Nan Zhong

Elevated plasma amyloid β-peptide 1–42 and onset of dementia in adults with Down syndrome

We compared levels of plasma amyloid beta-peptides Abeta1-42 and Abeta1-40 in 108 demented and nondemented adults with Down syndrome (DS) and 64 adults from the general population. Abeta1-42 and Abeta1-40 levels were significantly higher in adults with DS than in controls (P=0.0001). Compared to nondemented adults with DS, Abeta1-42 levels in demented adults with DS were selectively increased by 26% (28.2 pg/ml vs. 22.4 pg/ml, P=0.004). In addition, mean plasma levels of Abeta1-42 were 22% higher in DS cases with the apolipoprotein varepsilon4 allele than in DS subjects without an varepsilon4 allele (25.9 pg/ml vs. 21.2 pg/ml, P=0.01), while mean plasma levels of Abeta1-40 did not vary by APOE genotype. These results support the hypothesis that Abeta1-42 plays an important role in the pathogenesis of dementia associated with DS, as it does in Alzheimers disease, and that variations in plasma levels may be related to disease progression.

Fragile X founder effects and new mutations in Finland

The apparent associations between fragile X mutations and nearby microsatellites may reflect both founder effects and microsatellite instability. To gain further insight into their relative contributions, we typed a sample of 56 unrelated control and 37 fragile X chromosomes from an eastern Finnish population for FMR1 CGG repeat lengths, AGG interspersion patterns, DXS548, FRAXAC1, FRAXE and a new polymorphic locus, Alu-L. In the controls, the most common FMR1 allele was 30 repeats with a range of 20 to 47 and a calculated heterozygosity of 88%. A strong founder effect was observed for locus DXS548 with 95% of fragile X chromosomes having the 21 CA repeat (196 bp) allele compared to 17% of controls, while none of the fragile X but 69% of controls had the 20 repeat allele. Although the FRAXAC1 locus is much closer than DXS548 to FMR1 (7 kb vs. 150 kb), there was no significant difference between fragile X and control FRAXAC1 allele distributions. The FRAXE repeat, located 600 kb distal to FMR1, was found to show strong linkage disequilibrium as well. A newly defined polymorphism, Alu-L, located at approximately 40 kb distal to the FMR1 repeat, showed very low polymorphism in the Finnish samples. Analysis of the combined loci DXS548-FRAXAC1-FRAXE showed three founder haplotypes. Haplotype 21-19-16 was found on 27 (75%) of fragile X chromosomes but on none of controls. Three (8.4%) fragile X chromosomes had haplotypes 21-19-15, 21-19-20, and 21-19-25 differing from the common fragile X haplotype only in FRAXE. These could have arisen by recombination or from mutations of FRAXE. A second haplotype 21-18-17 was found in four (11.1%) fragile X chromosomes but only one (1.9%) control. This may represent a more recent founder mutation. A third haplotype 25-21-15, seen in two fragile X chromosomes (5.6%) and one (1.9%) control, was even less common and thus may represent an even more recent mutation or admixture of immigrant types. Analysis of the AGG interspersions within the FMR1 CGG repeat showed that 7/8 premutation chromosomes lacked an AGG whereas all controls had at least one AGG. This supports the hypothesis that the mutation of AGG to CGG leads to repeat instability and mutational expansion.

Reverse mutations in the fragile X syndrome.

Three females were identified who have apparent reversal of fragile X premutations. Based on haplotype analysis of nearby markers, they were found to have inherited a fragile X chromosome from their premutation carrier mothers, and yet had normal size FMR1 repeat alleles. The changes in repeat sizes from mother to daughter was 95 to 35 in the first, 145 to 43 in the second, and 82 to 33 in the third. In the first family, mutations of the nearby microsatellites FRAXAC2 and DXS548 were also observed. In the other two, only mutations involving the FMR1 repeats were found. We suggest differing mutational mechanisms such as gene conversion versus DNA replication slippage may underlie such reversions. We estimate that such revertants may occur among 1% or less of premutation carrier offspring. Our results indicate that women identified to be carriers by linkage should be retested by direct DNA analysis.

