David J. Amor

Beckwith-Wiedemann Syndrome and IVF: A Case-Control Study

To the Editor: A recent series of observations has suggested a linkbetween in vitro fertilization (IVF) and imprinting disorders, such as Beckwith-Wiedemann syndrome (BWS [MIM 130650]) and Angelman syndrome (MIM 105830). BWS is a model imprinting disorder and is characterized by prenatal and/or postnatal overgrowth, macroglossia, abdominal-wall defects, neonatal hypoglycemia, hemihypertrophy, ear abnormalities, and an increased risk of embryonal tumors (DeBaun et al. 2002). An analysis of BWS registries from three centers has shown the proportion of individuals with BWS conceived using IVF to be 3/65 (DeBaun et al. 2003), 6/149 (Maher et al. 2003), and 6/149 (Gicquel et al. 2003). These data suggest that ∼4% of individuals with BWS are conceived using IVF, a figure greater than the generally accepted usage of IVF in these centers. Further interpretation of these results has been limited because of a reliance by these studies on case records and questionnaire data to determine the method of conception in BWS cases, a lack of the use of appropriate controls, and a statistical significance that was either borderline (Gicquel et al. 2003; Maher et al. 2003) or not mentioned (DeBaun et al. 2003). A recent review of the epidemiology and molecular biology behind these and other related studies has highlighted the need for case-control studies in this area (Niemitz and Feinberg 2004). We report here the results of what we believe is the first case-control study done to test the null hypothesis that there is no difference between the rate of IVF in BWS cases and that in non-BWS controls, in an Australian population. The present study was possible because the State of Victoria, Australia, is serviced by a single clinical genetics service and laboratory providing molecular tests for BWS. This allowed complete ascertainment of children born in Victoria between 1983 and 2003 and diagnosed with BWS by a clinical geneticist. Only cases meeting the DeBaun criteria (DeBaun and Tucker 1998) were included in this study. Appropriate controls were obtained using data from the Victorian Perinatal Data Collection Unit, which registers all births of >19-wk gestation. For each BWS case, four live-born controls were randomly selected from babies born within 1 mo of that case, in which parity was 1 and the maternal age was within 1 year of the risk-set case. Manual record linkage was then used to determine if the BWS cases and the controls were recorded in the databases of the providers of IVF services in Victoria, with the use of maternal names and the dates of birth of mothers and babies. Ethics approval was obtained from all sites providing data. Statistical significance of differences in proportions between groups was assessed using Epi Info, with results expressed as odds ratios (ORs) and as Fishers-exact-test two-sided P values to account for cell sizes <5. Among ∼1,316,500 live births in Victoria between 1983 and 2003 (2003 data were estimated, as they were known to be very similar to 2002 data), 37 cases of BWS were detected, giving an overall BWS prevalence of ∼1/35,580 live births for this period. The average maternal age for BWS cases was 27.0 years. Record linkage of the 37 BWS cases and 148 matched controls identified IVF as the method of conception in 4 BWS cases (10.81%) and in 1 control (0.67%), giving an OR of 17.8 (95% CI 1.8–432.9), and Fishers-exact-test two-sided P=.006. The clinical and molecular features of the four patients with BWS conceived using IVF are listed in table 1, and the reasons for the use of IVF were varied (two unexplained infertility, one egg donation, and one oligospermia). Our results indicate that if a child has BWS, the odds that the child was conceived using IVF is ∼18 times greater than that for a child without BWS, although the magnitude of this OR should be cautiously interpreted, given the wide CI. During the study period (1983–2003), 14,894 babies were born as a result of an IVF procedure (excluding gamete intrafallopian transfer). Using our population-based data, we can then estimate the absolute risk of having a live-born baby with BWS when IVF is used as the means of conception to be 4/14,894. Table 1 Clinical Features of Four Patients Diagnosed with BWS Who Were Conceived Using IVF This study demonstrates that children conceived by IVF are significantly more likely to have BWS, compared with children conceived naturally. Our study design with a control group matched by maternal age has ensured that the rate of IVF procedures in the control (non-BWS) population is accurate for the entire study period, which encompasses a time from infrequent use of IVF (0.2% of pregnancies in 1983) to more frequent use (3% in 2003). We can quantify, for the first time, the risk of BWS in our IVF population as ∼1/4,000, or 9 times greater than in the general population. The mechanisms underlying this increased risk remain unclear, but this study and previous studies (DeBaun et al. 2003; Gicquel et al. 2003; Maher et al. 2003) have shown that patients with BWS conceived by IVF consistently show isolated hypomethylation at the maternal KVDMR1/LIT1 locus at 11p15.5. By comparison, this molecular mechanism is observed in only 46% of our overall BWS population, with the remainder of BWS cases resulting from uniparental disomy of chromosome 11 (16%), biparental methylation of H19DMR (7%), or an unidentified mutation (31%). The preponderance of BWS cases conceived by IVF that show hypomethylation of maternal KVDMR1/LIT1 suggests that collection of in vitro cultures might disturb methylation in the oocyte or early embryo, predisposing to maternal allele demethylation. The fact that the overall risk of BWS in children conceived using IVF remains low and that BWS is, in most cases, associated with a good long-term outcome makes it unlikely that this finding will deter couples from using IVF. Nor does it seem necessary to offer prenatal diagnosis for BWS to couples undergoing IVF. Questions remain, however, about potential effects of IVF on other regions of the genome that are subject to epigenetic regulation. In this context, the observation of a possible association between IVF and Angelman syndrome, another disorder resulting from hypomethylation of the maternal genome, is of some concern (Cox et al. 2002; Orstavik et al. 2003). Although long-term follow-up data of children conceived by IVF are generally reassuring, it remains possible that alterations in genomic imprinting might have other unrecognized health implications for children and adults who were conceived by IVF. Our data reinforce the need for long-term follow-up studies of children conceived by IVF.

