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The CGG repeat in the 5' untranslated region of the fragile X mental retardation 1 gene (FMR1) exhibits remarkable instability upon transmission from mothers with premutation alleles. A collaboration of 13 laboratories in eight countries was established to examine four issues concerning FMR1 CGG-repeat instability among females with premutation (approximately 55-200 repeats) and intermediate (approximately 46-60 repeats) alleles. Our central findings were as follows: (1) The smallest premutation alleles that expanded to a full mutation (200 repeats) in one generation contained 59 repeats; sequence analysis of the 59-repeat alleles from these two females revealed no AGG interruptions within the FMR1 CGG repeat. (2) When we corrected for ascertainment and recalculated the risks of expansion to a full mutation, we found that the risks for premutation alleles with100 repeats were lower than those previously published. (3) When we examined the possible influence of sex of offspring on transmission of a full mutation-by analysis of 567 prenatal fragile X studies of 448 mothers with premutation and full-mutation alleles-we found no significant differences in the proportion of full-mutation alleles in male or female fetuses. (4) When we examined 136 transmissions of intermediate alleles from 92 mothers with no family history of fragile X, we found that, in contrast to the instability observed in families with fragile X, most (99/136 [72.8%]) transmissions of intermediate alleles were stable. The unstable transmissions (37/136 [27.2%]) in these families included both expansions and contractions in repeat size. The instability increased with the larger intermediate alleles (19% for 49-54 repeats, 30.9% for 55-59, and 80% for 60-65 repeats). These studies should allow improved risk assessments for genetic counseling of women with premutation or intermediate-size alleles.

During hibernation, animals undergo metabolic changes that result in reduced utilization of glucose and oxygen. Fat is known to be the preferential source of energy for hibernating animals. Malonyldialdehyde (MDA) is an end product of fatty acid oxidation, and is generally used as an index of lipid peroxidation. We report here that peroxidation of lipids is increased in the plasma and in the membranes of red blood cells in black bears during hibernation. The plasma MDA content was about four fold higher during hibernation as compared to that during the active, non-hibernating state (P0.0001). Similarly, MDA content of erythrocyte membranes was significantly increased during hibernation (P0.025). The activity of Ca(2+)/Mg(2+)-ATPase in the erythrocyte membrane was significantly decreased in the hibernating state as compared to the active state. Na(+)/K(+)-ATPase activity was also decreased, though not significant, during hibernation. These results suggest that during hibernation, the bears are under increased oxidative stress, and have reduced activities of membrane-bound enzymes such as Ca(2+)/Mg(2+)-ATPase and Na(+)/K(+)-ATPase. These changes can be considered part of the adaptive for survival process of metabolic depression.

Two new observations have been made on human chromatid/chromosome ultrastructure using both scanning and transmission electron microscopy (SEM, TEM). A bipartite, apparently half-chromatid-like structure was observed in whole human chromosomes studied with SEM and in longitudinally sectioned chromosomes analyzed with TEM. In addition, we also observed a zipper-like configuration as the parallel sister chromatids separated likely due to the supercoiled structure of the chromosome and chromatid. It is possible that either or both of these new observations resulted from our (improved) method of preparing the chromosomes for SEM and TEM.

The Fmr1 gene knockout mouse is a model for the human Fragile X mental retardation syndrome. Fmr1 knockout mice with a C57BL/6-129/OlaHsd hybrid background have been reported to have only a very mild deficiency in learning the Morris water maze task. We compared the effect of this knockout mutation on learning in mice with either an FVB/N-129/OlaHsd hybrid background or a C57BL/6 background. When FVB-129 mice were tested in a cross-shaped water maze task, the knockout mice showed a pronounced deficiency in their ability to learn the position of a hidden escape platform in comparison to normal littermates. In contrast, knockout mice with a C57BL/6 background learned the maze just as well as their normal littermates. Fear conditioning did not reveal differences between knockout and normal mice in either background. These results show that silencing the Fmr1 gene clearly interfered with learning a specific visuospatial task in FVB/N-129 hybrid mice but not in C57BL/6 mice. The strain dependence may model the influence of genetic background in the human Fragile X syndrome.

The Fragile X protein FMRP is an RNA binding protein whose targets are not well known; yet, these RNAs may play an integral role in the disease's etiology. Using a biotinylated-FMRP affinity resin, we isolated RNAs from the parietal cortex of a normal adult that bound FMRP. These RNAs were amplified by differential display (DDRT–PCR) and cloned and their identities determined. Nine candidate RNAs were isolated; five RNAs, including FMR1 mRNA, encoded known proteins. Four others were novel. The specificity of binding was demonstrated for each candidate RNA. The domains required for binding a subset of the RNAs were delineated using FMRP truncation mutant proteins and it was shown that only the KH2 domain was required for binding. Binding occurred independently of homoribopolymer binding to the C-terminal arginine–glycine-rich region (RGG box), suggesting that FMRP may bind multiple RNAs simultaneously.

