Expansion of the neurodevelopmental phenotype of individuals with EEF1A2 variants and genotype-phenotype study.

Translation elongation factor eEF1A2 constitutes the alpha subunit of the elongation factor-1 complex, responsible for the enzymatic binding of aminoacyl-tRNA to the ribosome. Since 2012, 21 pathogenic missense variants affecting EEF1A2 have been described in 42 individuals with a severe neurodevelopmental phenotype including epileptic encephalopathy and moderate to profound intellectual disability (ID), with neurological regression in some patients. Through international collaborative call, we collected 26 patients with EEF1A2 variants and compared them to the literature. Our cohort shows a significantly milder phenotype. 83% of the patients are walking (vs. 29% in the literature), and 84% of the patients have language skills (vs. 15%). Three of our patients do not have ID. Epilepsy is present in 63% (vs. 93%). Neurological examination shows a less severe phenotype with significantly less hypotonia (58% vs. 96%), and pyramidal signs (24% vs. 68%). Cognitive regression was noted in 4% (vs. 56% in the literature). Among individuals over 10 years, 56% disclosed neurocognitive regression, with a mean age of onset at 2 years. We describe 8 novel missense variants of EEF1A2. Modeling of the different amino-acid sites shows that the variants associated with a severe phenotype, and the majority of those associated with a moderate phenotype, cluster within the switch II region of the protein and thus may affect GTP exchange. In contrast, variants associated with milder phenotypes may impact secondary functions such as actin binding. We report the largest cohort of individuals with EEF1A2 variants thus far, allowing us to expand the phenotype spectrum and reveal genotype-phenotype correlations.

Rare germline variants in POLE and POLD1 encoding the catalytic subunits of DNA polymerases ε and δ in glioma families.

Pathogenic germline variants in the DNA polymerase genes POLE and POLD1 cause polymerase proofreading-associated polyposis, a dominantly inherited disorder with increased risk of colorectal carcinomas and other tumors. POLE/POLD1 variants may result in high somatic mutation and neoantigen loads that confer susceptibility to immune checkpoint inhibitors (ICIs). To explore the role of POLE/POLD1 germline variants in glioma predisposition, whole-exome sequencing was applied to leukocyte DNA of glioma patients from 61 tumor families with at least one glioma case each. Rare heterozygous POLE/POLD1 missense variants predicted to be deleterious were identified in glioma patients from 10 (16%) families, co-segregating with the tumor phenotype in families with available DNA from several tumor patients. Glioblastoma patients carrying rare POLE variants had a mean overall survival of 21 months. Additionally, germline variants in POLD1, located at 19q13.33, were detected in 2/34 (6%) patients with 1p/19q-codeleted oligodendrogliomas, while POLE variants were identified in 2/4 (50%) glioblastoma patients with a spinal metastasis. In 13/15 (87%) gliomas from patients carrying POLE/POLD1 variants, features of defective polymerase proofreading, e.g. hypermutation, POLE/POLD1-associated mutational signatures, multinucleated cells, and increased intratumoral T cell response, were observed. In a CRISPR/Cas9-derived POLE-deficient LN-229 glioblastoma cell clone, a mutator phenotype and delayed S phase progression were detected compared to wildtype POLE cells. Our data provide evidence that rare POLE/POLD1 germline variants predispose to gliomas that may be susceptible to ICIs. Data compiled here suggest that glioma patients carrying POLE/POLD1 variants may be recognized by cutaneous manifestations, e.g. café-au-lait macules, and benefit from surveillance colonoscopy.

A second look at exome sequencing data: detecting mobile elements insertion in a rare disease cohort.