Neocentromeres: Role in human disease, evolution, and centromere study

The centromere is essential for the proper segregation and inheritance of genetic information. Neocentromeres are ectopic centromeres that originate occasionally from noncentromeric regions of chromosomes. Despite the complete absence of normal centromeric alpha-satellite DNA, human neocentromeres are able to form a primary constriction and assemble a functional kinetochore. Since the discovery and characterization of the first case of a human neocentromere in our laboratory a decade ago, 60 examples of constitutional human neocentromeres distributed widely across the genome have been described. Typically, these are located on marker chromosomes that have been detected in children with developmental delay or congenital abnormalities. Neocentromeres have also been detected in at least two types of human cancer and have been experimentally induced in Drosophila. Current evidence from human and fly studies indicates that neocentromere activity is acquired epigenetically rather than by any alteration to the DNA sequence. Since human neocentromere formation is generally detrimental to the individual, its biological value must lie beyond the individual level, such as in karyotype evolution and speciation.

A review of known imprinting syndromes and their association with assisted reproduction technologies

An association between assisted reproduction technologies (ART) and abnormal genomic imprinting in humans has been recognized for several years; however, the magnitude of this risk and the spectrum of imprinting syndromes to which the risk applies remains unknown. Nine human imprinting syndromes have been identified but current evidence links ART with only three: Beckwith-Wiedemann syndrome, Angelman syndrome and the newly described maternal hypomethylation syndrome. There is currently a lack of evidence linking ART with the remaining six imprinting syndromes: Prader-Willi syndrome, Russell-Silver syndrome, maternal and paternal uniparental disomy of chromosome 14, pseudohypoparathyroidism type 1b and transient neonatal diabetes. Evidence from clinical reports suggests that the association between imprinting syndromes and ART may be restricted to syndromes where the imprinting change takes the form of hypomethylation on the maternal allele. In contrast, studies of gametes and early embryos suggest that ART can be associated with hypermethylation as well as hypomethylation, with imprinting changes occurring on paternal as well as maternal alleles. The health effects of ART-associated imprinting changes may also extend beyond the nine recognized imprinting syndromes.

Extending the phenotype of recurrent rearrangements of 16p11.2: deletions in mentally retarded patients without autism and in normal individuals.

