Height discordance in monozygotic females is not attributable to discordant inactivation of X-linked stature determining genes.

We tested the hypothesis that X-linked genes determining stature which are subject to skewed or non-random X-inactivation can account for discordance in height in monozygotic female twins. Height discordant female monozygotic adult twins (20 pairs) were identified from the Australian Twin Registry, employing the selection criteria of proven monozygosity and a measured height discordance of at least 5 cm. Differential X-inactivation was examined in genomic DNA extracted from peripheral lymphocytes by estimating differential methylation of alleles at the polymorphic CAG triplet repeat of the Androgen receptor gene (XAR). There were 17/20 MZ pairs heterozygous at this locus and informative for analysis. Of these, 10/17 both had random X-inactivation, 5/17 showed identical X-inactivation patterns of non random inactivation and 2/17 (12%) showed discordant X-inactivation. There was no relationship between inactivation patterns and self-report chorionicity. We conclude that non-random X-inactivation does not appear to be a major contributor to intra-pair height discordance in female MZ twins.     

Trends in triplet stillbirth rates in Japan, 1975-1998.

Stillbirth rates of triplet births in the whole of Japan were analyzed using vital statistics from 1975 to 1998. Stillbirths were registered at 12 weeks gestation or later. The stillbirth rate was significantly higher in like- than in unlike-sex triplets for 1975-1998. During the 23-year period the stillbirth rate decreased from 342 to 49 per 1000 total births for like-sex and from 195 to 54 for unlike-sex triplets. The decrease in the stillbirth rate in the 23- year period was greater in both like- and unlike-sex triplets than in singleton and twin births. Risk factors for stillbirth in triplets were like-sex, youngest or oldest maternal age groups, shorter gestational age and lower birthweight. It is recommended that the optimum period to give birth for triplet pregnancies is 34-35 weeks of gestation for Japanese women.     

Body weight changes throughout pregnancy in the common marmoset Callithrix jacchus

Adult female common marmosets were weighed weekly for periods of 6-30 months. Of 27 animals, 25 were pregnant at some stage of the investigation. Patterns of body weight change throughout singleton, twin and triplet pregnancies were obtained and compared. Maternal weight increase was dependent on the number of young in utero; however, little or no change in weight was observed during the first 13 weeks of gestation, irrespective of the number of offspring delivered subsequently. The overall maternal:fetal body weight ratio was 1.00:0.21, ranging from 1.00:0.11 for singleton to 1.00:0.26 for triplet pregnancies. Lactation had little or no effect on maternal body weight during the first 4 weeks postpartum.     

Microstructural observations of villous and membrane histology in monochorionic triplet placenta after in vitro fertilization

The frequency of triplet gestation is low in humans, estimated at 1:6400 deliveries. Monochorionic gestations represent a subpopulation of approximately 10% of these triplet pregnancies. Hypertensive complications are known to occur with greater frequency in the context of multiple gestation. In this report we describe microscopic placental changes associated with pre-eclampsia and proteinuria in the setting of an uncommon monochorionic-triamniotic triplet pregnancy achieved via in vitro fertilization. Histologic features observed in this case include placental stromal fibrosis and increased syncytial nodularity (Tenney-Parker change). In this triplet delivery resulting from two consecutive fissions of a single embryo, chorion and amnion configuration are also characterized with a review of the literature discussing the potential relationship between in vitro culture conditions and monozygotic multiple gestation.     

Genetic influences on dentition and dental arch dimensions: a study of monozygotic and dizygotic triplets

Previous studies of the tooth and dental arch dimensions in twins have suggested that the size and form of the dentition is more under genetic control than the size and form of the dental arch. A study of dentition and dental arch was carried out on three sets of same-sex triplets, two sets being monozygotic and one set dizygotic. No significant differences were found in the dental arch dimensions in any set of the triplets. The tooth dimensions showed no significant differenes in the monozygotic triplet sets or in the monozygotic pair from the dizygotic triplet set, but showed significant differences between the monozygotic pair and the third member of the dizygotic set. This confirms the data obtained from twin studies.     