Late infantile neuronal ceroid lipofuscinosis, LINCL, is one of the most common pediatric neurodegenerative disorders. It is caused by mutations in the CLN2 gene, which encodes a lysosomal pepstatin-insensitive peptidase (LPIP). We have identified a novel mutation, T523-1G → A, by molecular analyses of three unrelated LINCL cases. The mutation was found to affect a 3′ intronic splicing acceptor site, resulting in an aberrant mRNA with an insertion of 146 bp of intronic sequence. This causes a frame shift, produces a nonfunctional truncated protein, and results in LINCL.

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.

Late infantile neuronal ceroid lipofuscinosis (LINCL) is one of the most common pediatric neuronal degenerative disorders. A candidate gene underlying this disease, designated CLN2, was recently cloned and the gene product was characterized as a lysosomal pepstatin-insensitive carboxypeptidase (LPIC). Four mutations were identified in CLN2 from three unrelated LINCL individuals. To investigate further the mutation frequency in LINCL, we screened 16 LINCL probands for these four mutations. The previously reported intronic mutation, T523-1 G-->C. was found in 56% (9/16) of the cases, of which two were homozygous and accounted for 34% (11/32) of LINCL chromosomes. The previously reported nonsense mutation, 636 C-->T leading to R208stop, was found in 31% (5/16) of the cases, including one homozygote and accounted for 19% (6/32) of LINCL chromosomes. Two previously described missense mutations, 1107 T-->C and 1108 G-->A, were not detected in any of these 16 probands. In total, the two observed mutations, T523-1 G-->C and 636 C-->T, accounted for 53% (17/32) of LINCL alleles. Thus, one or both mutations were seen in 11 (69%) cases and no mutation has yet been identified in five. Our finding that these two mutations are common in LINCL cases adds further evidence in support of the idea that dysfunction of LPIC underlies LINCL. Positive molecular testing can now complement clinical diagnosis of LPIC and will allow for pre-natal diagnosis for subsequent pregnancies.

SummaryWe examined premutation-female transmissions and premutation-male transmissions of the FMR1 CGG repeat to carrier offspring, to identify factors associated with instability of the repeat. First we investigated associations between parental and offspring repeat size. Premutation-female repeat size was positively correlated with the risk of having full-mutation offspring, confirming previous reports. Similarly, premutation-male repeat size was positively correlated with the daughter's repeat size. However, increasing paternal repeat size was associated also with both increased risk of contraction and decreased magnitude of the repeat-size change passed to the daughter. We hypothesized that the difference between the female and male transmissions was due simply to selection against full-mutation sperm. To test this hypothesis, we simulated selection against full-mutation eggs, by only examining premutation-female transmissions to their premutation offspring. Among this subset of premutation-female transmissions, associations between maternal and offspring repeat size were similar to those observed in premutation-male transmissions. This suggests that the difference between female and male transmissions may be due to selection against full-mutation sperm. Increasing maternal age was associated with increasing risk of expansion to the full mutation, possibly because of selection for smaller alleles within the offspring's soma over time; a similar effect of increasing paternal age may be due to the same selection process. Last, we have evidence that the reported association between offspring sex and risk of expansion may be due to ascertainment bias. Thus, female and male offspring are equally likely to inherit the full mutation.

Background: Hydroxysteroid (17beta) dehydrogenase X (HSD10) is a multifunctional protein encoded by the HSD17B10 gene at Xp11.2. In response to stress or hypoxia-ischemia its levels increase rapidly. Expression of this gene is also elevated significantly in colonic mucosa of the inactive ulcerative colitis patients. However, accurate information about its several transcripts is still lacking, and additional evidence for its escape from X-chromosome inactivation remains to be obtained in order to help settle a debate (He XY, Dobkin C, Yang SY: Does the HSD17B10 gene escape from X-inactivation? Eur J Hum Genet 2011, 19: 123-124). Results: Two major HSD17B10 transcription start sites were identified by primer extension at -37 and -6 as well as a minor start site at -12 nucleotides from the initiation codon ATG. Epigenetic analysis of the 5’-flanking region of the HSD17B10 gene showed that there was little 5-methylcytosine (

Two phenotypically abnormal liveborns in whom trisomy 16 mosaicism was diagnosed prenatally by amniocentesis are described. Analysis of a percutaneous umbilical blood sample in one case revealed a normal chromosomal complement. Ultrasound examinations performed at the time of amniocentesis were normal. Serial sonography during the late second and third trimesters demonstrated progressive intrauterine growth retardation (IUGR) in both fetuses and a cardiac defect in one. At birth, both infants had dysmorphic features and multiple congenital anomalies. Trisomy 16 mosaicism was confirmed postnatally in both infants in skin fibroblasts; however, peripheral blood samples contained only chromosomally normal cells. The two mosaic trisomy 16 cases described in this report, together with the five confirmed cases reported previously, demonstrate the need for caution in the counselling of patients when trisomy 16 mosaicism is diagnosed prenatally in amniotic fluid samples. Such cases potentially can result in the birth of dysmorphic infants with significant birth defects, growth retardation, and possible developmental disabilities.

