Birth of a normal girl after in vitro fertilization and preimplantation diagnostic testing for cystic fibrosis.

BACKGROUND: Cystic fibrosis is a common, severe autosomal recessive disease caused in a majority of cases by a three-nucleotide deletion (delta F508) in the cystic fibrosis transmembrane regulator gene. Current methods of prenatal diagnosis involve chorionic-villus sampling or amniocentesis. In vitro fertilization and diagnosis during embryonic development before implantation would allow only unaffected embryos to be selected for transfer to the uterus, thereby avoiding the need to terminate a pregnancy. METHODS: Preimplantation diagnosis of cystic fibrosis was attempted in the cases of three couples, both members of which carried the delta F508 deletion. In vitro fertilization techniques were used to recover oocytes from each woman and fertilize them with her husband's sperm. Three days after insemination, embryos in the cleavage stage underwent biopsy and removal of one or two cells for DNA amplification and analysis. RESULTS: Only two oocytes from one woman were fertilized normally; DNA analysis of one of the embryos failed and cystic fibrosis was diagnosed in the other (i.e., it was homozygous for delta F508), so neither was transferred. The oocytes of each of the other two women produced noncarrier, carrier, and affected embryos. Both couples chose to have one noncarrier embryo and one carrier embryo transferred. One woman became pregnant and gave birth to a girl free of the deletion in both chromosomes. CONCLUSIONS: Preimplantation diagnosis of the delta F508 deletion causing cystic fibrosis is possible through in vitro fertilization, biopsy of a cleavage-stage embryo, and amplification of DNA from single embryonic cells. This approach should be equally applicable to other single-gene diseases in which the defect has been identified. Analysis of a series of pregnancies, however, will be required to assess the method adequately.

Linkage studies and deletion screening in choroideremia.

Fourteen families with choroideremia (TCD) have been examined for linkage to nine genetic markers located on the proximal long arm of the X chromosome. Linkage to three markers (DXYS1, DXS72, DXS3) located in Xq21 was found with a four point lod score of 8.25. No evidence of submicroscopic deletions was observed using DXS233 and DXS232, both thought to lie within about 1 Mb of the TCD gene.

DXS165 detects a translocation breakpoint in a woman with choroideremia and a de novo X; 13 translocation.

The search for the gene for choroideremia (MIM 30310), a rare retinal dystrophy, has been of great interest due to the existence of several choroideremia patients with well-defined structural chromosome aberrations, thus providing the basis for a reverse genetics approach to the isolation of this disease gene. This report details our molecular studies of a woman with choroideremia and a de novo X; 13 translocation. Pulsed-field gel electrophoresis using a contour-clamped homogeneous electric field apparatus has allowed detection of the translocation breakpoint with the anonymous DNA marker p1bD5 (DXS165) and the mapping of this probe to within 120 kb of the breakpoint. In addition, we have used this probe to isolate a clone (pCH4) from a 100-kb jumping library which has crossed a rare-cutting restriction site (XhoI) between DXS165 and the choroideremia gene and detects the translocation breakpoint using this enzyme. Although DXS165 lies within 120 kb of the breakpoint and Cremers et al. (1987, Clin. Genet. 32: 421-423; 1989, PNAS 86: 7510-7514) have detected deletions of DXS165 in 3 of 30 choroideremia probands, we have detected no deletions of this marker or of pCH4 in 42 unrelated probands with this retinal disease.

Choroideremia and deafness with stapes fixation: a contiguous gene deletion syndrome in Xq21.

