereas the father contributes an allele without enzymatic activity.     

Mutational analysis of a patient with mucopolysaccharidosis type VII, and identification of pseudogenes.

PCR of cDNA produced from patient fibroblasts allowed us to determine the paternal mutation in the first patient reported with beta-glucuronidase-deficiency mucopolysaccharidosis type VII (MPS VII). The G-->T transversion 1,881 bp downstream of the ATG translation initiation codon destroys an MboII restriction site and converts Trp627 to Cys (W627C). Digestion of genomic DNA PCR fragments with MboII indicated that the patient and the father were heterozygous for this missense mutation in exon 12. Failure to find cDNAs from patient RNA which did not contain this mutation suggested that the maternal mutation leads to greatly reduced synthesis or reduced stability of mRNA from the mutant allele. In order to identify the maternal mutation, it was necessary to analyze genomic sequences. This approach was complicated by the finding of multiple unprocessed pseudogenes and/or closely related genes. Using PCR with a panel of human/rodent hybrid cell lines, we found that these pseudogenes were present over chromosomes 5-7, 20, and 22 and the Y chromosome. Conditions were defined which allowed us to amplify and characterize genomic sequences for the true beta-glucuronidase gene despite this background of related sequences. The patient proved to be heterozygous for a second mutation, in which a C-->T transition introduces a termination codon (R356STOP) in exon 7. The mother was also heterozygous for this mutation. Expression of a cDNA containing the maternal mutation produced no enzyme activity, as expected. Expression of the paternal mutation in COS-7 cells produced a surprisingly high (65% of control) level of activity. However, activity was 13% of control in transiently transfected murine MPS VII cells. The level of activity of this mutant allele appears to correlate with the level of overexpression, suggesting that high concentrations of mutant monomers can drive the folding and tetramerization of mutant enzyme to produce an active and stable enzyme.     

中国人Ⅱ型MPS家系IDS基因的一种新突变的鉴定

为了研究粘多糖贮积症Ⅱ型(MPSⅡ)患者发病的分子遗传学机制, 以便为今后的产前基因诊断等创造必要的前提条件, 文章先采用尿糖胺聚糖(GAGs)定性检测法对疑似MPSⅡ的先证者进行初诊, 然后采用PCR、PCR 产物直接测序法对先证者及其家系成员进行突变检测。在检出IDS基因c.876del2新突变后, 对随机采集的120例正常对照和其他非II型MPS患者包括MPSⅠ, Ⅳ, Ⅵ三型的病人共15例的IDS基因exon 6进行序列分析, 同时采用不同物种突变点序列的保守性分析法, 以及直接测定患儿及其家庭相关成员IDS酶活性的方法对该新突变进行致病性分析。结果显示: 先证者尿检呈强阳性(GAGs +++); 其IDS基因exon 6编码区内存在c.876-877 del TC新缺失突变, 为半合子突变, 而其母、其姐为杂合突变; 正常对照和其他非II型MPS患者的IDS基因exon 6的检测结果均未发现该突变; 不同物种氨基酸序列的同源性比对显示: c.876-877 del TC突变所在的位置即p.292-293的苯丙氨酸(F)谷氨酰胺(Q)高度保守; 酶活性测定的结果显示: 先证者的IDS酶活性仅为2.3 nmol/4 h/mL, 大大低于正常值, 而其父的为641.9 nmol/4 h/mL, 其母的血浆酶活性为95.8 nmol/ 4h/mL, 其姐的为103.2 nmol/4 h/mL。说明所发现的c.876-877 del TC缺失移码突变是一种新的病理性突变, 是该MPSⅡ患儿发病的根本内因。     

Analysis of the red cell membrane in a family with hereditary elliptocytosis — total or partial of protein 4.1

Summary In a 12-year-old boy carrying a clinically silent elliptocytosis, we observed a total lack of red cell membrane band 4.1. Band 4.1 was partially absent in the father who also displayed a clinically silent elliptocytosis and, remarkably, in the mother although she presented normal discocytes. Band (2 and 2.1) phosphorylation was sharply reduced in the three persons examined. In the propositus and his mother, but not in his father, a clearly phosphorylated band appeared at the level of band 4.2. We suggest that the father and the mother carry two distinct alleles affecting differently the interactions, within the spectrin-actin protein 4.1 complex. The father's allele is elliptocytogenic in the heterozygous state and, among other molecular alterations, prevents the attachment of protein 4.1. The mother's allele is morphologically silent in the heterozygous state, yet it also affects the binding of protein 4.1, possibly because the latter is shortened. The propositus, being doubly heterozygous, has the same morphological phenotype as his father, but his protein 4.1 electrophoretic phenotype is the addition of both parental phenotypes. The distinct phosphorylation patterns in the region of bands 4.1 and 4.2 are also consistent with the two-allele hypothesis.     