About 0.3% of all variants are due to de novo mobile element insertions (MEIs). The massive development of next-generation sequencing has made it possible to identify MEIs on a large scale. We analyzed exome sequencing (ES) data from 3232 individuals (2410 probands) with developmental and/or neurological abnormalities, with MELT, a tool designed to identify MEIs. The results were filtered by frequency, impacted region and gene function. Following phenotype comparison, two candidates were identified in two unrelated probands. The first mobile element (ME) was found in a patient referred for poikilodermia. A homozygous insertion was identified in the FERMT1 gene involved in Kindler syndrome. RNA study confirmed its pathological impact on splicing. The second ME was a de novo Alu insertion in the GRIN2B gene involved in intellectual disability, and detected in a patient with a developmental disorder. The frequency of de novo exonic MEIs in our study is concordant with previous studies on ES data. This project, which aimed to identify pathological MEIs in the coding sequence of genes, confirms that including detection of MEs in the ES pipeline can increase the diagnostic rate. This work provides additional evidence that ES could be used alone as a diagnostic exam.

Noninvasive Prenatal Screening for Trisomy 21 in Patients with a Vanishing Twin.

A vanishing twin (VT) occurs in up to 30% of early diagnosed twin pregnancies and is associated with an increased risk of fetal aneuploidy. Here, we describe our experience in a large VT population of 847 patients that underwent noninvasive prenatal testing (NIPT) for common fetal trisomies over a three-year period. All patients underwent an ultrasound examination prior to NIPT. Two comparison populations were included, namely, the singleton (n = 105,560) and the viable multiple gestation pregnancy samples (n = 9691) collected over the same period. All NIPT samples in the VT population received a result, of which 14 were high-risk for trisomy 21 (1.6%), nine for trisomy 18 (1.1%), and six for trisomy 13 (0.7%). Diagnostic testing confirmed the presence of trisomy 21 in 6/12 samples, giving a positive predictive value of 50%. One trisomy 18 case and no trisomy 13 cases were confirmed. The time between fetal demise and NIPT sampling did not appear to affect the number of true- or false-positive cases. In conclusion, NIPT is an effective screening method for trisomy 21 in the surviving fetus(es) in VT pregnancies. For trisomies 18 and 13, a positive NIPT should be interpreted carefully and ultrasound monitoring is preferrable over invasive diagnostic testing.

Case Report: How whole-genome sequencing-based cell-free DNA prenatal testing can help identify a marker mhromosome.

A supernumerary marker chromosome (SMC) is a structurally abnormal chromosome that cannot be characterized by conventional banding cytogenetics. Marker chromosomes are present in 0.075% of prenatal cases. They are associated with variable phenotypes, ranging from normal to severely abnormal, and the prognosis is largely dependent on the results of further cytogenomic analysis. Here, we report the identification and characterization of a marker chromosome following prenatal screening in a 39-year-old pregnant patient. The patient had a normal first trimester ultrasound but was high-risk for fetal chromosome anomalies based on the results of maternal serum parameters. Chorionic villus sampling was performed, and analysis of chorionic villi revealed the presence of two identical marker chromosomes. In the interest of a rapid identification of the markers, we performed noninvasive prenatal testing (NIPT) together with chorionic villus sampling. A pericentromeric 29 Mb duplication of chromosome 20: dup (20) (p13q11.21) was identified and thereafter confirmed by targeted metaphasic FISH. Whole-genome sequencing-based NIPT was instrumental in rapid characterization of the SMCs and allowed us to obviate the need for multiple expensive and time-consuming FISH analyses.

Genome Alert!: A standardized procedure for genomic variant reinterpretation and automated gene-phenotype reassessment in clinical routine.

