Shuyun Li

Prenatal diagnosis and carrier screening for fragile X by PCR

During the past three years, we have conducted fragile X DNA studies for carrier screening and prenatal diagnosis using a previously described PCR protocol that accurately resolves normal FMR1 alleles and premutations and detects most full mutations [Brown et al., JAMA 270:1569-1575, 1996]. A total of 344 pregnant women with a family history of mental retardation of unknown cause were screened and 6 fragile X carriers were identified: two had full mutations, and four had premutations. The mentally retarded relatives of two other women were found to be fragile X positive although the women themselves were not carriers. In all, 6 carriers and 8 fragile X families were identified by this screening. We have also screened 40 pregnant women who were members of previously identified fragile X families, but whose carrier status was unknown. Ten were found to be carriers and were offered prenatal diagnosis. Prospective prenatal testing of 84 carrier women correctly detected 31 fetal samples (19 females, 12 males) with full mutations and 6 with premutations (2 females, 4 males). No false positives but one false negative occurred early on due to undetected maternal cell contamination. In addition, screening of 806 males with developmental delays of unknown cause gave positive results in 33 (4.1%). Potential problems and pitfalls of direct DNA testing are discussed. Because of the proven success of fragile X screening with direct molecular analysis, screening of all undiagnosed individuals with mental retardation and at risk pregnant women should now be considered. The identification of fragile X carriers and prenatal diagnosis of their pregnancies should significantly reduce the prevalence of this syndrome.

Telomere shortening in T lymphocytes of older individuals with Down syndrome and dementia

Telomere shortening has been recently correlated with Alzheimers disease status. Therefore, we hypothesized that a possible association might exist for adults with Down syndrome (DS). Using blind, quantitative telomere protein nucleic acid FISH analyses of metaphase and interphase preparations from 18 age-matched trisomy 21 female study participants with and without dementia, we have observed increased telomere shortening in adults with DS and dementia (p < .01). From this initial study, we conclude that telomere shortening is associated with dementia in this high-risk population and suggest that additional research may show that telomere shortening may be a biological marker of dementia status.

Prenatal fragile X detection using cytoplasmic and nuclear-specific monoclonal antibodies

We have been carrying out studies aimed at improving prenatal detection of the fragile X chromosome/mutation. Our current protocol requires a turnaround time (TAT) of several days. In an attempt to reduce the TAT, we have turned to the use of monoclonal antibodies (mAbs). Monoclonal antibody 1A1 (provided by Dr. Mandel of INSERM) immunostaining was performed according to a modified three-step immunocytochemical procedure. We found that cytoplasmic staining intensities, using mAb 1A1/avidin biotinylated complex/diaminobenzidine, varied from light to heavy within each sample, with controls exhibiting a majority of heavily stained cells in both chorionic villus (CV) sample and amniotic fluid cultured cells. Using mAb 1A1 and a new nuclear-specific antibody, mAb 3F11, we found that CV cultured cells harboring the FMR1 full mutation could be distinguished from controls as early as 10 weeks of gestation in both male and female specimens. Western blot analysis showed that the antibodies have similar staining patterns but that mAb 3F11 has fewer background/nonspecific bands. Our results demonstrate that it is feasible to detect fragile X full mutations within one day after obtaining cells from CV specimens taken as early as 10 weeks of gestation.

Increased expression of β-catenin in brain microvessels of a segmentally trisomic (Ts65Dn) mouse model of Down syndrome

We examined the distribution of β-catenin and endogenous blood serum albumin at the ultrastructural level in blood microvessels (capillaries) from brains of control and trisomic Ts65Dn mice. Morphological examination revealed an increased immunolabeling for β-catenin in endothelial substructures of the capillary network, such as intercellular junctions, cytoplasm, and nuclei. These immunosignals were significantly increased in all endothelial substructures from trisomic mice. These changes, however, did not affect the blood–brain barrier function of the entire microvascular network, because the increased uptake of albumin by endothelial cells and the eventual escape of this protein (microleakage) into the perivascular neuropil were noted only in a few capillary profiles. Nevertheless, these findings suggest the involvement of some segments of the microvascular network in the brain pathology associated with DS.

Accelerated prenatal diagnosis of fragile X syndrome by polymerase chain reaction restriction fragment detection.