While studying founder chromosomes in the fragile X syndrome, we have unexpectedly found linkage equilibrium to FRAXAC2, an Alu-associated microsatellite within the defective gene, FMR-1. DNA sequencing of 265 chromosomes revealed 39 alleles and a complex microsatellite of form (GT)x-C-(TA)y-(T)z. A mutation rate of 3.3% was observed but only among fragile X maternally derived meioses. Finding a second mutable locus within FMR-1 suggests that the target for tandem repeat instability may not be confined to the (CGG)n repeat alone and raises the possibility of an FMR-1 mutation mechanism involving microsatellites.

The mutation that underlies the fragile X syndrome is presumed to be a large expansion in the number of CGG repeats within the gene FMR-1. The unusually GC-rich composition of the expanded region has impeded attempts to amplify it by the polymerase chain reaction (PCR). We have developed a PCR protocol that successfully amplifies the (CGG)n region in normal, carrier and affected individuals. The PCR analysis of several large fragile X families is presented. The PCR results agree with those obtained by direct genomic Southern blot analyses. These favorable comparisons suggest that the PCR assay may be suitable for rapid testing for fragile X mutations and premutations and genetic screening of at-risk individuals.

UNLABELLED Recently, we detected fra(X)(q27.3) in amniocyte cultures from female identical twins. The pregnant woman did not exhibit fra(X)(q27.3) in whole blood cultures but was the sister of 2 affected brothers. DNA marker analyses showed that she was a carrier of FRAXA. Amniotic fluid cultures (AFCs) from twins A and B exhibited the fragile X [fra(X)] chromosome, but the level of cytogenetic expression was very low in twin A's AFCs. DNA marker studies indicated both twins were carriers of FRAXA. Peripheral umbilical blood sample (PUBS) cultures exhibited fra(X)(q27.3) at a frequency of about 10% for both twins. DNA fingerprinting indicated that the twins were identical, confirming the clinical impression, with a very thin separating amniotic membrane. To our knowledge, this is the only report of prenatal fra(X)(q27.3) detection in female identical twins, and the second report of identical twin detection [Rocchi et al., 1985]. We have diagnosed prenatally fra(X)(q27.3) in 5 female fetuses using AFCs. The average fra(X) frequency was 4% for these positive female fetuses with a range of 0.5% to 8.5%. Follow-up whole blood studies confirmed our original results at an average fra(X) frequency of 25%. IN CONCLUSION 1. Low frequencies, perhaps 1 or 2%, or a few positive cells in AFCs, are likely to increase in magnitude when confirmed in whole blood cultures either pre- or postnatally. 2. It appears likely that the risk is low for false positive results in AFCs when low frequencies of fra(X)(q27.3) are encountered.

Human Xq28 is highly gene dense with over 27 loci. Because most of these genes have been mapped by linkage to polymorphic loci, only one of which (DXS52) is informative in most families, a search was conducted for new, highly polymorphic Xq28 markers. From a cosmid library constructed using a somatic cell hybrid containing human Xq27.3----qter as the sole human DNA, a human-insert cosmid (c346) was identified and found to reveal variation on Southern blot analyses with female DNA digested with any of several different restriction endonucleases. Two subclones of c346, p346.8 and p346.T, that respectively identify a multiallelic VNTR locus and a frequent two-allele TaqI polymorphism were isolated. Examination of 21 unrelated females showed heterozygosity of 76 and 57%, respectively. These two markers appeared to be in linkage equilibrium, and a combined analysis revealed heterozygosity in 91% of unrelated females. Families segregating the fragile X syndrome with key Xq28 crossovers position this locus (designated DXS455) between the proximal Xq28 locus DXS296 (VK21) and the more distal locus DXS374 (1A1), which is proximal to DXS52. DXS455 is therefore the most polymorphic locus identified in Xq28 and will be useful in the genetic analysis of this gene dense region, including the diagnosis of nearby genetic disease loci by linkage.