A survey of FRAXE allele sizes in three populations

FRAXE is a fragile site located at Xq27-8, which contains polymorphic triplet GCC repeats associated with a CpG island. Similar to FRAXA, expansion of the GCC repeats results in an abnormal methylation of the CpG island and is associated with a mild mental retardation syndrome (FRAXE-MR). We surveyed the GCC repeat alleles of FRAXE from 3 populations. A total of 665 X chromosomes including 416 from a New York Euro-American sample (259 normal and 157 with FRAXA mutations), 157 from a Chinese sample (144 normal and 13 FRAXA), and 92 from a Finnish sample (56 normal and 36 FRAXA) were analyzed by polymerase chain reaction. Twenty-seven alleles, ranging from 4 to 39 GCC repeats, were observed. The modal repeat number was 16 in the New York and Finnish samples and accounted for 24% of all the chromosomes tested (162/665). The modal repeat number in the Chinese sample was 18. A founder effect for FRAXA was suggested among the Finnish FRAXA samples in that 75% had the FRAXE 16 repeat allele versus only 30% of controls. Sequencing of the FRAXE region showed no imperfections within the GCC repeat region, such as those commonly seen in FRAXA. The smaller size and limited range of repeats and the lack of imperfections suggests the molecular mechanisms underlying FRAXE triplet mutations may be different from those underlying FRAXA.

Studies of atypical JNCL suggest overlapping with other NCL forms

In the United States, juvenile neuronal ceroid-lipofuscinosis (JNCL) is the most common form of NCL. This study analyzed 191 cases, diagnosed on the basis of age-at-onset, clinical symptomatology, and pathologic findings. Twenty percent (40/191) of these cases from 24/120 families manifested atypical clinical symptomatology and/or pathologic findings (typical revealed fingerprints and atypical revealed mixed inclusions, or only curvilinear or granular profiles) and, therefore, represent variant forms of JNCL. Those patients in the study with typical JNCL were a uniform group of cases, whereas the atypical were heterogenous and were divided into 8 subgroups based on the clinicopathologic findings. Forty-three families were analyzed (27 typical, 16 atypical) for the common 1.02 kb deletion and several pedigrees for novel mutations. In typical JNCL the common 1.02 kb deletion in both alleles (homozygous) were observed in 23/27, and only 1 allele (heterozygous) was exhibited in 4/27 families. In atypical JNCL families, 5/16 were heterozygous for the common 1.02 kb deletion. None of the remaining 11/16 families had the common 1.02 kb deletion in either allele, but in 9/11 cases the palmitoyl-protein thioesterase (PPT) levels were deficient. In cases where the mutation in CLN3 gene has not been identified, several possibilities may exist. The phenotype may be caused by a yet undefined mutation in CLN3 or may be due to overlapping with other forms of NCL.

FRAXAC1 and DXS548 polymorphisms in the Chinese population

The fragile X syndrome is the most common inherited form of mental retardation. Haplotype studies using FRAXAC1 and DXS548 polymorphic markers flanking the fragile site have demonstrated linkage disequilibrium at the FMR1 locus. We investigated the association of the FRAXAC1, DXS548 and CGG alleles between normal subjects and mentally retarded (MR) patients of unspecified cause who do have fragile X syndrome. We have evaluated the FRAXAC1 site in 390 normal subjects and 321 MR patients and the DXS548 site in 146 normal and 319 MR subjects. Both FRAXAC1 and DXS548 alleles were determined by application of the polymerase chain reaction. When compared with Caucasians, the normal Chinese population has a different FRAXAC1 allele distribution. There are more AC18 repeat alleles and fewer AC19 repeat alleles. The DXS548 allele distributions were similar between Chinese and Caucasians. The same distribution pattern of FRAXAC1 alleles was found in both normal subjects and MR patients, but there were significant differences in the distribution patterns of DXS548 alleles. The FMR1 CGG-DXS548 and FRAXAC1-DXS548 haplotype distribution between normal subjects and MR patients also differed significantly. Our results suggest a possible association between DXS548 alleles and non-FRAXA mental retardation.

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.

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.

Possible founder effects for FRAXE alleles.

To determine if FRAXE alleles may have haplotype associations with nearby microsatellites, we analyzed 149 unrelated control Caucasian X chromosomes for FRAXE GCC alleles along with five nearby microsatellites. The microsatellites included three that are new; GT25, CA4, and CA5 located approximately 24, approximately 48, and approximately 50 kb proximal to the FRAXE GCC repeat, and two that were identified previously: DXS8091 and DXS1691, located approximately 90 and approximately 5 kb distal. No significant correlations between haplotypes for the proximal microsatellites were found. Significant correlations of FRAXE GCC repeats and distal microsatellite allele sizes, DXS8091 (r = 0.24) and DXS1691 (r = -0.40), were found. One haplotype, 18-19 of DXS8091-DXS1691, was present on 57% of chromosomes with > or =22 FRAXE repeats but present on only 10% with <22 repeats. We conclude that this distal haplotype association likely reflects a FRAXE allele founder effect. The lack of association or founder effects seen for the three newly identified proximal markers, located within 50 kb of FRAXE GCC, may reflect an unusually high rate of mutation for these microsatellites or a higher rate of recombination in the proximal region.

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.