Maternal serum alpha-fetoprotein levels in multiple pregnancy.

Maternal serum alpha-fetoprotein (AFP) levels were higher in 10 twin pregnancies and one triplet pregnancy than in 22 control singleton pregnancies matched for maternal age, parity, and the time of gestation at which the serum sample was taken. In twin pregnancies the average AFP levels were double those found in singleton pregnancies and the level in the triplet pregnancy was even higher. Raised maternal serum AFP values due to multiple pregnancy should not cause unnecessary amniocentesis in the diagnosis of anencephaly or spina bifida if an ultrasound investigation is routinely performed first.     

The differential staining pattern of the X chromosome in the embryonic and extraembryonic tissues of postimplantation homozygous tetraploid mouse embryos.

(C57BL x CBA)F1 hybrid female mice were mated with hemizygous Rb(X.2)2Ad males to distinguish the paternal X chromosome. Homozygous tetraploids were produced by blastomere fusion at the 2-cell stage, and 161 of these were transferred to recipients and analysed on the 10th day of gestation. 59 implants contained resorptions and 76 contained either an embryo and/or extraembryonic membranes. 38 (20, XXXX and 18, XXYY) were analysed to investigate their X-inactivation pattern. Embryonic and yolk sac endodermally- and mesodermally-derived samples were analysed by G-banding and by Kanda analysis. In the XX and XY controls, the predicted pattern of X-inactivation was observed, though 12.2% of metaphases in the XX series displayed no X-inactivation. In the XY series the Y chromosome was seen in a high proportion of metaphases. In the XXXX tetraploids, 8 cell lineages were recognized with regard to their X-inactivation pattern, though most belonged to the following 3 categories: (XmXm)XpXp, Xm(XmXp)Xp and XmXm(XpXp). The other categories were only rarely encountered. In the embryonic and mesodermally-derived tissue the ratio of these groups was close to 1:2:1, whereas in the endodermally-derived tissue it was 1:4.11:4.88, due to preferential paternal X-inactivation. A significant but small proportion of all 3 tissues analysed displayed no evidence of X-inactivation. Indirect evidence suggests that this represents a genuine group because of the high efficiency of the Kanda staining. The presence of the Xm(XmXp)Xp category is consistent with the expectation that X-inactivation occurs randomly in 2 of the 4 X chromosomes present. The presence of small numbers of preparations with no evidence of X-inactivation and other unexpected categories suggests that these are probably selected against during development.     

Umbilical artery doppler sonography in Saanen goat fetuses during singleton and multiple pregnancies

The objective of this study was to evaluate the blood flow from the umbilical artery (UA) in healthy pregnant goats. Doppler sonography examinations were performed every two weeks in Saanen goats with a singleton (n = 5) or multiple (n = 4) pregnancy from 40 to 145 days of gestation. Fetal heart rates (FHR), pulsatility index (PI), and resistance index (RI) were recorded from the mid-cord site of the free-floating umbilical cord. FHR decreased gradually as the pregnancy progressed and significantly decreased during the last two examinations of all fetuses (P < 0.05). The mean PI level was dramatically different (P < 0.05) until 85 days of gestation, after which it reached a plateau level until parturition. Similar to PI, RI decreased by 85 days of gestation (P < 0.05), and decreased again by 130s gestation. No reverse or absent end-diastolic flow were observed in fetuses during any examinations. When comparing singleton and multiple pregnancies, there were no significant differences in UA pulsatility or resistance in fetuses seen. The middle of the second trimester was observed to be a threshold stage for indices in the pattern of caprine pregnancy.In conclusion, this work provides additional values that might be useful when evaluating singleton and multiple pregnancies, and may be evaluated in further studies regarding fetal monitoring.     

