Caroline E. Browne

Estimates of the frequency of chromosome abnormalities detectable in unselected newborns using moderate levels of banding.

Data on structural chromosome abnormalities identified during prenatal diagnosis were used to estimate the number of such abnormalities that would be detectable in an unselected series of newborns using moderate levels of banding (400 to 500 bands). These estimates were compared with the rates detected in nonbanded surveys of newborns. Between 1976 and 1990 prenatal diagnosis using banding techniques was carried out in our laboratory on 14,677 women aged 35 and over. Among these, we detected 112 structural rearrangements, 32 unbalanced and 80 balanced. These figures were adjusted by two methods to give an estimate of the frequency of structural abnormalities in the newborn. Our data suggest that the use of moderate levels of banding increases the frequency of unbalanced structural abnormalities from 0.052 to 0.061% and of balanced structural abnormalities from 0.212 to 0.522%. Thus, the total number of chromosome abnormalities detectable in the newborn is increased from 0.60% in unbanded preparations to 0.92% in banded preparations.

Inherited Interstitial Duplications of Proximal 15q: Genotype-Phenotype Correlations

We present the cytogenetic, molecular cytogenetic, and molecular genetic results on 20 unrelated patients with an interstitial duplication of the proximal long arm of chromosome 15. Multiple probes showed that the Prader-Willi/Angelman critical region (PWACR) was included in the duplication in 4/20 patients, each ascertained with developmental delay. The duplication was also found in two affected but not in three unaffected sibs of one of these patients. All four probands had inherited their duplication from their mothers, three of whom were also affected. Two of the affected mothers also carried a maternally inherited duplication, whereas the duplication in the unaffected mother and in an unaffected grandmother was paternal in origin, raising the possibility of a parental-origin effect. The PWACR was not duplicated in the remaining 16 patients, of whom 4 were referred with developmental delay. In the 14 families for which parental samples were available, the duplication was inherited with equal frequency from a phenotypically normal parent, mother or father. Comparative genomic hybridization undertaken on two patients suggested that proximal 15q outside the PWACR was the origin of the duplicated material. The use of PWACR probes discriminates between a large group of duplications of no apparent clinical significance and a smaller group, in which a maternally derived PWACR duplication is consistently associated with developmental delay and speech difficulties but not with overt features of either Prader-Willi syndrome or Angelman syndrome.

Xp deletions associated with autism in three females.

We report eight females with small deletions of the short arm of the X chromosome, three of whom showed features of autism. Our results suggest that there may be a critical region for autism in females with Xp deletions between the pseudoautosomal boundary and DXS7103. We hypothesise that this effect might be due either to the loss of function of a specific gene within the deleted region or to functional nullisomy resulting from X inactivation of the normal X chromosome.

Subtelomeric rearrangements: results from a study of selected and unselected probands with idiopathic mental retardation and control individuals by using high-resolution G-banding and FISH.

Abstract. The cause of mental retardation, present in approximately 3% of the population, is unexplained in the majority of cases. Recent reports have suggested that cryptic telomeric rearrangements resulting in segmental aneuploidy and gene-dosage imbalance might represent a significant cause of idiopathic mental retardation (IMR). Two groups of patients with unexplained developmental delay (unselected and selected) and a group of control individuals have been investigated to determine the frequency of submicroscopic telomeric rearrangements associated with IMR and the frequency within the normal population. In contrast to current thinking, our data have shown that true cryptic telomeric rearrangements are not a significant cause of IMR. No fully cryptic abnormalities were detected in our IMR groups, although a semi-cryptic unbalanced telomeric translocation was identified in one selected patient by high-resolution G-band analysis. This abnormality was confirmed and characterised by fluorescence in situ hybridisation (FISH) with telomere-specific probes. A further 13 cytogenetically detected subtle terminal rearrangements were characterised by using multi-telomere FISH. Seven of these had previously been reported as normal, three of which were shown to be interstitial deletions. These cases illustrate the importance of high-resolution analysis to exclude subtle but cytogenetically visible abnormalities prior to subtelomere FISH screening when determining the frequency of cryptic telomeric rearrangements. Unexpectedly, two cryptic telomeric abnormalities were detected among our control individuals, suggesting that submicroscopic telomeric abnormalities may be a not uncommon finding in the general population. Hence, our data have important implications when defining the significance of cryptic telomeric rearrangements detected during screening programmes.

