Alberto Plaja

Constitutional aneuploidy and cancer predisposition caused by biallelic mutations in BUB1B

Mosaic variegated aneuploidy is a rare recessive condition characterized by growth retardation, microcephaly, childhood cancer and constitutional mosaicism for chromosomal gains and losses. In five families with mosaic variegated aneuploidy, including two with embryonal rhabdomyosarcoma, we identified truncating and missense mutations of BUB1B, which encodes BUBR1, a key protein in the mitotic spindle checkpoint. These data are the first to relate germline mutations in a spindle checkpoint gene with a human disorder and strongly support a causal link between aneuploidy and cancer development.

Rapid prenatal diagnosis by QF-PCR: evaluation of 30,000 consecutive clinical samples and future applications.

Abstract:  Rapid prenatal diagnoses of major chromosome abnormalities can be performed on a large scale using highly polymorphic short tandem repeats (STRs) amplified by the quantitative fluorescent polymerase chain reaction (QF‐PCR). The assay was introduced as a preliminary investigation to remove the anxiety of the parents waiting for the results by conventional cytogenetic analysis using amniotic fluid or chorionic cells. However, recent studies, on the basis of the analyses of several thousand samples, have shown that this rapid approach has a very high rate of success and could reduce the need for cytogenetic investigations. Its high efficiency, for example, allows early interruption of affected fetuses without the need of waiting for completion of fetal karyotype. The main advantages of the QF‐PCR are its accuracy, speed, automation, and low cost that allows very large number of samples to be analyzed by few operators. Here, we report the results of using QF‐PCR in a large series of consecutive clinical cases and discuss the possibility that, in a near future, it may even replace conventional cytogenetic analyses on selected samples.

Assessment of QF-PCR as the First Approach in Prenatal Diagnosis

Quantitative fluorescent PCR (QF-PCR) has been used by many laboratories for prenatal diagnosis of the most common aneuploidies. QF-PCR is rapid, cost-effective, and suitable for automation and can detect most abnormalities diagnosed by conventional karyotyping. Whether QF-PCR should be used alone in most of the samples and in which karyotyping should also be offered is currently a topic of debate. We evaluated and compared the results obtained from 7679 prenatal samples in which conventional karyotype and QF-PCR had been performed, including 1243 chorionic villi and 6436 amniotic fluid samples. Concordant QF-PCR and karyotype results were obtained in 98.75% of the samples. An abnormal karyotype associated with adverse clinical outcome undetected by QF-PCR was found in 0.05% of samples. Therefore, QF-PCR can be used alone in a large number of samples studied in a prenatal laboratory, thereby reducing both the workload in cytogenetic laboratories and parental anxiety when awaiting results.

Two cases of tetrasomy 9p syndrome with tissue limited mosaicism

Tetrasomy of short arm of chromosome 9 constitutes a clinically recognizable chromosomal syndrome. Isochromosome 9p shows a strong propensity to tissue‐limited mosaicism. It occurs predominantly in peripheral blood cultures, often at a lower frequency or even absent in skin, amniotic fluid or chorionic villous cell cultures. Tissue‐limited nature of mosaicism may render prenatal detection of this condition very difficult. Herein, we report two new cases of mosaic tetrasomy 9p. Conventional cytogenetics (CC) and FISH studies demonstrated a differential expression of the mosaicism in several tissues. We review the literature and discuss the implications of these findings in cytogenetic prenatal diagnosis.

Prenatal diagnosis of a rare chromosomal instability syndrome: Variegated aneuploidy related to premature centromere division (PCD)

We report complete cytogenetic data of the first successful prenatal diagnosis of the variegated aneuploidy related to premature centromere division (PCD). Theclinical phenotypeof this condition comprisesmicrocephaly, CNS anomalies, mental retardation, severe preand postnatal growth retardation, and ahigh risk of malignancy. In blood and fibroblast cultures from these patients an impaired mitosis is expressed cytogenetically as an increased frequency of PCD (cells in division which have overcome a colchicine-induced metaphase block, and show split centromeres and splayed chromatids in all or most of the chromosomes) in combination with an increased number of cells with mosaic aneuploidies (‘‘variegated aneuploidy’’) [Scheres et al., 1986; Miller et al., 1990; Warburton et al., 1991; Kajii et al., 1998; Kawame et al., 1999; Limwongse et al., 1999; D’Agostino et al., 2000;Matsuuraet al., 2000;Kajii et al., 2001; Plaja et al., 2001a]. Amniocentesis was performed at the 14th week of pregnancy of a 34-year-old gravid 4 para 2, because a previous child diagnosed with PCD-related variegated aneuploidy [Plaja et al., 2001b]. Her husband was 32 years old; both were of Chinese origin and not consanguineous. The first child, an 8-year-old male, is reported to be normal by the parents, but no detailed clinical or cytogenetic examination has been performed. The second child, a female, was diagnosed postnatally with PCDrelated variegated aneuploidy and died from a rhabdomyosarcoma at the age of 18 months. Previous to this pregnancy, a gestationwas interrupted because of social reasons. At the 12th week of pregnancy a nuchal translucency of 3.2 mm was noted by ultrasonography. At 17 weeks, 2 days after the cytogenetic report, the pregnancy was spontaneously lost.Thepatientwasattended inanother hospital and follow-up was lost. Chromosome examination of three independent amniocyte cultures showed PCD in 43% of the metaphases (119/281). Cells not showing the PCD phenotype had a high frequency of gain and loss of chromosomes (Table I).Wehavenot foundanyPCDfigure in25 control amniotic cultures (200 metaphases evaluated in each case) and have not detected variegated aneuploidy in over 9,000 samples of amniotic fluid referred to our laboratory for prenatal cytogenetic analyses. To our knowledge, cytogenetic prenatal studies were performed previously in only two additional patients, but the condition was recognized only postnatally in both cases and the presence of PCD amniocyte cultures was not reported. Limwongse et al. [1999] reported an amniocentesis at 27 weeks of gestation because

