Preimplantation genetic diagnosis for spinal muscular atrophy type I.

OBJECTIVE: Couples with children who have spinal muscular atrophy type I (SMA) face a 25% risk of having affected offspring with spontaneous conception. Preimplantation genetic testing (PGT) is possible for the deletions in the survival motor neuron (SMN) gene that have been identified in 98% of SMA type I cases. PGT would provide new reproductive options for families at risk for SMA. METHODS: Three couples with previously affected children confirmed by DNA testing each underwent in vitro fertilization (IVF) and PGT of the resulting embryos. One or two blastomeres were biopsied from each embryo and analyzed for deletions in exons 7 and 8 of the SMN gene. RESULTS: Nine embryos were predicted to be unaffected, three to be affected, and one embryo could not be interpreted. One of three patients receiving transfer of unaffected embryos became pregnant with twins. CONCLUSIONS: Preimplantation genetic testing provides a means for couples at risk for spinal muscular atrophy type I to reduce their chance of initiating an affected pregnancy.

Maroteaux-Lamy syndrome in a large consanguineous kindred: biochemical and immunological studies.

We describe a large consanguineous German-Acadian ("Cajun") family from a rural area in Louisiana in which 11 persons in two generations had the Maroteaux-Lamy syndrome. The mutant arylsulfatase B enzyme in this family was similar to the mutant enzyme in previously studied families in its cross-reactivity with specific antibodies to the enzyme, but it differed in both its electrophoretic mobility and its residual enzymatic activity. These findings indicate that a different mutational event leading to Maroteaux-Lamy syndrome occurred in this family.

Improved sizing of fragile X CCG repeats by nested polymerase chain reaction.

We have developed an improved method for polymerase chain reaction (PCR)-based sizing of the CCG repeat region at the fragile X locus, FMR-1. This method is designed to optimize denaturation and replication of long repeats with high G + C content, which are otherwise refractory to amplification. The method utilizes nested PCR primers to increase sensitivity and specificity. Alkaline denaturation of the genomic template DNA, combined with addition of glycerol and deaza-dGTP, facilitates strand separation. Labeled PCR products are sized on denaturing polyacrylamide gels. For alleles in the normal-to-premutation size range, strong reproducible signals are routinely obtained from small amounts of rapidly prepared DNA. This allows precise determination of the CCG repeat number, providing data related to the expansion potential of the repetitive segment. Detection of large premutations and some full mutations is also enhanced by the improved procedure.

Mental retardation and Ullrich-Turner syndrome in cases with 45,X/46X,+mar: additional support for the loss of the X-inactivation center hypothesis.

Four cases having mosaicism for a small marker or ring [45,X/46,X,+mar or 45,X/46,X,+r] chromosome were ascertained following cytogenetic studies requested because of minor anomalies (cases 1, 3, and 4) and/or short stature (cases 2 and 4). While all 4 cases had traits typical of Ullrich-Turner syndrome (UTS), cases 1, 3, and 4 had manifestations not usually present in UTS, including unusual facial appearance, mental retardation/developmental delay (MR/DD) (cases 3 and 4), and syndactylies (case 1). The facial appearances of cases 1 and 3 were similar yet distinct from that of case 4. Using fluorescence in situ hybridization (FISH), each of the markers in these 4 cases was identified as having been derived from an X chromosome. The level of mosaicism for the mar/r(X) cell line in these cases varied from 70% (case 1) to 16% (case 4) but was not apparently correlated with the presence of MR/DD. Replication studies demonstrated a probable early replication pattern for the mar/r(X) in cases 1, 3, and 4, while the marker in case 2 was apparently late replicating. To date, 41 individuals having mosaicism for a small mar/r(X) chromosome have been described. Interestingly, most of the 14 individuals having a presumedly active mar/r(X) demonstrated clinical findings atypical of UTS, including abnormal facial changes (11) and MR/DD (13). MR was noted most frequently in those cases having at least 50% mosaicism for the marker or ring. In contrast, atypical UTS facial appearance or MR/DD was not noted in 14 of the 16 cases with UTS who carried a probable late replicating marker or ring. In conclusion, although the phenotype of 45,X/46,X,mar/r(X) individuals appears to be influenced by the genetic content and degree of mosaicism for the mar/r(X), the most significant factor associated with MR/DD appears to be the activity status of the mar/r(X) chromosome. Thus, our 4 cases provide further support for the hypothesis that a lack of inactivation of a small mar/r(X) chromosome may be a factor leading to the MR and other phenotypic abnormalities seen in this subset of individuals having atypical UTS.

