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Mitochondrial disorders
DiMauro S., Tanji K.
In this minireview, we attempt to survey the three main group of mitochondrial disorders, defects of nuclear DNA, defects of mitochondrial DNA, and defects of intergenomic signaling, with emphasis on recent contributions and pathogenetic mechanisms. In so doing, we have tried to point out some of the numerous unsolved problems in genotype/phenotype correlation and to indicate future directions of research.
Direct insertion of euchromatic material from chromosome y in the x-chromosome in hypogonadotropic hypogonadisms with crohn’s disease
Ramesh K.H., Qazi Q.H., Verma R.S.
The relationship between chromosomal abnormalities and Crohn’s disease has not been established. Crohn’s disease is associated with inflammation of the bowel, severe abdominal pain and chronic diarrhea. Its etiology is not known at present. A recessive gene with incomplete penetrance is thought to be a factor which does not follow simple mendelian inheritance. We report a case, where the euchromatin material of Y chromosome (p11.1 p11.2) has been directly inserted into the long arm of the X chromosome (q21.2), and is assumed to be the most likely cause of hypogonadotropic hypogonadism in this patient. It could also be that the function of the testis-determining factor (SRY) has been disrupted due to the insertion, causing loss of testicular development.
Molecular characterization of an unusual variant of the short arm of chromosome 15 by FISH-technique
Verma R.S., Kleyman S.M., Conte R.A.
One of the most frequent translocations involving the long arm of chromosome Y with autosomes is with the short arm of chromosome 15. The regions which are involved in this translocation fluoresce brightly, are highly heteromorphic and thus escape detection. Therefore, these abnormalities could not be fully characterized, especially in cases where parents are not available or paternity is disputed. Results from the employment of the selective staining techniques DA/DAPI and Q-banding have been inconclusive. FISH-technique using whole chromosome painting (WCP) probes should be used to decipher such translocations. We present a case where, even after using a battery of probes, the origin of extra material on chromosome 15p could not be identified though it was not a part of Yq.
Retrieval of aneuploidy by fish-technique in a case with 46,XX/47,XXX/47,XX,+8
Verma R.S., Gogineni S.K., Kleyman S.M., Mann D.N.
We report on a 46 year old female with a new chromosomal finding [46,XX/47,XXX/47,XX,+8] who was referred for ovarian failure. The clinical presentation was highly unusual and the patient does not exhibit the characteristic phenotype of trisomy 8 syndrome. Interphase cytogenetics using FISH-technique revealed discrepancies with a different population of cells when compared with its metaphase index. Therefore, it is advised that patients with mosaic karyotypes should be evaluated by analyzing metaphase as well as interphase nuclei labeled with chromosome specific molecular tags, especially in the situations where the incidence of a mosaic cell line is very low. Nevertheless, in a cost-conscious environment, we must exercise caution prior to making universal recommendations concerning the usefulness of medical devices which are increasing at a logarithmic rate.
Linkage and haplotype analysis of familial early-onset Alzheimer disease in Japanese population
Kamino K., Nagano K., Katsuya T., Nishiwaki Y., Takeda M., Tanabe H., Nishimura T., Ii K., Fujimoto K., Tsujimura R., Nonomura Y., Yoneda H., Sakai T., Nakajima T., Imagawa M., et. al.
Linkage and haplotype analysis of eleven early-onset Alzheimer disease (AD) families was performed in relation to D21S210 and microsatellite DNA polymorphisms localized on chromosome 14q24.3. Linkage analysis of eight informative families out of eleven early-onset AD families disclosed the highest LOD score of 3.45 (θ=0.00) at D14S77, while the locus of β/A4 amyloid protein precursor gene was formally excluded within 10 cM from D21S210, given the evidence of recombinations in five families. Transmission disequilibrium study between the patients and controls without dementia indicated significant differences at D14S43 (p=0.0001) and D14S71 (p=0.02). Association study between genotypes linked or related to onset of AD and those of control also revealed a significant difference at D14S43 (p<0.05), suggesting the existence of linkage disequilibrium. Moreover, the haplotypes at D14S43 linked with the onset of AD indicated a significant relationship with the mean age at onset. These results support that the major locus of earlyonset familial AD is located on 14q24.3, and its close linkage to D14S43 and the existence of allelic heterogeneity were suggested.
An apparent balanced translocation between chromosomes 7 and 13 [t(7;13) (p15;q32)] in a 47,XYY individual
Verma R.S., Giridharan R., Conte R.A., Luke S.
A Y-associated allele is shared among a few ethnic groups of Asia
Lin S.J., Tanaka K., Leonard W., Gerelsaikhan T., Dashnyam B., Nyamkhishig S., Hida A., Nakahori Y., Omoto K., Crawford M.H., Nakagome Y.
