Dorothy Warburton

De novo mutations in histone-modifying genes in congenital heart disease.

Congenital heart disease (CHD) is the most frequent birth defect, affecting 0.8% of live births. Many cases occur sporadically and impair reproductive fitness, suggesting a role for de novo mutations. Here we compare the incidence of de novo mutations in 362 severe CHD cases and 264 controls by analysing exome sequencing of parent–offspring trios. CHD cases show a significant excess of protein-altering de novo mutations in genes expressed in the developing heart, with an odds ratio of 7.5 for damaging (premature termination, frameshift, splice site) mutations. Similar odds ratios are seen across the main classes of severe CHD. We find a marked excess of de novo mutations in genes involved in the production, removal or reading of histone 3 lysine 4 (H3K4) methylation, or ubiquitination of H2BK120, which is required for H3K4 methylation. There are also two de novo mutations in SMAD2, which regulates H3K27 methylation in the embryonic left–right organizer. The combination of both activating (H3K4 methylation) and inactivating (H3K27 methylation) chromatin marks characterizes ‘poised’ promoters and enhancers, which regulate expression of key developmental genes. These findings implicate de novo point mutations in several hundreds of genes that collectively contribute to approximately 10% of severe CHD.

Holoprosencephaly due to mutations in ZIC2 , a homologue of Drosophila odd-paired

Holoprosencephaly (HPE) is the most common structural anomaly of the human brain and is one of the anomalies seen in patients with deletions and duplications of chromosome 13. On the basis of molecular analysis of a series of patients with hemizygous deletions of the long arm of chromosome 13, we have defined a discrete region in band 13q32 where deletion leads to major developmental anomalies (the 13q32 deletion syndrome). This approximately 1-Mb region 1 lies between markers D13S136 and D13S147. Patients in which this region is deleted usually have major congenital malformations, including brain anomalies such as HPE or exencephaly, and digital anomalies such as absent thumbs2. We now report that human ZIC2 maps to this critical deletion region and that heterozygous mutations in ZIC2 are associated with HPE. Haploinsufficiency for ZIC2 is likely to cause the brain malformations seen in 13q deletion patients.

Molecular Definition of 22q11 Deletions in 151 Velo-Cardio-Facial Syndrome Patients

Velo-cardio-facial syndrome (VCFS) is a relatively common developmental disorder characterized by craniofacial anomalies and conotruncal heart defects. Many VCFS patients have hemizygous deletions for a part of 22q11, suggesting that haploinsufficiency in this region is responsible for its etiology. Because most cases of VCFS are sporadic, portions of 22q11 may be prone to rearrangement. To understand the molecular basis for chromosomal deletions, we defined the extent of the deletion, by genotyping 151 VCFS patients and performing haplotype analysis on 105, using 15 consecutive polymorphic markers in 22q11. We found that 83% had a deletion and >90% of these had a similar approximately 3 Mb deletion, suggesting that sequences flanking the common breakpoints are susceptible to rearrangement. We found no correlation between the presence or size of the deletion and the phenotype. To further define the chromosomal breakpoints among the VCFS patients, we developed somatic hybrid cell lines from a set of VCFS patients. An 11-kb resolution physical map of a 1,080-kb region that includes deletion breakpoints was constructed, incorporating genes and expressed sequence tags (ESTs) isolated by the hybridization selection method. The ordered markers were used to examine the two separated copies of chromosome 22 in the somatic hybrid cell lines. In some cases, we were able to map the chromosome breakpoints within a single cosmid. A 480-kb critical region for VCFS has been delineated, including the genes for GSCL, CTP, CLTD, HIRA, and TMVCF, as well as a number of novel ordered ESTs.

Immunolocalization of CENP-A suggests a distinct nucleosome structure at the inner kinetochore plate of active centromeres

The trilaminar kinetochore directs the segregation of chromosomes in mitosis and meiosis. Despite its importance, the molecular architecture of this structure remains poorly understood [1]. The best known component of the kinetochore plates is CENP-C, a protein that is required for kinetochore assembly [2], but whose molecular role in kinetochore structure and function is unknown. Here we have raised for the first time monospecific antisera to CENP-A [3], a 17 kD centromere-specific histone variant that is 62% identical to the carboxy-terminal domain of histone H3 [4,5] and that resembles the yeast centromeric component CSE4 [6]. We have found by simultaneous immunofluorescence with centromere antigens of known ultrastructural location that CENP-A is concentrated in the region of the inner kinetochore plate at active centromeres. Because CENP-A was previously shown to co-purify with nucleosomes [7], our data suggest a specific nucleosomal substructure for the kinetochore. In human cells, these kinetochore-specific nucleosomes are enriched in alpha-satellite DNA [8]. However, the association of CENP-A with neocentromeres lacking detectable alpha-satellite DNA, and the lack of CENP-A association with alpha-satellite-rich inactive centromeres of dicentric chromosomes together suggest that CENP-A association with kinetochores is unlikely to be determined solely by DNA sequence recognition. We speculate that CENP-A binding could be a consequence of epigenetic tagging of mammalian centromeres.