Novel approach to the molecular diagnosis of Marfan syndrome: application to sporadic cases and in prenatal diagnosis.

Marfan syndrome is an autosomal dominant disorder affecting the skeletal, ocular, and cardiovascular systems. Defects in the gene that encodes fibrillin-1 (FBN1), the main structural component of the elastin-associated microfibrils, are responsible for the disorder. Molecular diagnosis in families with Marfan syndrome can be undertaken by using intragenic FBN1 gene markers to identify and track the disease allele. However, in sporadic cases, which constitute up to 30% of the total, DNA-based diagnosis cannot be performed using linked markers but rather requires the identification of the specific FBN1 gene mutation. Due to the size and complexity of the FBN1 gene, identification of a causative Marfan syndrome mutation is not a trivial undertaking. Herein, we describe a comprehensive approach to the molecular diagnosis of Marfan syndrome that relies on the direct analysis of the FBN1 gene at the cDNA level and detects both coding sequence mutations and those leading to exon-skipping, which are often missed by analysis at the genomic DNA level. The ability to consistently determine the specific FBN1 gene mutation responsible for a particular case of Marfan syndrome allows both prenatal and pre-implantation diagnosis, even in sporadic instances of the disease.

Molecular fragile X screening in normal populations.

In December, 1993, we initiated a pilot project in which DNA fragile X (fraX) testing was offered during routine prenatal or genetic counseling to all pregnant women seen at the Genetics & IVF Institute, most of whom were referred for the indication of advanced maternal age. A brochure on fragile X syndrome was sent to each patient prior to her appointment and was reviewed by a counselor or physician during the counseling session. As of June 1995, 3,345 patients were offered testing; 474 women with no identified family history of mental retardation or learning disability and 214 women with a positive family history accepted the test on a self-pay basis. The second population screened was 271 potential donors in our anonymous egg donor program. DNA from blood was tested by Southern blot using EcoRI/EagI and StB12.3. If an expansion was detected, CGG repeat number was determined by PCR-based analysis. Among the 474 patients with unremarkable family histories, three fraX carriers were identified (repeat sizes = 60+), whereas none were found in the 214 patients with a positive family history. Among the potential egg donors, two high borderline patients were identified (repeat sizes = between 50 and 59). Our ongoing study indicates that screening of pregnant or preconceptual populations for fraX carrier status using DNA testing is accepted by many patients and is an important addition to current medical practice.

Fragile X premutation is a significant risk factor for premature ovarian failure: the International Collaborative POF in Fragile X study--preliminary data.

The preliminary results of an international collaborative study examining premature menopause in fragile X carriers are presented. A total of 760 women from fragile X families was surveyed about their fragile X carrier status and their menstrual and reproductive histories. Among the subjects, 395 carried a premutation, 128 carried a full mutation, and 237 were noncarriers. Sixty-three (16%) of the premutation carriers had experienced menopause prior to the age of 40 compared with none of the full mutation carriers and one (0.4%) of the controls. Based on these preliminary data, there is a significant association between fragile X premutation carrier status and premature menopause.

Antibody to hepatitis B surface antigen in haemophiliacs on long-term therapy with Scottish factor VIII.

Thirty-five patients with haemophilia A were studied clinically and serologically between 1971-2 and 1975-6 for evidence of hepatitis B infection. One patient suffered from clinical hepatitis B, and a further eight patients showed antibody responses to hepatitis B surface antigen (HBsAg) consistent with exposure to HBsAg during this period. No evidence for HBsAg exposure was found in 14 patients, while the remaining 12 patients had high titres of antibody to HBsAg at both times and no inferences could be drawn about HBsAg exposure. All patients had received exclusively replacement factor VIII material prepared locally from HBsAg-screened voluntary Scottish blood donations. From the details of the therapy given we calculated that the rate of HBsAg seroconversion in these patients represented about 0.3 HBsAg-containing donations/1000 donations.

Hepatitis Be antigen and antibody in hepatitis B surface antigen positive blood donors.

In a study of 105 asymptomatic HBsAg positive blood donors, 9 (8.6%) were found to have HBeAg, 38 (36.2%) anti-HBe, and the remaining 58 (55.2%) neither marker detectable by gel diffusion. There was no correlation between HBeAg/anti-HBe status and HBsAg sub-types, Glm allotypes, the presence of anti-Gm, red cell antibodies, or rheumatoid factor. Rheumatoid factor activity could be removed from anti-HBe positive sera without removing anti-HBe activity, indicating that separate entities were involved. HBeAg was found only in donors under the age of 30 (P less than 0.005), while anti-HBe did not show an age-related trend. HBeAg was also found less commonly in donors of blood group A than in the total carrier population (P less than 0.05), indicating an apparent protection in carriers of group A. The blood group distribution for the 105 HBsAg positive donors was similar to that of the general population.