In our previous study, both of Y-associated alleles, Y1 and Y2, were detected in Japanese and Koreans, but only the Y1 allele was detected in each of other populations including Chinese in both Beijin and Guangzhou areas, Caucasians, Africans, and Jewish. In the present study, these observations were extended to other ethnic groups in East Asia. Evenks in central Siberia and Khalkhs in Mongolia had only the Y1 allele. On the other hand, two ethnic groups, Fo-lo and Hakka, in Taiwan had both of the Y1 and the Y2 alleles. Three of the eight Y2-positive men, 2 Fo-lo and a Hakka, shared family name Chen. Both Hakka people and ancesters of Chen families could be traced to the Province of Henan in northern China in early 4th century. They arrive din Fujian/Guangdong area in the south-east Chinavia various routes and then some of them migrated to Taiwan in the 18th century. It is tempting to speculate that the Y2 allele may be originated from an ancestral population in Henan from which, Japanese, Koreans, and some of the Taiwanese diverged.
Fluorescencein situ hybridization analysis of chromosomal localization of three human cytochrome P450 2C genes (CYP2C8, 2C9, and 2C10) at 10q24.1
Inoue K., Inazawa J., Suzuki Y., Shimada T., Yamazaki H., Guengerich F.P., Abe T.
Chromosomal localization of three human cytochrome P450 genes belonging to the CYP2C subfamily (CYP2C8, 2C9, and 2C10) was identified by fluorescencein situ hybridization (FISH). An original MP-8 clone was used as a DNA probe for the assignment of the CYP2C10 gene, while two cDNA probes, a 1.37 kb fragment of CYP2C8 and a 1.19 kb fragment (MP-20 and MP-4 clones, respectively) by polymerase chain reaction using a single human liver cDNA library. The results showed that three human CYP2C8, 2C9, and 2C10 cDNAs were located at the same subchromosomal region, 10q24.1.
Genetic services in the United States
Chen H., Wertelecki W.
Advances in medical genetics, including mapping of human genome, improved therapy for genetic disorders, and new screening tests for carrier detection and prenatal diagnosis, have created a growing demand for clinical genetic services in the United State. Such services (diagnosis, management, and genetic counseling) received support from state, federal, and private sources and were mostly based in academic medical centers. Gradually, such programs evolved into regional or state-wide activities with an emphasis on outreach clinics. Now, an increasing number of for-profit corporations have entered into this field. Clinical genetic teams usually include clinical geneticists and other professionals with expertise in the diagnosis and management of genetic conditions and skills in information presentation and family support. The American Board of Medical Genetics, the newest member of the American Board of Medical Specialties, provides certification for five categories of genetics professionals and sets standards for training programs. Based on personal experiences from the states of Alabama and Ohio and data from the Council of Regional Networks for Genetic Services, we show and compare trends of newborn screening programs and regional genetic services. The effects of economic and social trends as they impinge on genetic services are monitoredvia several databases in our center.
Isolation and characterization of a DNA fragment containing various kinds of repetitive sequences located on human chromosome 21
Yao R., Patterson D., Onodera K.
In order to investigate the repetitive sequences located on human chromosome 21, we have isolated DNA fragments containing Alu sequences. One of the clones, p1, was chosen for further study, because it contained repetitive sequences different from the Alu sequence. Nucleotide sequence analysis of p1 indicates that p1 contains L1 and O-family sequences. Interestingly, when the L1 sequence was used as a probe, a discrete band of 5 kb was seen in HindIII-digested DNA from somatic cell hydbrids containing human chromosome 21 as the sole human chromosome. The L1 sequence was rearranged and was interrupted by O-family sequence, which wasd flanked by 6 bp target site dup litations. Since all three repetitive sequences are known to act as retroposons, these results imply that there is an integration hot spot on human chromosome 21. The sequence was mapped within 21q11–21.
A linkage study with DNA markers (D4S95, D4S115, and D4S111) in Japanese Huntington disease families
Watanabe M., Kondo I., Nissato S., Wakisaka A., Toda T., Ikeda J., Wasmuth J.J., Gusella J.F., Kanazawa I.
Attempts to isolate the Huntington disease (HD) gene based on its position have been frustrated by apparently contradictory recombination events in HD pedigrees that have predicted two non-over-lapping candidate regions: 100 kb at the telomere of the short arm of chromosome 4, and a 2.2 Mb region located internally at 4p16.3. The proximal location is also supported by the detection of a linkage disequilibrium between HD and some restriction fragment length polymorphisms (RF-LPs) at the D4S95, D4S98, and D4S127 loci. In the present study, a proximal marker D4S95 showed tight linkage to the disease locus in Japanese pedigrees (Zmax=3.31,θ max=0.00), while distal markers D4S115 and D4S111 did not. Particularly, a two point linkage analysis between D4S111 and HD yielded a lod score −2.01 for θ=0.015. This result leads to the exclusion, as a possible region of localization of the HD gene, of more than 3 cM of the genome around D4S111 locus. At the same time our results favor aforementioned proximal location as a candidate location for the HD gene.