241 TZOUNTZOURIS et al. reported on apparent FMR1 allele instability of 9 developmentally delayed individuals with normal-sized alleles. This raises interesting questions and possible concerns regarding laboratory genetic testing of mutated Fragile X genes. The FMR1 CGG repeat is polymorphic ( , 10–55 repeats) in the normal population, with 30 repeats being the most common size (Brown et al., 1993). These normal alleles are generally considered to be very stable. However, their findings suggest that even normal sized alleles may, on occasion, have instability. Their documentation of this observation suggests laboratories doing such testing should be aware of the potential for this confusing finding to appear in their samples. Analysis of the repeat in normal males reveals AGG triplets embedded at regular intervals, most commonly at repeat positions of about 10 and 20 (Eichler et al., 1994; Kunst et al., 1994; Zhong et al., 1995). The alleles in Fragile X families differ in several ways. They have long repeats that are unstably transmitted from one generation to the next. Repeat expansions are gender-specific, with expansion to full-mutation alleles occurring only through females. In premutation females, the expansions are a function of repeat size, with larger repeats having a greater risk of expansion than smaller ones (Nolin et al., 1996, 1999). Analysis of premutation males indicates that Fragile X alleles usually have one or no AGG interruptions within the repeat and long stretches of pure CGG repeats in the 39 end. Despite intense interest in trinucleotide disorders, the mechanism of repeat expansion is poorly understood. For Fragile X, the expansion process is especially puzzling because the repeat may expand, for example, from 75 to more than 1,000 in one generation. The presence of long, uninterrupted CGG tracts in Fragile X families clearly distinguishes them from other families. The current favored model for large repeat expansions is slippage of Okazaki fragments during DNA replication of repeat regions (Brown et al., 1993; Richards and Sutherland, 1994). Expansions in Fragile X and the other trinucleotide disorders are not likely to be the result of defective DNA replication/repair proteins because the instability is limited to a single expanded repeat, and global microsatellite instability is not observed. Recent studies suggest that repeat instability is a consequence of abnormal localized DNA secondary structure (McMurray, 1999). Biochemical studies have demonstrated the ability of CGG tracts to form alternative DNA structures. Strand displacement of the repeat regions in Okazaki fragments may allow the formation of intramolecular hairpin and tetrahelical structures. These structures may defeat the DNA replication/repair machinery within a cell. Several DNA repair-related enzymes have been recently implicated in trinucleotide repeat instability. The FEN-1 enzyme is both an endonuclease and exonuclease involved in DNA replication and repair. One preferred substrate is branched DNA structures with a single-stranded 59 flap, such as Okazaki fragments generated from the lagging strand. In contrast, secondary structures such as loops and hairpins are resistant to cleavage by FEN-1. Impaired FEN-1 function due to inefficient cleavage of secondary structures in Okazaki fragments with long stretches of triplet repeats may lead to repeat expansion, whereas structures formed in the lagging strand during replication of CGG repeats can cause replication stalling (Goredin et al., 1997). The WRN gene is a DNA helicase responsible for Werner syndrome, an autosomal recessive disease characterized as a premature aging condition. It appears to be involved in DNA replication, both as a helicase and as a 39 to 59 exonuclease. The WRN helicase has been shown to unwind CGG/AGG oligomers more rapidly than uninterrupted CGG oligomers, suggesting that it is involved in repairing abnormal DNA structures such as DNA tetrahelices (Weisman-Shomer et al., 2000). Fragile X syndrome is a complex disorder that is poorly understood. Although the mutation is known to be an expansion of a CGG repeat, the factors leading to repeat expansion remain obscure. The findings in the current report (Tzountzouris et al., this issue) raise interesting questions regarding the mechanisms of normal allele length instability and imply that laboratories need to be on the look-out for their estimated 1% of cases that can lead to diagnostic confusion.

The reliable detection of fra(X)(q27.3) in prenatal samples is important for providing genetic counseling. We have identified 5 new cases of prenatal fragile X [fra(X)] detection in 3 chorionic villus sample (CVS) and 2 amniotic fluid (AF) cell cultures. In 4 of the 5 cases, either excess thymidine (THY) or a combination of THY and 5-fluorodeoxyuridine (FUdR) was clearly superior to FUdR alone as fra(X) inducers. Amniocytes from one case were cultured only in RPMI-1640 and later exposed to FUdR or THY separately. They showed only 2% fra(X) while parallel cultures initiated in Chang medium and incubated in RPMI for at least 7 days (recovery) before fra(X) induction exhibited strikingly increased fra(X) frequencies. Chang medium alone will not allow fra(X) induction in AF (Jenkins EC, Brown WT [1986]: “Genetic Disorders and the Fetus: Diagnosis, Prevention and Treatment.” New York: Plenum Press, pp 185–204). Now, using CVS cells, we report that only 1% and 0% fra(X) were detected using FUdR or THY in cells cultured in RPMI for 4 days after removal from Chang medium. Cells with 7 days “recovery” in RPMI exhibited increases from 2 to 6%. Therefore, we have found that Chang medium is very helpful when the appropriate recovery time in another medium is allowed before fra(X) induction. Some false negative reports can be attributed to: induction in Chang medium alone; lack of sufficient recovery time after initiating cells in Chang before induction; and unavailability of the excess THY fra(X) induction system. In conclusion, 1) multiple induction systems, including a combination of FUdR/THY, and 2) initial culture of cells in Chang medium with more than a 4 day recovery period in RPMI or another appropriate medium, enhance optimal conditions for prenatal fra(X) detection. These modifications should reduce the possibility of false negatives thereby improving the reliability of prenatal fra(X) detection.