The distribution of chromosomal genotypes associated with Turner's syndrome: livebirth prevalence rates and evidence for diminished fetal mortality and severity in genotypes associated with structural X abnormalities or mosaicism

SummaryThe proportions of chromosomal genotypes associated with the Turner syndrome genotype (excluding those with a Y chromosome) in embryonic and fetal deaths, in fetuses diagnosed prenatally, and in living individuals were reviewed. The ratio of apparent non-mosaic 45,X to 45,X/46,XX mosaics was notably higher in a New York City series of embryonic and fetal deaths, 13.5 to 1, than in living individuals reported to the New York State Chromosome Registry, 3.6 to 1. The ratios of 45,X cases to those with 46,Xi(Xq) was 5.7 to 1 in living individuals, but was 112 to 0 in embryonic and fetal deaths, an even greater disparity, indicating the marked fetoprotective effect of more than one dose of some locus or loci on the long arm of the X chromosome. The results of review of data pertinent to the livebirth prevalence of the (apparent non-mosaic) 45,X geno-type suggest a rate of about 5.7 per 100,000 livebirths (11.8 per 100,000 females) with 95% confidence limits of 2.6 per 100,000 to 10.8 per 100,000. The rate in fetuses diagnosed prenatally is 8/27,202, about 30 per 100,000. As a large proportion of these, perhaps 75%, would undergo spontaneous fetal death if not terminated electively, these figures are consistent with the direct estimate of livebirth rates. The rate of all those with X chromosome abnormalities (without a Y chromosome) associated with signs or symptoms that eventually lead to referral for cytogenetic study was estimated at a minimum of 10.7 per 100,000 livebirths, (22.2 per 100,000 females).

The genetic architecture of Down syndrome phenotypes revealed by high-resolution analysis of human segmental trisomies

Down syndrome (DS), or trisomy 21, is a common disorder associated with several complex clinical phenotypes. Although several hypotheses have been put forward, it is unclear as to whether particular gene loci on chromosome 21 (HSA21) are sufficient to cause DS and its associated features. Here we present a high-resolution genetic map of DS phenotypes based on an analysis of 30 subjects carrying rare segmental trisomies of various regions of HSA21. By using state-of-the-art genomics technologies we mapped segmental trisomies at exon-level resolution and identified discrete regions of 1.8–16.3 Mb likely to be involved in the development of 8 DS phenotypes, 4 of which are congenital malformations, including acute megakaryocytic leukemia, transient myeloproliferative disorder, Hirschsprung disease, duodenal stenosis, imperforate anus, severe mental retardation, DS-Alzheimer Disease, and DS-specific congenital heart disease (DSCHD). Our DS-phenotypic maps located DSCHD to a <2-Mb interval. Furthermore, the map enabled us to present evidence against the necessary involvement of other loci as well as specific hypotheses that have been put forward in relation to the etiology of DS—i.e., the presence of a single DS consensus region and the sufficiency of DSCR1 and DYRK1A, or APP, in causing several severe DS phenotypes. Our study demonstrates the value of combining advanced genomics with cohorts of rare patients for studying DS, a prototype for the role of copy-number variation in complex disease.

DRINKING DURING PREGNANCY AND SPONTANEOUS ABORTION

The frequency of drinking alcohol among 616 women who aborted spontaneously (cases) was compared with that among 632 women who delivered after at least 28 weeks gestation (controls). 17.0% of cases reported drinking twice a week or more during pregnancy whereas among controls, only 8.1% of women reported drinking twice a week or more. The hypothesis that drinking during pregnancy is associated with spontaneous abortion was tested by maximum-likelihood logistic regression analysis. The adjusted-odds ratio for this association was 2.62. We estimate that more than 1/4 of pregnant women drinking twice a week or more are likely to abort, compared with about 14% among women who drink less often. Consideration of wine, beer, and spirits suggested that the minimum harmful dosage was 1 ounce of absolute alcohol. Several potentially confounding variables, including maternal age, gestation, prior spontaneous abortions, smoking, and nausea/vomiting, were controlled in the analysis. The association between drinking during pregnancy and spontaneous abortion did not vary with these factors. Even moderate alcohol consumption during pregnancy is a risk factor for, and may be a cause of, spontaneous abortion. Among the possible mechanisms, acute fetal poisoning seems the most likely, although chronic poisoning is also possible.