The study of contiguous gene deletion syndromes by using reverse genetic techniques provides a powerful tool for precisely defining the map location of the genes involved. We have made use of individuals with overlapping deletions producing choroideremia as part of a complex phenotype, to define the boundaries on the X chromosome for this gene, as well as for X-linked mixed deafness with perilymphatic gusher (DFN3). Two patients with deletions and choroideremia are affected by an X-linked mixed conductive/sensorineural deafness; one patient, XL-62, was confirmed at surgery to have DFN3, while the other patient, XL-45, is suspected clinically to have the same disorder. A third choroideremia deletion patient, MBU, has normal hearing. Patient XL-62 has a cytogenetically detectable deletion that was measured to be 7.7% of the X chromosome by dual laser flow cytometry; the other patient, XL-45, has a cytogenetically undetectable deletion that measures only 3.3% of the X chromosome. We have produced a physical map of the X-chromosome region containing choroideremia and DFN3 by using routine Southern blotting, chromosome walking and jumping techniques, and long-range restriction mapping to generate and link anonymous DNA sequences in this region. DXS232 and DXS233 are located within 450 kb of each other on the same SfiI and MluI fragments and share partial SalI fragments of 750 and greater than 1,000 kb but are separated by at least one SalI site. In addition, DXS232, which lies outside the MBU deletion, detects the proximal breakpoint of this deletion. We have isolated two new anonymous DNA sequences by chromosome jumping from DXS233; one of these detects a new SfiI fragment distal to DXS233 in the direction of the choroideremia gene, while the other jump clone is proximal to DXS233 and detects a new polymorphism. These data refine the map around the loci for choroideremia and for mixed deafness with stapes fixation and will provide points from which to isolate candidate gene sequences for these disorders.

Linkage analysis of a large Latin-American family with X-linked retinitis pigmentosa and metallic sheen in the heterozygote carrier.

An extended linkage analysis was performed on the large Latin-American kindred with X-linked retinitis pigmentosa (XLRP) and metallic sheen in the heterozygous carrier studied and reported previously by R.L. Nussbaum et al. (1985, Hum. Genet. 70:45-50) and on a smaller family with the same XLRP variant. In these kindreds the XLRP locus shows close linkage with Xp21 marker loci OTC and DXS206. The results of this linkage analysis agree with the observations made by Nussbaum et al. (1985) that an XLRP locus is distal to DXS7.

Isolation of anonymous DNA sequences from within a submicroscopic X chromosomal deletion in a patient with choroideremia, deafness, and mental retardation.

Choroideremia, an X-chromosome linked retinal dystrophy of unknown pathogenesis, causes progressive nightblindness and eventual central blindness in affected males by the third to fourth decade of life. Choroideremia has been mapped to Xq13-21 by tight linkage to restriction fragment length polymorphism loci. We have recently identified two families in which choroideremia is inherited with mental retardation and deafness. In family XL-62, an interstitial deletion in Xq21 is visible by cytogenetic analysis and two linked anonymous DNA markers, DXYS1 and DXS72, are deleted. In the second family, XL-45, an interstitial deletion was suspected on phenotypic grounds but could not be confirmed by high-resolution cytogenetic analysis. We used phenol-enhanced reassociation of 48,XXXX DNA in competition with excess XL-45 DNA to generate a library of cloned DNA enriched for sequences that might be deleted in XL-45. Two of the first 83 sequences characterized from the library were found to be deleted in probands from family XL-45 as well as from family XL-62. Isolation of these sequences proves that XL-45 does contain a submicroscopic deletion and provides a starting point for identifying overlapping genomic sequences that span the XL-45 deletion. Each overlapping sequence will be studied to identify exons from the choroideremia locus.

Multipoint linkage analysis of loci in the proximal long arm of the human X chromosome: application to mapping the choroideremia locus.

Choroideremia (McK30310), an X-linked retinal dystrophy, causes progressive night blindness, visual field constriction, and eventual central blindness in affected males by the third to fourth decade of life. The biochemical basis of the disease is unknown, and prenatal diagnosis is not available. Subregional localization of the choroideremia locus to Xq13-22 was accomplished initially by linkage to two restriction-fragment-length polymorphisms (RFLPs), DXYS1 (Xq13-q21.1) and DXS3 (Xq21.3-22). We have now extended our linkage analysis to 12 families using nine RFLP markers between Xp11.3 and Xq26. Recombination frequencies of 0%-4% were found between choroideremia and five markers (PGK, DXS3, DXYS12, DXS72, and DXYS1) located in Xq13-22. The families were also used to measure recombination frequencies between RFLP loci to provide parameters for the program LINKMAP. Multipoint analysis with LINKMAP provided overwhelming evidence for placing the choroideremia locus within the region bounded by DXS1 (Xq11-13) and DXS17 (Xq21.3-q22). At a finer level of resolution, multipoint analysis suggested that the choroideremia locus was proximal to DXS3 (384:1 odds) rather than distal to it. Data were insufficient, however, to distinguish between a gene order that puts choroideremia between DXS3 and DXYS1 and one that places choroideremia proximal to both RFLP loci. These results provide linkage mapping of choroideremia and RFLP loci in this region that will be of use for further genetic studies as well as for clinical applications in this and other human diseases.