Selected aspects of collared peccary (Dicotyles tajacu) reproductive biology in a captive texas herd

Collared peccaries (Dicotyles tajacu) from 32 litters were born in captivity during the period from September, 1981 through July, 1983. Incidence of singleton, twin, and triplet litters was 28%, 66%, and 6%, respectively. Mean (± SE) weight of young from singleton, twin, and triplet litters was 751 (± 35) gm, 698 (± 17) gm, and 612 (± 74) gm, respectively. Sex ratio among all litters was 56:44 (M:F). Neonatal mortality amounted to 9% of the young born. Mean duration of estrus was 2.60 days and mean length of gestation was 145.6 days. Evidence indicated that multiple copulations increased ovulation rate and the chance for conception.     

Pigmentary mosaicism in hypomelanosis of Ito

We report on a female with mental and motor retardation, facial dysmorphism, abnormal pigmentation reminiscent to hypomelanosis of Ito (HI), and karyotypic mosaicism involving a small supernumerary marker chromosome. The marker chromosome was defined by fluorescence in situ hybridisation (FISH) as a ring X chromosome with breakpoints in the juxtacentromeric region. FISH analysis showed that the ring does not include the XIST locus at the X-inactivation centre and, therefore, may not be subject to X inactivation. X-inactivation studies with the HUMARA (human androgen receptor) and FMR1 assay showed a skewed X-inactivation pattern (85:15) with preferential inactivation of the paternal X chromosome. These results are discussed with respect to the role of functional disomy of Xp in the pathogenesis of HI. Received: 16 February 1998 / Accepted: 17 July 1998     

Two cases with dichorionic diamniotic triplet pregnancy and early cord entanglement.

Within a period of 6 years (1994-99) we registered 29 triplet deliveries out of a total of 13,969 hospital deliveries (0.02%). Since there is limited information about specific problems of chorionicity in triplet pregnancies, we analysed the 29 cases according to origin of pregnancy and chorionicity. We here report on two cases with a high risk according to chorionicity with dichorionic (DC) diamniotic (DA) triplet pregnancies. Out of the two cases, one pregnancy was spontaneous and one originated after in-vitro fertilization. In both pregnancies, cord entanglement was detected early in pregnancy (at 10 and 15 weeks) by color Doppler velocimetry demonstrating different heart rates within the segment of the entangled umbilical branches. The pregnancies were followed by documenting fetal behavior and color Doppler velocimetry of umbilical and fetal arteries at weekly intervals. In both cases, a primary Cesarean section was performed after detection of lung maturity. In the first case, one of the MA triplets had a transposition of the great arteries and abnormal lung vein drainage, which was the reason for neonatal death three weeks postnatally. Although early cord entanglement has been described in MA twins, this series demonstrates that it can as well be demonstrated in MA triplets. The early detection allows for extensive surveillance of the a priori high risk triplet pregnancy.     

A new assay for the analysis of X-chromosome inactivation based on methylation-specific PCR

The pattern of X-chromosome inactivation in females is currently evaluated by assays of differential methylation in the genes between the active and the inactive X chromosomes, with methylation-sensitive enzymes. We report a new assay in the human androgen receptor (HUMARA) locus involving a methylation-specific polymerase chain reaction (M-PCR) technique, independent of the use of restriction enzymes. The assay involves the chemical modification of DNA with sodium bisulfite and subsequent PCR. By using the assay with specific primers for the methylated allele, we obtained an X-inactivation pattern based on the ratio of the maternal inactive X to the paternal inactive X. These patterns were consistent with those obtained by conventional PCR assay at the same locus in 48 female cases. We also obtained another X-inactivation pattern based on the ratio of the maternal active X to the paternal active X by using specific primers for the unmethylated allele. The latter pattern was complementary to the former pattern, and a combination of these patterns produced a reliable X-inactivation pattern. The assay revealed that 12 (11%) of the 105 normal females had non-random inactivation patterns (>80:20 or <20:80). Four patients with an X; autosome translocation showed extremely non-random patterns, and these results were consistent with those obtained by previous molecular/cytogenetic studies. We conclude that M-PCR provides an accurate assay for X-inactivation and that it can be performed on various DNA samples unsuitable for restriction digestion. Received: 3 September 1998 / Accepted: 10 October 1998     