Incidence of infection with hepatitis B virus in 56 patients with haemophilia A 1971-1979.

During each of the four-year periods 1971-1975 and 1975-1979, the annual incidence of hepatitis B infection has been assessed in 56 patients with haemophilia A by measuring plasma HBsAg, anti-HBs and anti-HBc levels. Infection rates of 7% and 9.5% per annum respectively were observed for each four-year period despite the screening of individual blood donations for HBsAg by techniques up to the sensitivity of reversed passive haemagglutination. The highest incidence of seroconversion was amongst severe haemophiliacs many of whom had received treatment predominantly with cryoprecipitate. Of the 16 patients in whom serological evidence of hepatitis B infection was obtained only one had an accompanying clinical episode of hepatitis. We conclude that haemophiliacs are still at high risk of infection by hepatitis B virus despite the screening of individual blood donors for HBsAg.

Pregnancy rates in sequential in vitro fertilization cycles by oocyte donors(1).

OBJECTIVE: To evaluate the clinical outcome of in vitro fertilization (IVF) treatment cycles from individual oocyte donors who underwent multiple sequential donations. METHODS: We reviewed clinical outcome data from sequential anonymous oocyte donation cycles using donors who underwent multiple IVF stimulations. Donors were grouped by the interval between cycles and the cycle number (rank). The primary outcome measure was delivery rate by individual donor per retrieval from the combined derivative fresh and frozen embryo transfers. RESULTS: Duration and amount of gonadotropin therapy and the fertilization rates did not correlate significantly with the interval between cycles or cycle rank. Cumulative delivered pregnancy rates for cycles 1-6 were 51.5%, 54.6%, 50.5%, 51.5%, 51.1%, and 57.6%, respectively. Delivered pregnancy rates did not vary by interval between cycles. CONCLUSION: Young healthy presumed or proven fertile women can reliably donate oocytes for at least six cycles with the expectation of consistently high pregnancy rates.

A community health promotion partnership model: the South Carolina health connection.

Over a five year period, the South Carolina Health Connection Project has evolved to multi-site, multi-organization community-base collaborative initiative. From this project over $60,000.00 in funds have been secured. However, when costing the human resources and many other in-kind contributions involved in the SCHC Projects activities, the Project can modestly be valued at nearly $200,000.00. The efforts of a few have been shared with others, who also shared the resources with others, and the health promotion empowerment cycle continues. We believe the South Carolina Health Connection is an exemplary of a Community Health Promotion Partnership Model. We hope you will agree!

Preimplantation genetic diagnosis.

Preimplantation genetic diagnosis now represents an alternative reproductive option for parents at high risk of having offspring affected with certain genetic diseases. Progress in the past year has included increasing reliability in embryo sexing by both polymerase chain reaction and fluorescent in situ hybridization techniques; delivery of babies free of specific diseases such as cystic fibrosis, Lesch-Nyhan syndrome, and Tay-Sachs disease; and successful development of molecular techniques for detecting common diseases such as fragile-X syndrome. In addition, sperm separation in combination with preimplantation genetic diagnosis appears to be an exciting advance in yielding more in vitro fertilization female embryos for transfer and subsequent pregnancy in families at risk for X-linked diseases. Accumulated world experience can now be reviewed to provide couples considering preimplantation genetic diagnosis with observed pregnancy rates and accuracy of diagnosis.

Characterization of the cytoplasmic fibrils of Treponema refringens (Nichols).

The cytoplasmic fibrils of Treponema refringens were studied in situ by electron microscopy of thin sectioned and negatively stained cells. From 5 to 21 parallel fibrils ran through the cell in a band adjacent to the inner side of the cytoplasmic membrane, on the inner sides of the curves of the spirochete. The nuclear areas of cells were adjacent to the fibrils. Cross sections of fibrils isolated from cells which had been lysed were polygonal and not uniformly electron dense. Polyacrylamide gel electrophoresis of partially purified fibril preparations indicated their main component to be a protein with a molecular weight of 97,000. Fibrils were solubilized by 1% trypsin, 1% pronase, 6 M urea, 1 N HCl, 0.005 N NaOH or 1.3% sodium dodecyl sulfate. By electron microscopy of negatively stained isolated fibrils, each fibril was found to be a complex arrangement of strands rather than a single tubule.