Thymidylate synthase-deficient Chinese hamster cells: a selection system for human chromosome 18 and experimental system for the study of thymidylate synthase regulation and fragile X expression.

Chinese hamster lung (CHL) V79 cells already deficient in hypoxanthine phosphoribosyltransferase were exposed to uv light and selected for mutations causing deficiency of thymidylate synthase (TS) by their resistance to aminopterin in the presence of thymidine and limiting amounts of methyl tetrahydrofolate. Three of seven colonies chosen for initial study were shown to be thymidylate synthase deficient (TS-) by enzyme assay, thymidine auxotrophy, and their inability to incorporate labeled deoxyuridine into their DNA in vivo. Complementation analysis of human X TS- hamster hybrids revealed that TS activity segregated with human chromosome 18. Southern analysis of a panel of 14 human X hamster hybrids probed with complementary DNA from mouse TS confirmed the chromosome assignment of TS to human chromosome 18; quantitative Southern blotting using unbalanced human cell lines further localized the gene to 18q21.31----qter. Another hybrid was generated that contained a human X chromosome with the Xq28 folate-dependent fragile site as its only human chromosome in a hamster TS- background. The fragile site could be easily and reproducibly expressed in this hybrid without the use of antimetabolites simply by removing exogenous thymidine from the medium. These TS-deficient cells are useful for: somatic cell genetics as a unique selectable marker for human chromosome 18, studies on regulation of the TS gene, and analysis of the fragile (X) chromosome and other folate-dependent fragile sites.

Choroideremia is linked to the restriction fragment length polymorphism DXYS1 at XQ13-21.

Choroideremia (McK30310), an X-linked hereditary retinal dystrophy, causes night-blindness, progressive peripheral visual field loss, and, ultimately, central blindness in affected males. The location of choroideremia on the X chromosome is unknown. We have used restriction fragment length polymorphisms from the X chromosome to determine the regional localization of choroideremia by linkage analysis in families with this disease. One such polymorphic locus, DXYS1, located on the long arm (Xq) within bands q13-q21, shows no recombination with choroideremia at lod = 5.78. Therefore, with 90% probability, choroideremia maps within 9 centiMorgans (cM) of DXYS1. Another polymorphic locus, DXS11, located within Xq24-q26, also shows no recombination with choroideremia, although at a smaller lod score of 1.54 (90% probability limit theta less than 30 cM). This linkage with DXS11, a marker that is distal to DXYS1, suggests that the locus for choroideremia is also distal to DXYS1 and lies between these two markers in the region Xq13-q24. These results provide regional mapping for the disease that may be useful for prenatal diagnosis and, perhaps ultimately, for isolating the gene locus for choroideremia.

Mapping X-linked ophthalmic diseases: II. Linkage relationship of X-linked retinitis pigmentosa to X chromosomal short arm markers.

X-linked retinitis pigmentosa (XLRP) is a series of hereditary dystrophic diseases of the retina that occur in three clinically distinguishable variants: the classic form (McK31360), a type known as choroidoretinal dystrophy (McK30330), and a variant with golden-metallic or "tapetal" reflex in the heterozygote (McK30320). Controversy exists as to whether these phenotypic differences are due to clinical variability in disease expression, heterogeneity in disease alleles at a single locus, or a multiplicity of loci for XLRP. We have studied a single large kindred segregating for XLRP with the metallic fundus reflex in the heterozygote with restriction fragment length polymorphisms (RFLPs) from the short arm of the human X chromosome, and found measurable linkage to DXS7 (theta = 12.5 cMorgans at LOD = 2.5), the same RFLP previously shown by others to be tightly linked to the other forms of XLRP at theta = 3 cM. Although these estimates appeared to be different, each fell just within the 95% probability interval of the other and, therefore, were insufficient to prove or disprove that the metallic sheen form of XLRP is allelic with other forms of XLRP. Additional RFLPs at the DXS43 and the ornithine transcarbamoylase loci provided three-point crosses for determining the relative positions of DXS7 and XLRP, and supported an order that placed this form of XLRP distal to DXS7 on the Xp. Until the question of genetic heterogeneity is resolved, careful phenotypic characterization of the clinical type of XLRP present in families being used for linkage analyses is advisable.