Chromosome 15q11-13 abnormalities and other medical conditions in individuals with autism spectrum disorders.

Objectives The frequency of abnormalities of 15q11-q13 and other possibly causal medical disorders including karyotypic abnormalities was investigated in an unselected series of children who were referred to one of two autism assessment centres. Methods Two hundred and twenty-one cases were assessed using the Autism Diagnostic Interview and Observation Schedule and, where appropriate, standardized tests of intelligence and language abilities. Medical histories and notes were reviewed, and molecular and cytogenetic investigations used to detect chromosomal abnormalities. Results One hundred and eighty-one cases were diagnosed according to International Classification of Diseases – version 10 criteria as having an autism spectrum disorder (autistic-like Pervasive Developmental Disorder) and 40 cases as having other disorders. Twenty-one (11.6%) of the children with autism spectrum disorders had a possibly causal condition compared with six (15%) of the children with other diagnoses. One child with an autism spectrum disorder had a paternally inherited familial duplication of 15q11-13. The pattern of genotype–phenotype correlation within the family indicated that this form of abnormality might carry a risk for developmental difficulties, although the risk did not appear to be specific for autism spectrum disorders. Conclusion The overall rate of possibly causal medical and cytogenetic conditions in children with autism spectrum disorders was low and no different from the rate of disorder in children with other developmental/neuropsychiatric disorders that attended the same clinics. Further research is required to determine whether paternal duplication of 15q11-13 gives rise to adverse developmental outcomes.

Neurofibromatosis pseudogene amplification underlies euchromatic cytogenetic duplications and triplications of proximal 15q

Cytogenetically visible interstitial duplications of proximal 15q, which lack the Prader-Willi Angelman critical region (PWACR) frequently segregate in families without phenotypic effect, but the nature of the extra euchromatin has remained unclear. We used comparative genome hybridisation to confirm that the extra material in a cytogenetic triplication originated from proximal 15q. A PAC clone containing sequences specific for the type-1 neurofibromatosis (NF-1) pseudogenes, which map to 15q11.2, hybridised along the length of the enlarged region between the PWACR and the centromere. Computerised measurement of the fluorescent signal from the enlarged and normal chromosomes gave an average ratio of 9.85:1, consistent with amplification. In a second family, an amplified P1–4 signal co-segregated with a cytogenetic duplication and the average ratio between amplified and normal signals in the proband was 8.22:1. Ratios in non-carrier family members and control individuals were close to unity in most cases, but significantly greater than one in at least one instance. Our results provide a novel explanation for cytogenetic variation in 15q11.2. They also suggest that NF-1 pseudogene copy number may be polymorphic in the normal population, and that high copy numbers can produce G bands which do not reflect those of the normal constitutional karyotype.

Investigation of a cryptic interstitial duplication involving the Prader-Willi/Angelman syndrome critical region

Abstract. A 3-year-old female referred with developmental delay, hypotonia and seizures was found to have a cryptic interstitial duplication of the Prader-Willi/Angelman critical region (PWACR). Her clinical features form part of a common phenotype characteristic of PWACR duplications including developmental delay, behavioural problems and speech difficulties. Microsatellite analysis showed that the duplication had arisen de novo,was maternal in origin and involved the entire 4-Mb PWACR between the common deletion breakpoints. The existence of cryptic rearrangements emphasises the need for molecular tests alongside conventional cytogenetics when investigating abnormalities involving this imprinted region.