Expression of fragile X [fra(X)] (q27.3) and en-doreduplicated metaphases have been reported in methotrexate-treated (MTX) fra(X) cultures (Kerem B, Biotein R, Schaap T [1988]: Chromosoma 97: 6–10). Further, new data (Kimchi-Sarfaty C, Goitein R, Kerem B, Werner M, Medan B, Schaap T [1991]: Am J Med Genet, this issue) indicate that MTX may specifically induce polyploidy and endo-reduplication in cells with the fra(X) mutation. To confirm and extend these results, we have studied short-term lymphocyte cultures incubated in M199, a folate deficient system, and RPMI-1640 in the presence and absence of 5-fluorodeoxyuridine (FUdR) exposure during the last day of a 4 day culture. No endo-reduplicated cells were seen under these conditions and there was no change in the level of polyploidy. We also studied the distribution of polyploid and endoreduplicated cells in amniotic fluid and chorionic villus sample cultures from one fra(X) positive and 4 at-risk specimens. No increase in the incidence of polyploidy or endoreduplication was observed in cultures exposed to MTX for both 24 and 48 hours from a fra(X) positive amniotic fluid case. Cytogenetic results were fra(X) negative for the remaining 4 cases tested. There was significant discordance between our findings and those expected based on MTX-induced increased frequencies of polyploidy and endoreduplication. Thus, our studies do not confirm the reported correlation between the presence of FRAXA and increased frequencies of polyploidy and endoreduplication in MTX-exposed amniocyte cultures and there was no evidence for increased levels of polyploidy and endoreduplication in short-term fra(X) lymphocyte cultures exposed to non-MTX fra(X) induction.

The aging process and resulting senescence seem likely to have an underlying basis that is in large part encoded in our genes. This is reflected by the wide range of maximal life-span potentials that animal species have attained. They vary from about 1 day in the adult form of the May fly (Ephemera sp, imago form) to more than 150 years in some turtles (Tedudo summeri) (Lints, 1978). Even among mammals, approximately a 100-fold range is seen, from about 1 year in the smokey shrew (Sorex furneus) (Hamilton, 1940) to about 120 years in humans (Russell, 1988). This 100- to 50,000-fold variation is undoubtedly due to underlying differences in the genetic constitution of species. Analyses of the degree of genetic complexity underlying longevity have suggested it may be encoded by a few genes, perhaps 20–50, which have a major gene effect on aging (Cutler, 1980; Martin, 1977; Sacher, 1980). Therefore, to understand the genetic basis of aging, the most direct approach may be to study appropriate genetic mutations that appear to affect longevity. As such, the genetic diseases of premature aging can serve as useful models for the study of senescence.

Mitral valve prolapse, the most common inherited cardiovascular condition, has been associated with a variety of signs, symptoms and electrocardiographic abnormalities, but the true spectrum of the mitral prolapse syndrome remains in doubt because clinical findings often contribute to patient identification and their prevalence in patient groups may be overstated because of ascertainment bias. Accordingly, clinical findings in 88 patients with echocardiographic mitral prolapse were compared with those in 81 of their adult first degree relatives with mitral prolapse (a group free of ascertainment bias) and in two control groups without mitral prolapse: 172 first degree relatives and 60 spouses. Comparison of relatives with and without mitral prolapse demonstrated true associations between mitral prolapse and clicks or murmurs, or both (67 versus 9%, p less than 0.001), thoracic bony abnormalities (41 versus 16%, p less than 0.001), systolic blood pressure less than 120 mm Hg (53 versus 31%, p less than 0.001), body weight 90% or less of ideal (31 versus 14%, p less than 0.005) and palpitation (40 versus 24%, p less than 0.01). In contrast, relatives with mitral prolapse showed no significant increase over normal relatives or spouses without mitral prolapse in prevalence of chest pain, dyspnea, panic attacks, high anxiety or repolarization abnormalities, but these features were all more common in women than in men (p less than 0.01 to less than 0.001). Thus, the true spectrum of the mitral prolapse syndrome encompasses a midsystolic click and late systolic murmur, thoracic bony abnormalities, low body weight and blood pressure and palpitation. Other suggested clinical features, including nonanginal chest pain, dyspnea, panic attacks and electrocardiographic abnormalities, have appeared to be associated with mitral valve prolapse because of ascertainment bias and an erroneous classification of differences between men and women as being due to mitral valve prolapse.