The ornithine aminotransferase gene in gyrate,atrophy of the retina: Analysis of expression and gross structure of this gene in cultured fibroblasts

Summary Gyrate atrophy (GA), a degenerative disease of the human chorioretina, is associated with a deficiency of ornithine aminotransferase (OAT) activity, hyperornithinemia, and ornithinuria. We have characterized a cDNA clone for OAT (HLOAT) that was isolated from a cDNA library constructed from mRNA prepared from Hep G2, cells, a human hepatoma cell line. We have used HLOAT and a nearly full length OAT cDNA clone isolated from, a rat liver library (RLOAT) to examine in cultured fibroblasts from individuals with GA and control individuals, the expression of OAT mRNA and the gross structure of the OAT gene. Northern blot analyses of total cellular RNA indicated that 3 of 3 control cell lines and 5 of 6 GA cell lines are capable of expressing an OAT related mRNA of approximately 2100 bases, the size of OAT mRNA. To date, this is the only case of GA in which a complete lack of OAT mRNA has been observed. Southern blot analyses of DNA isolated from these cell lines indicated that the gross structure of the OAT gene is usually not detectably altered in individuals with GA. However, a unique pattern, of restriction fragments was observed upon digestion with Eco RI or Hind III of DNA from the GA cell line that does not express OAT mRNA. These unique Eco RI and Hind III fragments arise from the OAT structural gene and will serve as useful molecular markers that allow this particular defective OAT allele to be identified. When the cellular DNAs were digested with Hinf I and examined with a probe that corresponds to at least a portion of the active site of the enzyme, i. e., the pyridoxal phosphate binding site, identical patterns of fragments were detected in all samples. Therefore, it appears unlikely that the loss of OAT activity associated with these GA cases, 4 of which are pyridoxal phosphate responders, is the result of insertions or deletions in this region of the OAT gene. This study indicates that the lack of OAT enzyme activity associated with GA is the result of a variety of different molecular defects within the OAT gene. This project was initiated in the laboratory of H. C. P. and was supported by grants CA07175, CA22484, and 5 T32 CA09020 from the National Cancer Institute and Postdoctoral Fellowship PF-2414 from the American Cancer Society. The continuing work in the laboratory of J. D. S. was supported by grants CA36727 and HD24189 from the National, Institutes of Health, grants SIG-16, ACS-IN165A, and a Junior Faculty Research Award (JFRA-227) from the American Cancer Society, and by University of Nebraska Medical Center Seed Research Grant 88-10.     

Molecular, immunological, enzymatic and biochemical studies of coproporphyrinogen oxidase deficiency in a family with hereditary coproporphyria.

A 27-year-old woman who had recurrent pain in renal bed since 1998 with increasing character, was stationary admitted. The patient showed dark urine, complained of hair loss and took since 1994 a hormonal oral contraceptive. No photosensitivity was observed. Determinations of urinary porphyrin metabolites in 1998 revealed a porphyria cutanea tarda like excretion pattern with elevations of uro- (1767 nmol/24 hr, normal <29 nmol/24 hr) and heptacarboxyporphyrin (568 nmol/24 hr; normal <4 nmol/24 hr). Follow-up studies in feces showed the characteristics of a hereditary coproporphyria with dominance of coproporphyrin isomer III (total= 1470 nmol/g, isomer III= 93%), (normal: <37 nmol/g, isomer III = 25-35%). The excretion of porphyrin precursors (delta-aminolevulinic acid and porphobilinogen) was increased by taking an ethinylestradiol-cyproteronacetate-preparation, but acute and/or chronic manifestations were not observed. Coproporphyrinogen oxidase activity was decreased to 35% in the patient (normal=138+/-21 pkat/g protein; x+/-s), whereas the activity of red cell uroporphyrinogen decarboxylase was normal. Her mother and both sisters could be verified as heterozygous gene carriers of hereditary coproporphyria by their urinary and fecal excretion parameters and because of reduced coproporphyrinogen oxidase activity up to 50%. The father was normal with respect to his genotype. Molecular analysis revealed a hitherto unknown mutation with the transversion of a cytosine to thymine at nucleotide position 854 in exon 4 of the coproporphyrinogen oxidase gene. The gene defect was confirmed by DGGE in the mother and her three daughters. The investigation of the immunological nature of the defective coproporphyrinogen oxidase gene from the whole family revealed decreased concentrations of coproporphyrinogen oxidase protein in the patient, her mother and her two sisters.