Trisomy Recurrence: A Reconsideration Based on North American Data

Few reliable data exist concerning the recurrence risk for individual trisomies or the risk for recurrence of trisomy for a different chromosome. We collected records from two sources: (1) prenatal diagnoses performed at the Hopital Sainte-Justine in Montreal and (2) karyotype analyses performed at Genzyme. Using the standardized morbidity ratio (SMR), we compared the observed number of trisomies at prenatal diagnosis with the expected numbers, given maternal age-specific rates (by single year). SMRs were calculated both for recurrence of the same trisomy (homotrisomy) and of a different trisomy (heterotrisomy). After all cases with an index trisomy 21 were combined, the SMR for homotrisomy was 2.4 (90% CI 1.6-3.4; P=.0005). For women with both the index trisomy and subsequent prenatal diagnosis at age <30 years, the SMR was 8.0; it was 2.1 for women with both pregnancies at age >/=30 years. For the other index viable trisomies (13, 18, XXX, and XXY) combined, the SMR for homotrisomy was 2.5 (90% CI 0.7-8.0). For heterotrisomy, the SMR after an index trisomy 21 was 2.3 (90% CI 1.5-3.8, P=.0007); the SMR did not vary with maternal age at the first trisomy. When all cases with index viable trisomies were combined, the SMR for heterotrisomy was 1.6 (90% CI 1.1-2.4; P=.04). For prenatal diagnoses following a nonviable trisomy diagnosed in a spontaneous abortion (from Genzyme data only), the SMR for a viable trisomy was 1.8 (90% CI 1.1-3.0; P=.04). The significantly increased risk for heterotrisomy supports the hypothesis that some women have a risk for nondisjunction higher than do others of the same age.

Chromosomal Aberrations in Cord Blood Are Associated with Prenatal Exposure to Carcinogenic Polycyclic Aromatic Hydrocarbons

Molecular and traditional epidemiology studies have indicated a possible relationship between in utero environmental exposures and increased risk for childhood cancers, especially acute leukemias. Chromosomal aberrations have been associated with environmental exposures and cancer risk in adults. In order to more clearly define the association between prenatal exposures to carcinogenic polycyclic aromatic hydrocarbons (PAH) and chromosomal aberrations, chromosomal aberration frequencies were measured in a subset of 60 newborns from the Columbia Center for Childrens Environmental Health (CCCEH) Prospective Cohort Study. The subset was composed of African American and Dominican, nonsmoking mother-newborn pairs residing in low-income neighborhoods of New York City, who were exposed to varying levels of airborne PAHs. Prenatal exposure was assessed by questionnaire, personal air monitoring during the third trimester, and PAH-DNA adducts in umbilical cord blood. Chromosomal aberrations were measured in cord blood lymphocytes by fluorescence in situ hybridization. PAH-DNA adducts were not associated with chromosomal aberrations. However, airborne PAHs were significantly associated with stable aberration frequencies in cord blood (P < 0.01). Moreover, stable aberration frequencies were significantly higher among African American newborns compared with Dominican, despite no significant differences in PAH exposure. These results show for the first time an association between prenatal exposure to airborne carcinogenic PAHs and chromosomal aberrations in cord blood, suggesting that such prenatal exposures have the potential to cause cytogenetic damage that has been related to increased cancer risk in other populations. If confirmed, this finding may open new avenues for prevention.

Trisomic Pregnancy and Earlier Age at Menopause

We tested the hypothesis that the connection between advanced maternal age and autosomal trisomy reflects the diminution of the oocyte pool with age. Because menopause occurs when the number of oocytes falls below some threshold, our hypothesis is that menopause occurs at an earlier age among women with trisomic pregnancies than it does among women with chromosomally normal pregnancies. To determine their menstrual status, we interviewed women from our previous study of karyotyped spontaneous abortions who, in 1993, were age >/=44 years. Premenopausal women completed interviews every 4-5 mo, until menopause or until the study ended in 1997. The primary analyses compare 111 women whose index pregnancy was a trisomic spontaneous abortion with two groups: women whose index pregnancy was a chromosomally normal loss (n=157) and women whose index pregnancy was a chromosomally normal birth (n=226). We used a parametric logistic survival analysis to compare median ages at menopause. The estimated median age at menopause was 0.96 years earlier (95% confidence interval -0.18 to 2.10) among women with trisomic losses than it was among women with chromosomally normal losses and chromosomally normal births combined. Results were unaltered by adjustment for education, ethnicity, and cigarette smoking. Our results support the hypothesis that trisomy risk is increased with decreased numbers of oocytes. Decreased numbers may indicate accelerated oocyte atresia or fewer oocytes formed during fetal development.