At least 35 cases of prenatal fra(X) diagnosis have been confirmed and reported. Amniotic fluid, fetal blood and chorion -ic villus samples have exhibited fra(X) (q27.3) in cultures from 26 males and 9 females. Here we have detected fra(X) in female and male amniotic fluid specimens, AF1/fra(X),X and AF2/fra(X), Y, respectively, and a male CVS/fra (X), Y1 using both FUdR and excess thymidine (THY) to demonstrate the marker chromosome. Both FUdR and THY detected fra(X) and usually FUdR was superior to THY with the exception of placental cultures. It was important to examine more than one culture per protocol since no fra(X) was observed in one AF2 FUdR culture while another exhibited 19.2% expression. Similarly, confirmation studies in lung fibroblast cultures for AF2 exhibited 4.3% fra(X) in one 1ab while another found negative results. A similar observation in whole blood cultures was also made recently by us. In addition, we have recently experienced our first false negative fra(X), X prenatal diagnosis. We have observed another case where only one cellin 300 exhibited fra(X) where the male fetus was 50% at-risk and was referred to us after the 20th week of gestation by sonography. On the basis of our experience we recommend the following: (1) the excess THY fra(X) induction system is effective but not superior to FUdR; (2) at least two duplicate cultures per induction system should be analyzed forthe marker chromosome to avoid the possibility of false-negative diagnosis; (3) where fra(X) is not demonstrated or is present in very low frequencies in CVS and/or amniotic fluid cultures, complementary DNA marker studies and/or fetal blood cultures must be made available; (4) gestational age dating by ultrasonography is recommended as early as possible.

A chromosome 21 (ch. 21) flow-sorted library was screened for the presence of unique DNA segments which are specific for the 21st chromosome. By combining the techniques of somatic cell genetics and in situ hybridization, we have identified several of these recombinant probes and have regionally mapped one of them to the distal half of the long arm of chromosome 21 (q21.1-qter). This represents the first report of the sublocalization of a unique DNA segment to chromosome 21 by in situ hybridization.

The fra(X) expression in heterozygotes varies considerably from family to family. In family D23, 5 carriers express the fra(X) regardless of age. DNA studies using 3 markers were inconclusive as to whether cytogenetic testing is more reliable in this family than in others. III-2 and III-5 are the critical individuals in the pedigree; further study with closer probes should resolve this question.

We have attempted the prenatal detection of the fra(X) 9 times. Three fra(X) positive fetuses have been diagnosed: 2 males and one female. The diagnosis on the 2 males has been confirmed. The testes of the 2 fra(X) positive fetuses appeared large for gestational age. However, results of anthropometric, bone age, anatomical and neurohistological studies were normal. Normal outcome was confirmed after birth in 2 males and one female on the basis of whole blood fra(X) studies. A presumptively positive female and a presumptively negative female await confirmation. Two presumptively negative males remain unborn. Further experience is needed to establish the reliability of the prenatal detection of fra(X) (q27).

The effect of cell density on expression of fra(X) was studied. A lymphoblast cell line from one fra(X) individual and whole blood from another individual was tested at various cell densities using RPMI-1640 with FUdR (0.1 microM) 24 hrs before harvest. Densities from 0.25 X 10(5) to 2 X 10(6) cells/ml were tested. Chromosomes were G-banded and analyzed for fra(X) frequency. Increased density caused fra(X) frequency to decline in lymphoblasts and whole blood. In the established line low density fra(X) frequency was 51.2% and decreased to 6.5% at the high density. In blood fra(X) frequency was 34.7% at low density and decreased to 18% fra(X) in high density. We suspect that decay of the FUdR effect may explain the results. Our results suggest that to maximize fra(X) frequency, cultures should be inititated at low density. This may be important in analysis of low-percentage fra(X) patients, nonexpressing female carriers, and obligate nonpenetrant males.

Using the FUdR system for fragile X induction, we have observed no triradial or bisatellited configurations at fra (X) (q27.3) in over 5,000 fra(X) chromosomes examined from over 150 fra(X) individuals. Based on our observations, and those of Turner and Jacobs (1983) and Daniel et al (1984), we hypothesize that triradial configurations may not occur at Xq27 with FUdR induction. To test this hypothesis we cultured whole blood simultaneously in parallel folate-deficient and FUdR fra(X) induction systems, and systematically examined fra(X) chromosomes for triradials. Neither autosomes nor X chromosomes exhibited any apparent triradial figures in the FUdR system, while 1.4% of the fra(X) chromosomes in TC 199 exhibited a triradial. Also we observed one autosomal triradial at 4q35. We conclude that triradial configurations occur in low frequencies in the folate deficient system and seldom if ever in the FUdR system.