PURPOSE: Retrospective interpretation of sequenced data in light of the current literature is a major concern of the field. Such reinterpretation is manual and both human resources and variable operating procedures are the main bottlenecks. METHODS: Genome Alert! method automatically reports changes with potential clinical significance in variant classification between releases of the ClinVar database. Using ClinVar submissions across time, this method assigns validity category to gene-disease associations. RESULTS: Between July 2017 and December 2019, the retrospective analysis of ClinVar submissions revealed a monthly median of 1247 changes in variant classification with potential clinical significance and 23 new gene-disease associations. Re-examination of 4929 targeted sequencing files highlighted 45 changes in variant classification, and of these classifications, 89% were expert validated, leading to 4 additional diagnoses. Genome Alert! gene-disease association catalog provided 75 high-confidence associations not available in the OMIM morbid list; of which, 20% became available in OMIM morbid list For more than 356 negative exome sequencing data that were reannotated for variants in these 75 genes, this elective approach led to a new diagnosis. CONCLUSION: Genome Alert! (https://genomealert.univ-grenoble-alpes.fr/) enables systematic and reproducible reinterpretation of acquired sequencing data in a clinical routine with limited human resource effect.

Pathogenic variants in KCNQ2 cause intellectual deficiency without epilepsy: Broadening the phenotypic spectrum of a potassium channelopathy.

High-throughput sequencing (HTS) improved the molecular diagnosis in individuals with intellectual deficiency (ID) and helped to broaden the phenotype of previously known disease-causing genes. We report herein four unrelated patients with isolated ID, carriers of a likely pathogenic variant in KCNQ2, a gene usually implicated in benign familial neonatal seizures (BFNS) or early onset epileptic encephalopathy (EOEE). Patients were diagnosed by targeted HTS or exome sequencing. Pathogenicity of the variants was assessed by multiple in silico tools. Patients' ID ranged from mild to severe with predominance of speech disturbance and autistic features. Three of the four variants disrupted the same amino acid. Compiling all the pathogenic variants previously reported, we observed a strong overlap between variants causing EOEE, isolated ID, and BFNS and an important intra-familial phenotypic variability, although missense variants in the voltage-sensing domain and the pore are significantly associated to EOEE (p < 0.01, Fisher test). Thus, pathogenic variants in KCNQ2 can be associated with isolated ID. We did not highlight strong related genotype-phenotype correlations in KCNQ2-related disorders. A second genetic hit, a burden of rare variants, or other extrinsic factors may explain such a phenotypic variability. However, it is of interest to study encephalopathy genes in non-epileptic ID patients.

Strategy for Use of Genome-Wide Non-Invasive Prenatal Testing for Rare Autosomal Aneuploidies and Unbalanced Structural Chromosomal Anomalies.

Atypical fetal chromosomal anomalies are more frequent than previously recognized and can affect fetal development. We propose a screening strategy for a genome-wide non-invasive prenatal test (NIPT) to detect these atypical chromosomal anomalies (ACAs). Two sample cohorts were tested. Assay performances were determined using Cohort A, which consisted of 192 biobanked plasma samples-42 with ACAs, and 150 without. The rate of additional invasive diagnostic procedures was determined using Cohort B, which consisted of 3097 pregnant women referred for routine NIPT. Of the 192 samples in Cohort A, there were four initial test failures and six discordant calls; overall sensitivity was 88.1% (37/42; CI 75.00-94.81) and specificity was 99.3% (145/146; CI 96.22-99.88). In Cohort B, there were 90 first-pass failures (2.9%). The rate of positive results indicating an anomaly was 1.2% (36/3007) and 0.57% (17/3007) when limited to significant unbalanced chromosomal anomalies and trisomies 8, 9, 12, 14, 15, 16, and 22. These results show that genome-wide NIPT can screen for ACAs with an acceptable sensitivity and a small increase in invasive testing, particularly for women with increased risk following maternal serum screening and by limiting screening to structural anomalies and the most clinically meaningful trisomies.

Report of the first patient with a homozygous OTUD7A variant responsible for epileptic encephalopathy and related proteasome dysfunction.