PIAS4 is associated with macro/microcephaly in the novel interstitial 19p13.3 microdeletion/microduplication syndrome

Array comparative genomic hybridization (aCGH) is a powerful genetic tool that has enabled the identification of novel imbalances in individuals with intellectual disability (ID), autistic disorders and congenital malformations. Here we report a ‘genotype first’ approach using aCGH on 13 unrelated patients with 19p13.3 submicroscopic rearrangement (11 deletions and 2 duplications) and review cases in the literature and in public databases. Shared phenotypic features suggest that these patients represent an interstitial microdeletion/microduplication syndrome at 19p13.3. Common features consist of abnormal head circumference in most patients (macrocephaly with the deletions and microcephaly with the duplications), ID with developmental delay (DD), hypotonia, speech delay and common dysmorphic features. The phenotype is associated with at least a ~0.113 Mb critical region harboring three strong candidate genes probably associated with DD, ID, speech delay and other dysmorphic features: MAP2K2, ZBTB7A and PIAS4, an E3 ubiquitin ligase involved in the ubiquitin signaling pathways, which we hypothesize for the first time to be associated with head size in humans.

Comparative Genomic Hybridization Analysis Reveals New Different Subgroups in Early-stage Bladder Tumors

OBJECTIVES To classify bladder tumors according to their genomic imbalances and evaluate their association with patients outcome. METHODS Sixty-three superficially and minimally invasive bladder tumors were analyzed by conventional comparative genomic hybridization. Subtelomeric screening in 15 of these tumors was performed by multiplex ligation-dependent probe amplification. RESULTS Losses of 9q and 9p (32% and 25% of all cases, respectively) as well as gains of chromosomes Xq and Xp (28% and 25%, respectively) were the most frequent chromosome imbalances. Losses of 8p and gains in 1q and 8q were detected in >20% of cases. Tumors were classified into 3 groups according to their individualized pattern of gains and losses. The largest group was characterized by few chromosome imbalances, presenting 77% and 49% of the Ta and T1 tumors, respectively. Another group characterized by chromosomal gains, was composed of equal number of Ta and T1 tumors, with +1q and +17q gains being the most common imbalances. A minority group was characterized by chromosomal losses on 11q, 5q, and 6q. The multiplex ligation-dependent probe amplification study showed good correlation with comparative genomic hybridization results. With regard to the biological significance of this classification, a remarkable fact is that this minority group composed mainly of T1 tumors, showed a significant decrease in patient overall survival. CONCLUSIONS Our data suggest that superficial carcinomas of the bladder can be subdivided into a larger number of subclasses than had previously been expected. Our results also demonstrate a decreased survival among patients whose tumors show more genomic losses than gains.

Supernumerary Ring Chromosome: An Etiology for Pallister-Killian Syndrome?

Characterization of marker chromosomes before the introduction of array CGH (aCGH) assays was only based on their banding patterns (G, C, and NOR staining) and fluorescent in situ hybridization techniques. The use of aCGH greatly improves the identification of marker chromosomes in some cases. We describe an atypical case of Pallister-Killian syndrome (PKS) detected at prenatal diagnosis with a very unusual cytogenetic presentation: a supernumerary ring chromosome including two copies of 12p. A similar anomaly described in a postnatal patient suggests ring chromosome as a possible cause of PKS. Extra ring chromosomes might be a more common etiology for PKS than previously thought, given the difficulty in their characterization before the advent of aCGH.

Trisomy 18p caused by a supernumerary marker with a chromosome 13/21 centromere: a possible recurrent chromosome aberration.

We present a clinical and molecular cytogenetic characterization of two new patients with a complex supernumerary marker consisting of the entire short arm of chromosome 18 with a chromosome 13/21 centromere. One patient is a girl with a nonsyndromic intellectual disability and the second is a prenatally diagnosed fetus. To our knowledge, these are the fourth and fifth such cases to be described in the literature, suggesting the existence of a possible recurring constitutional structural chromosome abnormality.

Intrachromosomal 3p Insertion as a Cause of Reciprocal Pure Interstitial Deletion and Duplication in Two Siblings: Further Delineation of the Emerging Proximal 3p Deletion Syndrome

Very few cases of constitutional interstitial deletions of the proximal short arm of chromosome 3 have been reported; however, the proximal 3p deletion is emerging as a clinically recognizable syndrome. We present an intrachromosomal insertion of 3p12.3p14.1 in a phenotypic normal man (46,XY,ins(3)(p25p12.3p14.1)) which is responsible for the unbalanced karyotype in 2 affected offspring, one with a 3p12.3p14.1 interstitial deletion and the other with a reciprocal duplication. The exceptionality of these 2 reciprocal recombinants contributes to a better definition of the proximal 3p deletion syndrome and its duplication counterpart.