Prenatal diagnosis of fragile X syndrome requires detection of the full FMR1 mutation in chorionic villus or amniotic fluid cell samples. Although analysis of genomic DNA restriction fragment pattern is a highly reliable technique for identification of the full FMR1 mutation, standard Southern blot determination of this pattern requires significantly more genomic DNA than is initially available from a prenatal sample. To overcome this limitation we developed a method that determines the diagnostic pattern of genomic restriction fragments from a fraction of a prenatal specimen. The prenatal DNA sample is first digested with EcoRI and EagI, and after agarose gel electrophoresis, the 2- to 10-kb region of the gel is serially sectioned and amplified by polymerase chain reaction. Analysis of prenatal samples from an unaffected male and from a full mutation male showed that this approach generated a diagnostic pattern comparable with a Southern blot of 100-fold more material. This innovation enables laboratories to prenatally diagnose the full FMR1 mutation sooner than standard techniques.

Mitotic index in Down's syndrome with and without dementia.

Previously, we reported reduced mitotic indices (MIs) in skin fibroblast cultures from five individuals with Alzheimer’sDisease (AD) [Jenkins et al., 1998].Wehave now found reduced MIs in short-term whole blood cultures from 12 of 15 individuals with trisomy 21 and dementia vs. 13 other non-demented trisomy 21 individuals who were generally ageand sex-matched—X(1) Yates corrected1⁄4 11.69, P< 0.01 (Table I). A MI 25 suggested dementia while a MI>25 suggested nondementia. Only one of the 13 non-demented trisomy 21 individuals exhibited aMI of<25. When the two sets of data in Table I were compared using the t-test (STATISTICA 6.0), a P value of <0.0002 was obtained. The individuals in Table I ranged in age from 50 to 66 years. The mean age for individuals with dementia was 56.95 while it was 55.95 years for those without dementia. All classifications of dementia were consistent with criteria recommended by the Working Group for the Establishment of Criteria for the Diagnosis of Dementia in Individuals with Intellectual Disability, of the ‘‘American Association on Mental Retardation’’ and the ‘‘International Association for the Scientific Study of Intellectual Disability’’ [Aylward et al., 1997] and the World Health Organization [1992]. These individuals were participants in a longitudinal study of DS. The present study was conducted from archived, fixed specimens used to confirm trisomy 21 Down’s syndrome in the longitudinal study. Results of additional preliminary studies shown in Table II also exhibited reduced MIs in cultures from people not yet classified as having dementia but who were exhibiting mild declines. Similar to the results in Table I, an MI 25 suggested mild declines for individuals listed in Table II while anMI>25 suggested nondementia (Fisher’s Exact test, P<0.04 two-tailed). These individuals ranged in age from 50 to 73 years and the mean age of females with cognitive decline was 54.60 years while that for females with no cognitive decline was 53.4 and, similar to Table I were generally ageand sex-matched. They were classified (as were those listed in Table I) based upon evaluations of the health, functional, and cognitive status carried out for each subject at 14–18 month intervals to determine if cognitive declines consistent with dementia were present. These evaluations included a comprehensive review of clinical records, interviews with knowledgeable informants providing objective descriptions of participants’ adaptive skills, health status and problem behaviors, and direct assessments of cognition explicitly designed for use with this population. Assessment of cognition was carried out using a battery of tests including: the Wisniewski and Hill [1985] modification of the Mini-Mental State Exam [Folstein et al., 1975]; the Test for Severe Impairment [Albert and Cohen, 1992]; a mental status evaluation developed by Haxby [1989] and Haxby and Schapiro [1992]; the Peabody Picture Vocabulary Test [Dunn and Dunn, 1981]; an adaptation of theMcCarthy [1972] test of verbal fluency; the Beery Visual Motor Integration Test [Beery and Bukenia, 1989]; and an 8-item version of the Selective Reminding Test [Buschke, 1973]. The human subjects in Table I exhibited either definite dementia, indicating that ICD-10 criteria [International Statistical Classification of Diseases; World Health Organization, 1992] were met and substantial declines over time were documented or they were not demented indicating that ICD-10 criteria for dementia were not met. The human subjects listed in Table IIwere eithernot demented or theywere classified as questionable, indicating the presence of some evidence consistent with mild functional or cognitive decline, but not of sufficient severity to suggest a diagnosis of dementia (mild declines). Our findings provide a preliminary indication that reduced MI is associated with dementia and cognitive decline in adults with DS. In light of our preliminary results, this association is likely to be strong, and given that we are focusing on adults with DS, this association is likely to be caused by the progression of AD/dementia. Wewill now evaluate additional subjects with dementia or with preliminary signs that may lead to dementia, Grant sponsor: NYS Office of Mental Retardation and Developmental Disabilities, Alzheimer’s Association Grant; Grant number: IIRG-99-1598; Grant sponsor: NIH; Grant numbers: PO1 HD35897, HD37425, RO1 AG014673.