Timing of the absence of <Emphasis Type="Italic">FMR1</Emphasis> expression in full mutation chorionic villi

Fragile X syndrome is caused by the expansion of the CGG repeat in the 5' untranslated region of the FMR1 gene. This expansion leads to methylation of the FMR1 promoter region thereby blocking FMR1 protein (FMRP) expression. Prenatal diagnosis can be performed on chorionic villi samples (CVS) by Southern blot analysis. Alternatively, for males, an immunohistochemical method has been introduced for CVS. In this study, we have used this immunocytochemical method for CVS in full mutation male fetuses at different times of gestational age, varying from 10.0-12.5 weeks, and in two cases of full mutation female fetuses (>13 weeks). FMRP expression studies in CVS from full mutation male fetuses (10.0-12.5 weeks) illustrate the timing of the disappearance of FMRP expression in these CVS. Until approximately 10 weeks of gestation, FMRP is expressed normally in full mutation male CVS, whereas FMRP is completely absent at 12.5 weeks of gestation. FMRP expression in full mutation female CVS (>13 weeks) is completely absent in a number of villi, whereas other villi show normal FMRP expression. Unlabelled villi can only be present in the absence of the expression of the full mutation FMR1 gene on one X-chromosome together with the X-inactivation of the normal X allele. FMRP positive villi can be explained by an active normal X allele. The presence of both positive and negative villi indicates that X-inactivation in human CVS is a random process. No villi are found with a mixture of both FMRP-expressing and non-FMRP-expressing cells. This indicates that X-inactivation occurs very early in development, before the villi start to proliferate, and that X-inactivation in villi is a clonal process. In addition, our results indicate that the timing of both X-inactivation and full mutation FMR1 allele inactivation is different, i.e. X-inactivation occurs earlier in development than inactivation of the full mutation.     

Chlorophyll triplet states associated with Photosystem I and Photosystem II in thylakoids of the green alga Chlamydomonas reinhardtii

The analysis of FDMR spectra, recorded at multiple emission wavelengths, by a global decomposition technique, has allowed us to characterise the triplet populations associated with Photosystem I and Photosystem II of thylakoids in the green alga Chlamydomonas reinhardtii. Three triplet populations are observed at fluorescence emissions characteristic of Photosystem II, and their zero field splitting parameters have been determined. These are similar to the zero field parameters for the three Photosystem II triplets previously reported for spinach thylakoids, suggesting that they have a widespread occurrence in nature. None of these triplets have the zero field splitting parameters characteristic of the Photosystem II recombination triplet observed only under reducing conditions. Because these triplets are generated under non-reducing redox conditions, when the recombination triplet is undetectable, it is suggested that they may be involved in the photoinhibition of Photosystem II. At emission wavelengths characteristic of Photosystem I, three triplet populations are observed, two of which are attributed to the P(700) recombination triplet frozen in two different conformations, based on the microwave-induced fluorescence emission spectra and the triplet minus singlet difference spectra. The third triplet population detected at Photosystem I emission wavelengths, which was previously unresolved, is proposed to originate from the antenna chlorophyll of the core or the unusually blue-shifted outer antenna complexes of this organism.     