Array CGH (comparative genomic hybridization) screening of large patient cohorts with mental retardation and/or multiple congenital anomalies (MR/MCA) has led to the identification of a number of new microdeletion and microduplication syndromes. Recently, a recurrent copy number variant (CNV) at chromosome 16p11.2 was reported to occur in up to 1% of autistic patients in three large autism studies. In the screening of 4284 patients with MR/MCA with various array platforms, we detected 22 individuals (14 index patients and 8 family members) with deletions in 16p11.2, which are genomically identical to those identified in the autism studies. Though some patients shared a facial resemblance and a tendency to overweight, there was no evidence for a recognizable phenotype. Autism was not the presenting feature in our series. The assembled evidence indicates that recurrent 16p11.2 deletions are associated with variable clinical outcome, most likely arising from haploinsufficiency of one or more genes. The phenotypical spectrum ranges from MR and/or MCA, autism, learning and speech problems, to a normal phenotype.

De novo mutations in the mitochondrial ND3 gene as a cause of infantile mitochondrial encephalopathy and complex I deficiency

Both nuclear and mitochondrial DNA mutations can cause energy generation disorders. Respiratory chain complex I deficiency is the most common energy generation disorder and a frequent cause of infantile mitochondrial encephalopathies such as Leighs disease and lethal infantile mitochondrial disease. Most such cases have been assumed to be caused by nuclear gene defects, but recently an increasing number have been shown to be caused by mutations in the mitochondrially encoded complex I subunit genes ND4, ND5, and ND6. We report the first four cases of infantile mitochondrial encephalopathies caused by mutations in the ND3 subunit gene. Three unrelated children have the same novel heteroplasmic mutation (T10158C), only the second mutation reported in ND3, and one has the previously identified T10191C mutation. Both mutations cause disproportionately greater reductions in enzyme activity than in the amount of fully assembled complex I, suggesting the ND3 subunit plays an unknown but important role in electron transport, proton pumping, or ubiquinone binding. Three cases appear to have a de novo mutation, with no mutation detected in maternal relatives. Mitochondrial DNA disease may be considerably more prevalent in the pediatric population than currently predicted and should be considered in patients with infantile mitochondrial encephalopathies and complex I deficiency.

DLX3 mutation associated with autosomal dominant amelogenesis imperfecta with taurodontism

Amelogenesis imperfecta hypoplastic‐hypomaturation with taurodontism (AIHHT) is an autosomal dominant (AD) trait associated with enamel defects and enlarged pulp chambers. In this study, we mapped an AIHHT family to human chromosome 17 q21‐q22 (lod score 3.3) and identify a two basepair deletion (CT) at nucleotide 560 in DLX3 associated with the disease. This mutation causes a frameshift altering the last two amino acids of the DNA‐binding homeodomain introducing a premature stop codon truncating the protein by 88 amino acids. This is the first report of a mutation within the homeodomain of DLX3. Previous studies have shown a DLX3 mutation outside the homeodomain associated with tricho‐dento‐osseous syndrome (TDO) suggesting TDO and some forms of AIHHT are allelic. copy; 2005 Wiley‐Liss, Inc.

Further molecular and clinical delineation of co-locating 17p13.3 microdeletions and microduplications that show distinctive phenotypes

Background Chromosome 17p13.3 contains extensive repetitive sequences and is a recognised region of genomic instability. Haploinsufficiency of PAFAH1B1 (encoding LIS1) causes either isolated lissencephaly sequence or Miller–Dieker syndrome, depending on the size of the deletion. More recently, both microdeletions and microduplications mapping to the Miller–Dieker syndrome telomeric critical region have been identified and associated with distinct but overlapping phenotypes. Methods Genome-wide microarray screening was performed on 7678 patients referred with unexplained learning difficulties and/or autism, with or without other congenital abnormalities. Eight and five unrelated individuals, respectively, were identified with microdeletions and microduplications in 17p13.3. Results Comparisons with six previously reported microdeletion cases identified a 258 kb critical region, encompassing six genes including CRK (encoding Crk) and YWHAE (encoding 14-3-3ε). Clinical features included growth retardation, facial dysmorphism and developmental delay. Notably, one individual with only subtle facial features and an interstitial deletion involving CRK but not YWHAE suggested that a genomic region spanning 109 kb, encompassing two genes (TUSC5 and YWHAE), is responsible for the main facial dysmorphism phenotype. Only the microduplication phenotype included autism. The microduplication minimal region of overlap for the new and previously reported cases spans 72 kb encompassing a single gene, YWHAE. These genomic rearrangements were not associated with low-copy repeats and are probably due to diverse molecular mechanisms. Conclusions The authors further characterise the 17p13.3 microdeletion and microduplication phenotypic spectrum and describe a smaller critical genomic region allowing identification of candidate genes for the distinctive facial dysmorphism (microdeletions) and autism (microduplications) manifestations.

