Targeted extended cystic fibrosis mutation testing on known and at-risk patients and relatives

This paper reports mathematically derived residual risks of being a carrier or being affected with cystic fibrosis following various screening scenarios to assist in interpreting test results and advising patients. While parental screening with 23 American College of Medical Genetics (ACMG) cystic fibrosis mutations defines the 64% of affected U.S. Caucasian fetuses with two detectable mutations, newborn screening for elevated immunoreactive trypsinogen (IRT) and sweat chloride identifies an additional 36% of affected newborns with zero or one detected mutation. The relatives of these affected newborns with less than two detectable mutations have higher posterior (after) 23 mutation-negative test risks of carrying undetected mutations. These calculations emphasize how knowledge of the mutations in the related affected patient substantially improves upon the quality of after-test advice to patients. Furthermore, negative tests of the partner without a family history and/or more extensive cystic fibrosis transmembrane conductance regulator (CFTR) gene testing also increases the likelihood that a negative report is truly negative. When a newborn patient with zero or one detected CFTR mutation has an inconclusive sweat test result, the sweat test should be repeated before ordering additional often unnecessary CFTR gene sequencing. Given the same composite mutation panel test accuracy, a higher proportion of reported test results would be correct during parental screening than when testing at-risk fetuses or symptomatic newborns. Prenatal and newborn screening would be enhanced substantially by medical professionals offering copies of all positive parental and newborn test reports to the parents to share with their relatives. These principles are likely to be applicable to other genetic diseases as the most common mutation frequencies are reported.     

Existence of glucose-6-phosphate dehydrogenase-like locus on chromosome 17.

Hybridization of DNA samples prepared from flow-sorted human chromosomes with a cDNA probe for the X-linked glucose-6-phosphate dehydrogenase (G6PD) suggested the existence of the G6PD-like locus on chromosome 17. Southern hybridization analysis of endonuclease-digested DNA samples from the human-mouse hybrid cell line with human chromosome 17, and from control human and mouse cells, proved that not only X chromosomes, but also chromosome 17, contain DNA sequences that are hybridizable with the G6PD cDNA probe. The G6PD-like locus on chromosome 17 could be a putative pseudogene or a functional gene for the fetal brain-specific G6PD isozyme or other protein.     

Multicolor in situ hybridization and linkage analysis order Charcot-Marie-Tooth type I (CMTIA) gene-region markers.

This study demonstrates a clear and current role for multicolor in situ hybridization in expediting positional cloning studies of unknown disease genes. Nine polymorphic DNA cosmids have been mapped to eight ordered locations spanning the Charcot-Marie-Tooth type 1 (CMT1A) disease gene region in distal band 17p11.2, by multicolor in situ hybridization. When used with linkage analysis, these methods have generated a fine physical map and have firmly assigned the CMT1A gene to distal band 17p11.2. Linkage analysis with four CMT1A pedigrees mapped the CMT1A gene with respect to two flanking markers (8B10-5 cM[LOD 5.2]-CMT1A-3.5 cM[LOD 5.3]-10E4). Additional loci were physically mapped and ordered by in situ hybridization and analysis of phase-known recombinants in CMT1A pedigrees. The order determined by multicolor in situ hybridization was 17cen-LEW301-8B10-5H5/6A9-VAW409- 5G7-6G1-4A11-VAW412-10E4-pter. Two ordered probes, 4A11 and 6G1, reside on the same 440-kb partial SfiI restriction fragment. These data demonstrate the ability of in situ hybridization to resolve loci within 0.5 Mb on early-metaphase chromosomes. Multicolor in situ hybridization also excluded the possibility of pericentric inversions in two unrelated patients with CMT1 and neurofibromatosis type 1. When used with pulsed-field gel electrophoresis, multicolor in situ hybridization can establish physical location, order, and distance in closely spaced chromosome loci.     

Evolutionary implications of the human aldolase-A, -B, -C, and -pseudogene chromosome locations.

The aldolase genes represent an ancient gene family with tissue-specific isozymic forms expressed only in vertebrates. The chromosomal locations of the aldolase genes provide insight into their tissue-specific and developmentally regulated expression and evolution. DNA probes for the human aldolase-A and -C genes and for an aldolase pseudogene were used to quantify and map the aldolase loci in the haploid human genome. Genomic hybridization of restriction fragments determined that all the aldolase genes exist in single copy in the haploid human genome. Spot-blot analysis of sorted chromosomes mapped human aldolase A to chromosome 16, aldolase C to chromosome 17, the pseudogene to chromosome 10; it previously had mapped the aldolase-B gene to chromosome 9. All loci are unlinked and located on to two pairs of morphologically similar chromosomes, a situation consistent with tetraploidization during isozymic and vertebrate evolution. Sequence comparisons of expressed and flanking regions support this conclusion. These locations on similar chromosome pairs correctly predicted that the aldolase pseudogene arose when sequences from the aldolase-A gene were inserted into the homologous aldolase location on chromosome 10.     