Chlorophyll triplet states associated with Photosystem I and Photosystem II in thylakoids of the green alga Chlamydomonas reinhardtii

The analysis of FDMR spectra, recorded at multiple emission wavelengths, by a global decomposition technique, has allowed us to characterise the triplet populations associated with Photosystem I and Photosystem II of thylakoids in the green alga Chlamydomonas reinhardtii. Three triplet populations are observed at fluorescence emissions characteristic of Photosystem II, and their zero field splitting parameters have been determined. These are similar to the zero field parameters for the three Photosystem II triplets previously reported for spinach thylakoids, suggesting that they have a widespread occurrence in nature. None of these triplets have the zero field splitting parameters characteristic of the Photosystem II recombination triplet observed only under reducing conditions. Because these triplets are generated under non-reducing redox conditions, when the recombination triplet is undetectable, it is suggested that they may be involved in the photoinhibition of Photosystem II. At emission wavelengths characteristic of Photosystem I, three triplet populations are observed, two of which are attributed to the P₇₀₀ recombination triplet frozen in two different conformations, based on the microwave-induced fluorescence emission spectra and the triplet minus singlet difference spectra. The third triplet population detected at Photosystem I emission wavelengths, which was previously unresolved, is proposed to originate from the antenna chlorophyll of the core or the unusually blue-shifted outer antenna complexes of this organism.     

Risk factors for heart disease associated with abnormal sidedness

BACKGROUND: The purpose of this study is to obtain information on potential familial and environmental risk factors for liveborn cases of heart disease associated with abnormal visceral and vascular sidedness, heterotaxy heart disease, so that hypotheses about this congenital cardiovascular malformation (CCVM) and its risk factors can be generated. We describe the characteristics of infants with heterotaxy heart malformations and case-control comparisons of interview data obtained on parental socio-demographic characteristics, occupational and household environmental exposures. METHODS: Cases and controls are drawn from the Baltimore Washington Infant Study (BWIS) a population based case control study of CCVM diagnosed in the region from 1981-89. RESULTS: Maternal diabetes (OR = 5.5, 95% CI = 1.6-19.1) and family history of malformations (OR = 5.1, 95% CI = 2.0-12.9) are strongly associated with cardiac disorders of sidedness. Cocaine use by mothers during the first trimester is associated with heterotaxy heart disease with odds of 3.7 (95% CI = 1.3-10.7). Cases of isolated dextrocardia shared risk factors with other heterotaxy malformations. The odds of a twin proband having heterotaxy heart disease is 4.8 (95% CI = 1.9-11.8) compared to singleton births. Twin probands are predominantly monozygotic twins in contrast to twin probands in other congenital cardiovascular malformations. CONCLUSIONS: Our findings are consistent with a role for multiple genetic factors in the development of left-right axis formation and with variable cardiac phenotypes according to gene expression and possible gene-environment interactions. Association with monozygotic twinning and with parental cocaine use may point to additional mechanistic clues for future research.     

Mouse singletons and twins developed from isolated diploid blastomeres supported with tetraploid blastomeres.

The aim of this study was to obtain mice, hopefully identical multiplets, from single diploid blastomeres isolated at the 4-cell stage, or from pairs of sister blastomeres isolated at the 8-cell stage. To this end isolated blastomeres were aggregated with one or two tetraploid carrier embryos produced by electrofusion of 2-cell embryos. Diploid embryos were albino and homozygous for the "a" allele of glucose-phosphate isomerase (GPI-1a1a) and tetraploid embryos were pigmented and GPI-1b1b. The aggregates were cultured in vitro up to the blastocyst stage. Each quartet (occasionally triplet or doublet) of chimaeric blastocysts was transplanted to the oviduct of a separate pseudopregnant recipient. Altogether 62 blastocysts were transplanted to 17 recipients. Eight full-term foetuses (two singletons and three pairs of twins) were rescued by Caesarian section on day 19, 20 or 21 of pregnancy. Three young (one singleton and twins) were successfully reared by foster mothers and proved to be normal and fertile females. All foetuses and animals were albino. In five individuals only the 1-A form of GPI (characteristic for 2n blastomere) was found. In one adult female traces of the 1-B form of GPI (characteristic for 4n carrier blastomeres) were detected in the heart and the lungs while 4 other organs contained only the 1-A form. These observations strongly suggest that the majority of foetuses/animals produced according to our experimental system are 'pure' diploids rather than 2n/4n chimaeras, and that the described method can be used in future to produce twins, triplets and quadruplets in the mouse. Our study confirms earlier work by Kelly (1975, 1977) that 'quarter' blastomeres of the mouse are still totipotent.     