Estimate of the prevalence of chromosome 15q11‐q13 duplications

We refer to the recent letter in which Moeschler et al. [2002] estimated a frequency of 1 in 200 for chromosome 15q11-q13 duplications. The figure was derived from a molecular screen of 400patients referredwith suspected fragile X. As part of a 5 year study to characterize structural abnormalities of chromosome 15, we recently published our findings on 16 interstitial duplications [Roberts et al., 2002]. Collecting these patients took considerable effort and in our experience a frequency of 1 in 200 seems an overestimate We first reported that cytogenetically visible duplications of proximal 15q could be divided into those which included the critical region for Prader–Willi and Angelman syndromes (PWACR) in 15q11-q13 [Browne et al., 1997] and those caused by more proximal pseudogene expansions [Barber et al., 1998]. Routine cytogenetic analysis during the previous 3 years identified a PWACR positive duplication in approximately 1 in 600 individuals referred with developmental delay. In support of this estimate we re-assessed samples referred to the Wessex Regional Genetics Laboratory (Salisbury, UK) for cytogenetic analysis over the period January 1997 to September 2002 (Table I). Karyotypes were determined for all patients by analysis of G banded metaphase chromosomes, harvested from peripheral blood, at an ISCN band level of 550. During this time we received 2,982 samples referred with developmental delay or suspected fragile X and 327 samples with behavioral problems suspected to fall within an Autistic Spectrum Disorder (ASD). The majority of the ASD referrals had developmental delay and were tested to exclude fragile X. From this total of 3,309 individuals we identified four who carried a cytogenetically visible 15q11-q13 duplication (Table II). Not all PWACR duplications are visible cytogenetically [Roberts et al., 2002] and so a cytogenetic screen alone may underestimate the frequency. In order to detect cryptic duplications we screened a subset of the cytogenetic referrals using a combination of two PCR methods: microsatellite analysis at 4–6 loci within the PWACR and quantitative fluorescent PCR at a further 2 loci [Roberts et al., 2002]. Almost all duplications are of a uniform size and include the entire 4 Mb PWACR cassette. Approximately two thirds of interstitial duplications arise from an interchromosomal event and would be recognized by the presence of three different alleles. The remaining one third of duplications are intrachromosomal in origin and would require assessment of thepeakheights ofmicrosatellite products.Both categories would be detected by quantitative PCR. Thus we are unlikely to miss any standard duplications. Using both PCRmethods we tested a total of 457 cytogenetic referrals and an additional 83 autistic individuals, who underwent an Autism Diagnostic Interview, forwhomwereceived onlyaDNAsample.Themolecular screen identified one additional interstitial duplication carrier and one individual with a cryptic maternal deletion of 15q11-q13 resulting in a diagnosis of AS (Table I). Both abnormalities were confirmed by FISH. Although we found no abnormalities in 83 patients with confirmed autism (molecularly) and 327with a suspectedASD (cytogenetically), imbalances of thePWACR represent one of the most frequent chromosome disorders found in autistic samples. From patients attending an autism clinic Cook et al. [1997] estimated that collectively chromosome 15 rearrangements could account for up to 3% of cases. Full details of the five duplication carriers are reported in Roberts et al. [2002] but a summary is given in Table II. The twopatients in familyfivewerehalf-sisters but considered as independent referrals because one sister carried two separate duplications, one inherited from each parent. The study of Moeschler et al. [2002] screened patients with developmental delay and reported one patient with an interstitial duplication, one patient with a supernumerarymarker chromosome, SMC(15), and one patient with an apparent duplication who also carried an unidentified marker chromosome. (Approximately 50% of SMC(15) are derived from chromosome 15 [Buckton et al., 1985; Blenow et al., 1995] and it was not indicated whether FISH analysis was performed to demonstrate that the additional PWACR copies were interstitial rather than carried on the SMC.) *Correspondence to: Dr. N. Simon Thomas, Ph.D., Wessex Regional Genetics Laboratory, Salisbury District Hospital, Salisbury, SP2 8BJ, United Kingdom. E-mail: dr.s.thomas@salisbury.nhs.uk