Assignment of the human cytochrome P-450 nifedipine oxidase gene (CYP3A4) to chromosome 7 at band q22.1 by fluorescencein situ hybridization
Inoue K., Inazawa J., Nakagawa H., Shimada T., Yamazaki H., Guengerich F.P., Abe T.
We have used a full length cDNA clone (2.2 kb) for the human cytochrome P-450 nifedipine oxidase (CYP3A4) enzyme as a probe to determine its chromosome localization by fluorescencein situ hybridization. CYP3A4 was mapped on R-banded human prometaphase chromosomes, and the precise localization of CYP3A4 on chromosome 7 was further confirmed by a delineation of G-banded pattern on the same prometaphase chromosomes through a combination of UV-filter. We assigned CYP3A4 to chromosome 7 at q22.1.
Yeast artificil chromosome (YAC) clones and sequence tagged site (STS) markers anchored at human chromosome 21
Eki T., Yokoyama K., Tashiro H., Ozawa K., Murakami Y., Watkins P.C., Soeda E.
Isoelectric focusing studies in Brazilian Indians —uncovering variation of ORM, AHSG and IF—
Salzano F.M., Umetsu K., Yuasa I., Black F.L., Suzuki T.
Allele frequencies for the orosomucoid 1 (ORM1), orosomucoid 2 (ORM2), alpha-2-HS-glycoprotein (AHSG) and complement component I (IF) loci were studied in 393 individuals of three Brazilian Indian tribes. In the ORM1 locus only two alleles were observed among the Urubu-Kaapor, while five were found among the Pacaás Novos. The frequency ofORM1 *1 was similar in these two tribes (0.734 and 0.715, respectively) but departed more markedly among the Parakanã (0.870). Variation for ORM2 locus was found among the Pacaás Novos only, withORM2 *3 being observed in just three individuals. A new variant (AHSG * PN) was found in the AHSG system. Frequency forAHSG *1 was unexpectedly low in the three tribes, especially, among the Pacaás Novos, where the prevalence (0.145) is the lowest considering other data reported thus far. For IF locus, variability was also restricted to only one trible (Urubu-Kaapor) and attributed to a new polymorphic allele,IF * A3.
Isolation of A Y chromosomal DNA sequence and its clinical application
Tsukahara M., Matsuura S., Kishi F., Yoshida A., Kajii T.
A 4.6 kb long, Y-specific DNA fragment was isolated from a flow-sorted human Y chromosomal library, and its male specificity was confirmed by Southern blot analysis. The fragment, designated as pY-80, was proven with anin situ hybridization experiment to have originated from the Yp11.2-Ypter region. Its 2,808 bp section was sequenced. The polymerase chain reaction proceeded with oligonucleotides flanking a 666 bpPstI-EcoRI fragment of the sequence as primers and a male genomic DNA as a template, but not with a female genomic DNA. Preliminary tests of samples of various sources successfully detected the Y-specific fragment in male-derived samples, including mouth wash, single hair roots, urinary epithelial cells, dried blood spots and amniotic fluid cells.
Assignment of the vascular smooth muscle actin geneACTSA to human chromosome 10
Ueyama H., Bruns G., Kanda N.
Human vascular smooth muscle actin gene (ACTSA) was cloned and its unique sequence was used as the hybridization probe for Southern blot analysis of DNAs from 18 rodent-human somatic cell hybrids; the gene was assigned to human chromosome 10. Regional mapping byin situ hybridization showed that the gene is located on the long arm (q22–q24) of the chromosome. Thus, the gene is on a different chromosome from the other four actin genes so far examined.
Human type II collagen gene (COL2A1) assigned to chromosome 12q13.1-q13.2 byin situ hybridization with biotinylated DNA probe
Takahashi E., Hori T., Lawrence J.B., McNeil J., Singer R.H., O'Connell P., Leppert M., White R.
We have made a regional assignment of the type II collagen gene (COL2A1) on human chromosome 12 by means of anin situ hybridization technique with a biotinylated DNA probe. The precise localization of the signal was mapped to the band 12q13.1-q13.2. This result was in agreement with the previous mapping by isotopicin situ hybridization technique (12q13.1-q13.2), but not with the result of Southern hybridization analysis using somatic cell hybrids (12q14.3).