Flanking sequence effects within the pyrimidine triple-helix motif characterized by affinity cleaving.

Nearest neighbor interactions affect the stabilities of triple-helical complexes. Within a pyrimidine triple-helical motif, the relative stabilities of natural base triplets T.AT, C + GC, and G.TA, as well as triplets, D3.TA and D3.CG, containing the nonnatural deoxyribonucleoside 1-(2-deoxy-beta-D-ribofuranosyl)-4-(3-benzamido)phenylimidazole (D3) were characterized by the affinity cleaving method in the context of different flanking triplets (T.AT, T.AT: T.AT, C + GC: C + GC, T.AT: G + GC, C + GC). The to be insensitive to substitutions in either the 3' or 5' directions, while the relative stabilities of triple helices containing C + GC triplets decreased as the number of adjacent C + GC triplets increased. Triple helices incorporating a G.TA interaction were most stable when this triplet was flanked by two T.AT triplets and were adversely affected when a C + GC triplet was placed in the adjacent 5' direction. Similarly, complexes containing D3.TA or D3.CG triplets were most stable when the triplet was flanked by two T.AT triplets but were destabilized when the adjacent 3' neighbor position was occupied with a C + GC triplet. This information regarding sequence composition effects in triple-helix formation establishes a set of guidelines for targeting sequences of double-helical DNA by the pyrimidine triple-helix motif.     

Development of multiple embryos in polyembryonic insertional mutant OsPE of rice

A T-DNA insertional mutant OsPE of rice gives twin and triplet seedlings in up to 20?% of the seeds. Detailed cytological and histological analysis of OsPE indicated normal male and female gametogenesis in the OsPE mutant. Confocal laser scanning microscopic (CLSM) analysis of the developing seeds of OsPE showed multiple embryo development in up to 60?% of the ovules. The multiple embryos, mostly twins and triplets, and rarely quadruplets, developed through sequential cleavage from a single zygotic embryo in each ovule. The reduced number of multiple seedlings compared with multiple embryos observed in CLSM study may be attributed to their inability to develop further due to competition in a single embryo sac. Key message Multiple seedlings in the OsPE mutant are due to sequential proliferation and cleavage of the zygotic embryos. The nucellar tissue was not involved in multiple embryo development.     

Expansion of GAA triplet repeats in the human genome: unique origin of the FRDA mutation at the center of an Alu

Friedreich ataxia is caused by expansion of a GAA triplet repeat (GAA-TR) in the FRDA gene. Normal alleles contain <30 triplets, and disease-causing expansions (66-1700 triplets) arise via hyperexpansion of premutations (30-65 triplets). To gain insight into GAA-TR instability we analyzed all triplet repeats in the human genome. We identified 988 (GAA)(8+) repeats, 291 with >or=20 triplets, including 29 potential premutations (30-62 triplets). Most other triplet repeats were restricted to <20 triplets. We estimated the expected frequency of (GAA)(6+) repeats to be negligible, further indicating that GAA-TRs have undergone significant expansion. Eighty-nine percent of (GAA)(8+) sequences map within G/A islands, and 58% map within the poly(A) tails of Alu elements. Only two other (GAA)(8+) sequences shared the central Alu location seen at the FRDA locus. One showed allelic variation, including expansions analogous to short Friedreich ataxia mutations. Our data demonstrate that GAA-TRs have expanded throughout primate evolution with the generation of potential premutation alleles at multiple loci.