Human and Mouse Mutations in WDR35 Cause Short-Rib Polydactyly Syndromes Due to Abnormal Ciliogenesis

Defects in cilia formation and function result in a range of human skeletal and visceral abnormalities. Mutations in several genes have been identified to cause a proportion of these disorders, some of which display genetic (locus) heterogeneity. Mouse models are valuable for dissecting the function of these genes, as well as for more detailed analysis of the underlying developmental defects. The short-rib polydactyly (SRP) group of disorders are among the most severe human phenotypes caused by cilia dysfunction. We mapped the disease locus from two siblings affected by a severe form of SRP to 2p24, where we identified an in-frame homozygous deletion of exon 5 in WDR35. We subsequently found compound heterozygous missense and nonsense mutations in WDR35 in an independent second case with a similar, severe SRP phenotype. In a mouse mutation screen for developmental phenotypes, we identified a mutation in Wdr35 as the cause of midgestation lethality, with abnormalities characteristic of defects in the Hedgehog signaling pathway. We show that endogenous WDR35 localizes to cilia and centrosomes throughout the developing embryo and that human and mouse fibroblasts lacking the protein fail to produce cilia. Through structural modeling, we show that WDR35 has strong homology to the COPI coatamers involved in vesicular trafficking and that human SRP mutations affect key structural elements in WDR35. Our report expands, and sheds new light on, the pathogenesis of the SRP spectrum of ciliopathies.

Tumour surveillance in Beckwith–Wiedemann syndrome and hemihyperplasia: A critical review of the evidence and suggested guidelines for local practice

Abstract:  There is strong evidence for an association between overgrowth disorders such as Beckwith–Wiedemann syndrome and the development of neoplasia. An increased cancer risk has also been observed in individuals with isolated hemihyperplasia. We critically review the evidence for tumour surveillance in Beckwith–Wiedemann syndrome and isolated hemihyperplasia and suggest local practice guidelines.

Adverse obstetric and perinatal outcomes in subfertile women conceiving without assisted reproductive technologies.

OBJECTIVE To determine whether adverse perinatal outcomes are increased in subfertile women. DESIGN Cohort study. SETTING Two tertiary assisted reproductive technologies (ART) centers; Victorian births register. PATIENT(S) Records of women who registered with the clinics (1991-2000), but did not have an infant using ART, were linked to the birth register (1991-2004) to identify singleton non-ART births within 5 years of registration (N = 2171). Controls, matched by maternal age and year of infants birth, were selected randomly from birth records (N = 4363). INTERVENTIONS None. MAIN OUTCOME MEASURE(S) Adverse obstetric and perinatal outcomes. RESULT(S) After adjusting for confounders, compared with controls, subfertile women had increased odds of hypertension or preeclampsia (adjusted odds ratio [OR] 1.29, 1.02-1.61), antepartum hemorrhage (adjusted OR 1.41, 1.05-1.89), perinatal death (adjusted OR 2.19, 1.10-4.36), low birth weight (adjusted OR 1.44, 1.11-1.85), preterm birth <37 weeks (adjusted OR 1.32, 1.05-1.67) or <31 weeks (adjusted OR 2.37, 1.35-4.13), and cesarean delivery (adjusted OR 1.56, 1.37-1.77). There was weak evidence for increased birth defects (adjusted OR 1.30, 0.98-1.72) and gestational diabetes (adjusted OR 1.25, 0.96-1.63). No increased risk was found for prelabor rupture of membranes, small for gestational age, or postpartum hemorrhage. CONCLUSION(S) Subfertile women with singleton births are at increased risk of several adverse outcomes. These risks should be considered during their antenatal care and when analyzing adverse effects of ART.