Congenital adrenal hyperplasia in monozygotic twins with variable clinical manifestations
Kanjilal D., Verma R.S., Glass L., Babu A., Ramazanoglu F., Popescu S.
The first cases of congenital adrenal hyperplasia III with variable clinical manifestations in female monozygotic twins are presented. Twin “A” revealed severe hypertrophy of the clitoris, labial fusion and a visible introitus. However, twin “B” manifested moderate clitoral hypertrophy, a visible introitus and no labial fusion. Neither infant had palpable gonads.
Assignment of the myeloperoxidase geneMPO to human chromosome 17 using somatic cell hybrids and flow-sorted chromosomes
Kudoh J., Minoshima S., Hashinaka K., Nishio C., Yamada M., Shimizu Y., Shimizu N.
A cDNA coding for human myeloperoxidase (MPO) was used as a probe to study MPO gene structure and to determine the chromosomal location of the gene in the human genome. Southern blot hybridization of restriction endonuclease digests of human DNA with the MPO cDNA probe showed that a single gene for human MPO was present in the human genome. Southern blot hybridization experiments with human-mouse cell hybrid DNAs containing various subsets of human chromosomes revealed that the human MPO gene is located on chromosome 17. This conclusion was supported by DNA spot-blot hybridization using flow-sorted human chromosomes.
Determination of chromosomal area of double minute chromosomes and a homogeneously staining region in HL-60 human leukemia cells by the use of a color image analyzer
Misawa S., Abe T., Takamatsu T., Fujita S., Testa J.R.
The surface area of chromosomes was measured in two sublines of HL-60 human leukemia cells using a color image analyzer. One subline, HL-60 (dmin), had double minute chromosomes (dmin) and the other line, HL-60 (HSR), showed an abnormal chromosome #8 (8q+) which contained a homogeneously staining region (HSR) within the long arm at band q24. Dmin were not seen in the latter cells. The area of individual chromosomes analyzed from metaphase plates of Giemsa-stained photographic prints proved to be reasonably accurate and reproducible. Moreover, the relative area of each chromosome correlated well both with the relative length of corresponding chromosomes and the relative DNA content. The results indicate that the mean area of a single dmin represents 0.16% of the total genomic area of HL-60 (dmin) cells. The average number of dmin per cell was 8.14, and the total area of all dmin in a representative cell was 1.32% of the entire genome. The 8q+ chromosome had 1.46 times the area of the normal homologous chromosome #8, and the HSR itself had 1.30% of the total chromosomal area of HL-60 (HSR) cells. Thus, perhaps coincidentally, the relative amount of DNA in the HSR in an HL-60 (HSR) cell was similar to the total found in multiple dmin in HL-60 (dmin) cells.
Unusual staining property of human Y-chromosome
Verma R.S., Rodriguez J., Babu K.A.
Genetic studies of familial amyloid polyneuropathy in the Arao district of Japan
Ueji M., Suzuki T., Higa S., Sakoda S., Kishimoto S., Titani K., Takio K., Hayashi A., Takaba Y., Nakajima A.
The predominant amyloid fibril proteins isolated from kidneys of four patients with familial amyloid polyneuropathy (FAP) from three genealogically independent families in the Arao district of Japan have been analysed for the primary structure. Irrespective of the patient or the family, the major protein isolated consisted of some components of a prealbumin variant, in which an amino acid substitution of methionine for valine occurred at position 30, with a heterogenous N-terminus caused by some degradation of N-terminal amino acids in the prealbumin subunit. It is likely that this prealbumin variant is concerned with the process of this hereditary disease, rather than being a genetic polymorphism of prealbumin. Further, we conclude that the FAP families of the Arao focus may have a common ancestor.
A rapid semi-quantitative determination of phenylalanine, glycine and cystine from urine and phenylalanine from serum
Lee M., Toke D.A., Wang T.
Based on our previously described method of amino acid detection with fluorescamine, we described here a semi-quantitative determination of amino acid concentrations from urine and blood. The method is simple and rapid and is applicable for at least the urinary phenylalanine, glycine, cystine and the serum phenylalanine.
A micro thin layer chromatography technique for urinary and serum amino acids using fluorescamine as a staining reagent
Wang T., Lee M., Toke D.A.
A rapid, sensitive, inexpensive micro technique for thin layer chromatography of amino acids from urine and blood is described. The method which requires only 0.1 μl of sample, uses a fluorescent reagent, fluorescamine, as a staining agent, and commercially available cellulose plates, Polygram Cel 300, as a supporting matrix. The great majority of amino acids can be detected when concentration is greater than 0.25mm. The application of this technique toward the identification of various abnormal amino acid metabolisms is discussed. A method of deproteinization of serum, modified from a previously published technique, is also reported.
Genetics in clinical oncology
Chaganti R.S.