Isolation and characterization of Y chromosome DNA probes.

A sorted, cloned Y chromosome phage library was screened for unique Y chromosome sequences. Of the thousands of plaques screened, 13 did not hybridize to radiolabeled 46,XX total chromosomal DNA. Three plaques were characterized further. Clone Y1 hybridized to multiple restriction enzyme fragments in both male and female DNA with more intense bands in male DNA. Clone Y2, also found in female and male DNA, is probably located in the pseudosutosomal region because extra copies of either the X or Y chromosomes increased Y2 restriction enzyme fragment intensity in total cellular DNA. Clone Y5 was male specific in three of four restriction enzyme digests although in the fourth a light hybridizing band was observed in both male and female DNA. Clone Y5 was sublocalized to band Yq 11.22 by hybridization to a panel of cellular DNA from patients with Y chromosome rearrangements. Clone Y5 can be used to test for retention of the proximally long arm Y suggested to cause gonadal cancer in carrier females. The long series of GA repeats in Y5, anticipated to be polymorphic, may provide a sensitive means to follow Y chromosome variation in human populations.     

Mutation analysis in Rett syndrome

Rett syndrome is an X-linked dominant neurodevelopmental disorder caused by mutations in the MECP2 gene. Mutations have been demonstrated in more than 80% of females with typical features of Rett syndrome. We identified mutations in the MECP2 gene and documented the clinical manifestations in 65 Rett syndrome patients to characterize the genotype-phenotype spectrum. Bidirectional sequencing of the entire MECP2 coding region was performed. We diagnosed 65 patients with MECP2 mutations. Of these, 15 mutations had been reported previously and 13 are novel. Two patients have multiple deletions within the MECP2 gene. Eight common mutations were found in 43 of 65 patients (66.15%). The majority of patients with identified mutations have the classic Rett phenotype, and several had atypical phenotypes. MECP2 analysis identified mutations in almost all cases of typical Rett syndrome, as well as in some with atypical phenotypes. Eleven (20.4%) of the 54 patients with defined mutations and in whom phenotypic data were obtained did not develop acquired microcephaly. Hence, microcephaly at birth or absence of acquired microcephaly does not obviate the need for MECP2 analysis. We have initiated cascade testing starting with PCR analysis for common mutations followed by sequencing, when necessary. Analysis of common mutations before sequencing the entire gene is anticipated to be the most efficacious strategy to identify Rett syndrome gene mutations.     

Structural organization of the beta-globin locus of B-haplotype sheep

Goats and some sheep synthesize a juvenile hemoglobin, Hb C (alpha 2 beta C2), at birth and produce this hemoglobin exclusively during severe anemia. Sheep that synthesize this juvenile hemoglobin are of the A haplotype. Other sheep, belonging to a separate group, the B haplotype, do not synthesize hemoglobin C and during anemia continue to produce their adult hemoglobin. To understand the basis for this difference we have determined the structural organization of the beta- globin locus of B-type sheep by constructing and isolating overlapping genomic clones. These clones have allowed us to establish the linkage map 5' epsilon I-epsilon II-psi beta I-beta B-epsilon III-epsilon IV- psi beta II-beta F3' in this haplotype. Thus, B sheep lack four genes, including the BC gene, and have only eight genes, compared with the 12 found in the goat globin locus. The goat beta-globin locus is as follows: 5' epsilon I-epsilon II-psi beta X-beta C-epsilon III-epsilon IV-psi beta Z-beta A-epsilon V-epsilon VI-psi beta Y-beta F3'. Southern blot analysis of A-type sheep reveals that these animals have a beta- globin locus similar to that of goat, i.e., 12 globin genes. Thus, the beta-globin locus of B-haplotype sheep resembles that of cows and may have retained the duplicated locus of the ancestor of cows and sheep. Alternatively, the B-sheep locus arrangement may be the result of a deletion of a four-gene set from the triplicated locus.      

Rare McArdle disease locus polymorphic site on 11q13 contains CpG sequence

Summary When probes throughout the McArdle disease (myophosphorylase) gene region were used to search for DNA polymorphisms, only an MspI polymorphism was found in 94 enzyme-probe combinations. Along with an insertion/deletion polymorphism more 3 to the gene locus, these polymorphisms will be informative in 75% of at-risk patients. These results contrast strikingly to the six polymorphic sites detected in 15 enzyme-probe combinations in the homologous Her's disease (liver phosphorylase) gene region. This single MspI polymorphic site includes a CpG sequence of known increased mutability suggesting that DNA regions with rare polymorphisms will have most polymorphic sites at sequences with enhanced mutability. Fluorescence in situ hybridization sublocalized this gene to proximal band 11q13, establishing a point of cross-reference between the physical and genetic maps.