Chromosome 21 is the only human chromosome in the mouse human hybrid, WA17. WA17 cells were treated with 0.1 μg/ml Colcemid, lysed, and mitotic chromosomes were separated based on their sizes in sucrose velocity gradients. The position of chromosome 21 on the gradient was determined in two ways. First, 1% of each fraction was denatured, blotted onto nitrocellulose, and quantitatively probed for a repetitive sequence found only in human DNA. Of the 76 fractions, fractions 44 through 62 contained human Alu sequences. Second, fractions containing human DNA were pooled in groups of three, Southern blotted, and probed with a chromosome 21-specific probe, 21.159. Pool E-DNA (fractions 54–56) hybridized most strongly to the 21.59 probe, while the control H DNA (fractions 34–36) and mouse L cell DNA failed to hybridize. Based on densitometric scans of autoradiograms, we estimate chromosome 21 DNA in pool E is 15-fold purified with respect to WA17 DNA.

When cultures of fibroblast-like cells from inbred mouse strains RBC/Dn and AEJ/GnRk were exposed to 5-fluoro-deoxyuridine (FUdR), non-random strain-specific distributions of chromosome gaps, breaks and exchanges were observed. Throughout the genomes there appeared to be specific sites at which lesions occurred preferentially. Two strain-specific fragile sites were identified in strain RBC/Dn at G-band 15A2, and at G-band 19B in strain AEJ/GnRk. Constitutive fragile sites at G-bands 12A2 and 18A2 were identified in both strains. A strain-specific marker at G-band 9B was found in strain AEJ/GnRk. The fragile sites reported here provide an animal model for the study of chromosome fragility as well as polymorphic markers for linkage studies.

Familial Alzheimer Disease (FAD) presents as a diagnostically unique disorder having an autosomal dominant form of inheritance with an age-dependent penetrance. Recent mapping of the FAD and beta-amyloid protein (AP) genes to human chromosome 21 has raised questions as to the role of AP in the development of FAD. The involvement of gene(s) on chromosome 21 in FAD was suggested because of the observation that individuals with Down Syndrome (DS) over the age of 40 all develop the neuropathological changes associated with AD. Refinement of the AP mapping has eliminated this locus as being the site of the primary genetic defect causing FAD. This leads to speculation as to the mutation(s) responsible for and the role of gene(s) on chromosome 21 involved in FAD.

At the present time, the molecular nature of the fragile site at Xq27.3 is not well understood. To examine the sensitivity of this region to DNAase I, in situ nick translation was performed on metaphase chromosomes from a fragile X (fra(X] positive individual. In this technique DNAase I is used to nick regions of chromosomal DNA that are in "open" conformation. Biotinylated dUTP was incorporated by nick translation at these sites. The incorporation was identified by double antibody labeling and avidin-horseradish peroxidase staining. Spreads, which had been stained with this technique, were photographed and subsequently trypsin-Giemsa G banded (post-GTG banded) for chromosome identification. In 36 of 44 (82%) fra(X) positive male cells, the region distal to fra(X) (q27.3) was prominently stained in contrast to its light staining appearance in GTG preparations. The fragile site itself was outlined more clearly than can be achieved by GTG or homogeneous staining. When autosomal fragile sites were induced by the addition of 1.5 microM aphidicolin 17 hours prior to harvest, 24 of 27 (89%) fragile sites on the ends of autosomes were prominently stained in regions distal to the break. Because the fra(X) and autosomal fragile regions behaved similarly, this suggests that they have a similar conformation. Thus, while autosomal and Xq27.3 fragile sites are strongly induced by different means, the organization of these sites and the regions distal to them appear to be similar.

We screented 183 autistic males for the fra(X) and found 24 (13.1%) to be positive. Adding the subjects of this study to those of 11 other surveys, of which 6 were positive and 5 were negative, a total of 614 autistis males have been screened. Overall 47 (7.7%) were positive. Based on this estimate and the prevalence of autism and fra(X), we estimate that 12.3% of fra(X) males are autistic. We have found that 17.3% of our fra(X) males were autistic and overall a 21.2% frequency has been reported, these higher figures are most likely due to biases in age and ascertainment. With an overall 7.7% frequency of fra(X) among autistic males and an estimated 12.3% of autism among fra(X) males, we conclude there is likely to be a significant association of fra(X) with autism. Because fra(X) appears to be the single most common cause of the condition, chromosomal testing is recommended for any autistic person with undiagnosed etiology.

Induction of some fragile sites including fragile X [fra(X)] depends on the depletion of thymidine monophosphate (TMP) from the culture medium. This can be accomplished by use of inhibitors such as 5-fluorodeoxyuridine (FUdR) and by culturing cells in medium deficient in folate and TMP. FUdR inhibits the activity of thymidylate synthase (TS), thereby depleting cells of TMP. To determine the degree of FUdR inhibition of TS under routine cytogenetic culture conditions, we modified the tritiated dUMP TS method for use in short-term whole blood cultures stimulated with phytohemagglutinin. TS inhibition was highly variable across whole blood cultures from 30 individuals exposed to FUdR during the last 24 hours of a 4 day culture. If an additional dose of FUdR was added 12 hours before harvest, TS inhibition usually increased. These findings have a potential impact on the use of FUdR for the diagnosis of the fra(X) syndrome.

Infectious fractions prepared from scrapie-infected hamster brains contain a protein, PrP 27–30, which shares antigenic determinants with polypeptides found in similarly prepared fractions from patients with Creutzfeldt-Jakob disease. cDNA sequences encoding the hamster PrP 27–30 identified homologous sequences in the human genome as well as in normal human brain mRNA preparations. Antibodies raised against the mouse PrP's identified antigenically related peptides in both normal hamster and human brain as well as in scrapie-infected hamster brain and CJD-affected human brain. By using in situ hybridization we localized the homologous human genomic sequences on the short arm of chromosome 20. Our results indicate that the reportedly unique proteins detected in human CJD preparations derive from normal human gene products.

Fragile X is the most common inherited cause of intellectual disability and is frequently associated with autism. The syndrome is due to mutations of the FMR1 gene that result in the absence of fragile X mental retardation protein (FMRP). We have developed a rapid, highly sensitive method for quantifying FMRP from dried blood spots and lymphocytes. This assay uses two new antibodies, a bacterially expressed abbreviated FMRP standard, and a Luminex platform to quantify FMRP. The assay readily distinguished between samples from males with fragile X full mutations and samples from normal males. It also differentiated mosaic from nonmosaic full-mutation male samples. This assay, because of its methodology and minimal cost, could be the basis for newborn or population screening.

Summary The location of human superoxide dismutase has been identified on 2-D gels of cultured fibroblast extracts. Human superoxide dismutase was located on 2-D gels of a mouse-human somatic cell hybrid containing only human chromosome 21, and was absent in the mouse parental cell line. The native enzyme is a diner composed of two identical monomers present on 2-D gels as a single spot of molecular weight approximately 16,000 and isoelectric point of pH 6.2. Comparison of the density of the SOD spot on gels of a trisomy 21 cell strain to that of a monosomy 21 cell strain showed a 3 fold increase indicating agene dosage effect was present. This approach demonstrates the utility of quantitative 2-0 analysis for gene dosage studies.

Using pulsed-field gradient-gel electrophoresis (PFGE) we compared two fragile X (fra(X] chromosomes from individuals in families that exhibit linkage heterogeneity between fra(X) and coagulation factor IX (F9). The analysis of very large restriction fragments indicated that there is a structural difference in the interval between fra(X) and F9 near the locus DXS105. Differences were observed in the Sfi I partial digestion analysis and in the Mlu I pattern of the DXS105 region. Digestion with Nru I and Sst II also showed differences between these alleles. The analyses suggest that the alleles differ in a region of greater than 200 kb. Analysis of other normal and fra(X) chromosomes will be necessary to determine whether the observed difference is a normal population variant or if it may be responsible for the linkage heterogeneity observed between these loci.

Subjects with mitral-valve prolapse (MVP) have been observed to have an asthenic body build. To determine whether body-weight differed between individuals with inherited MVP and normal subjects, 177 relatives of 45 patients with MVP were studied, 35 female and 19 male relatives had MVP, and 51 female and 72 male relatives did not. There was no difference in mean height between relatives with and without MVP, but those with MVP weighed significantly less. Blood pressure was also significantly lower in relatives with MVP than in normal relatives. It is suggested that the lower blood pressure and the possible beneficial effects of lower weight on other cardiovascular risk factors may provide a selective advantage to carriers of the MVP gene, explaining its high prevalence in the general population. These findings may provide the first example of a common inherited condition which is systematically associated with changes in body-weight and blood pressure.

The fragile X chromosome has been identified in specimens from 17 male and 10 female fetuses in 11 laboratories throughout the world, obtained from at least 79 fetuses at increased risk for the fra(X) syndrome. Of these, 19 were confirmed, 6 were pending, 1 was negative and 1 could not be confirmed. Twenty-five of the 79 cases were studied in our laboratory (Institute for Basic Research [IBR]) and resulted in fra(X) demonstration in specimens from 3 male and 5 female fetuses. All 3 males and 2 of the 5 females have been confirmed. When amniocytes from the two confirmed female fetuses were exposed to FUdR after culturing in Chang medium, fra(X) frequencies were virtually negative indicating that Chang medium should not be used in fragile X studies at least when FUdR is used to induce fragility. Finally, amniocytes from a fra(X) male fetus studied in 3 different laboratories exhibited strikingly different frequencies. To date, we have experienced no false-positives or negatives, but the latter case was controversial. It is recommended that laboratories undertaking fra(X) prenatal detection use a combination of at least two different proven induction systems as well as complementary DNA marker studies to prevent false negative diagnosis.