Heterozygous microdeletions of chromosome 15q13.3 (MIM: 612001) show incomplete penetrance and are associated with a highly variable phenotype that may include intellectual disability, epilepsy, facial dysmorphism and digit anomalies. Rare patients carrying homozygous deletions show more severe phenotypes including epileptic encephalopathy, hypotonia and poor growth. For years, CHRNA7 (MIM: 118511), was considered the candidate gene that could account for this syndrome. However, recent studies in mouse models have shown that OTUD7A/CEZANNE2 (MIM: 612024), which encodes for an ovarian tumor (OTU) deubiquitinase, should be considered the critical gene responsible for brain dysfunction. In this study, a patient presenting with severe global developmental delay, language impairment and epileptic encephalopathy was referred to our genetics center. Trio exome sequencing (tES) analysis identified a homozygous OTUD7A missense variant (NM_130901.2:c.697C>T), predicted to alter an ultraconserved amino acid, p.(Leu233Phe), lying within the OTU catalytic domain. Its subsequent segregation analysis revealed that the parents, presenting with learning disability, and brother were heterozygous carriers. Biochemical assays demonstrated that proteasome complex formation and function were significantly reduced in patient-derived fibroblasts and in OTUD7A knockout HAP1 cell line. We provide evidence that biallelic pathogenic OTUD7A variation is linked to early-onset epileptic encephalopathy and proteasome dysfunction.

Deciphering exome sequencing data: Bringing mitochondrial DNA variants to light.

The expanding use of exome sequencing (ES) in diagnosis generates a huge amount of data, including untargeted mitochondrial DNA (mtDNA) sequences. We developed a strategy to deeply study ES data, focusing on the mtDNA genome on a large unspecific cohort to increase diagnostic yield. A targeted bioinformatics pipeline assembled mitochondrial genome from ES data to detect pathogenic mtDNA variants in parallel with the "in-house" nuclear exome pipeline. mtDNA data coming from off-target sequences (indirect sequencing) were extracted from the BAM files in 928 individuals with developmental and/or neurological anomalies. The mtDNA variants were filtered out based on database information, cohort frequencies, haplogroups and protein consequences. Two homoplasmic pathogenic variants (m.9035T>C and m.11778G>A) were identified in 2 out of 928 unrelated individuals (0.2%): the m.9035T>C (MT-ATP6) variant in a female with ataxia and the m.11778G>A (MT-ND4) variant in a male with a complex mosaic disorder and a severe ophthalmological phenotype, uncovering undiagnosed Leber's hereditary optic neuropathy (LHON). Seven secondary findings were also found, predisposing to deafness or LHON, in 7 out of 928 individuals (0.75%). This study demonstrates the usefulness of including a targeted strategy in ES pipeline to detect mtDNA variants, improving results in diagnosis and research, without resampling patients and performing targeted mtDNA strategies.

Molecular Characterization of a Familial 13.6-Mb 20p11.1p12.1 Duplication without Clinical Consequence.

Chromosomal microarray (CMA) is currently considered as a first-tier test in the genetic assessment of patients presenting with intellectual disability and/or multiple congenital abnormalities. The distinction between pathogenic CNVs, polymorphisms, and variants of unknown significance can be a diagnostic dilemma for cytogeneticists. The size of the CNV has been proposed as a useful criterion. We herein report the characterization of a 13.6-Mb interstitial duplication 20p11.1p12.1, found in a child presenting with mild global developmental delay, by standard karyotype and CMA. Unexpectedly, the same CNV was detected in the patient's mother and pregnant sister, who were healthy. On the basis of these results, an implication of this CNV in the neurological problems observed in the proband was considered to be unlikely. This report underlines the complexity of genetic counseling concerning rare chromosomal abnormalities, when little information is available either in the literature or in international cytogenetic databases.

2.5 years' experience of GeneMatcher data-sharing: a powerful tool for identifying new genes responsible for rare diseases.

PURPOSE: Exome sequencing (ES) powerfully identifies the molecular bases of heterogeneous conditions such as intellectual disability and/or multiple congenital anomalies (ID/MCA). Current ES analysis, combining diagnosis analysis restricted to disease-causing genes reported in OMIM database and subsequent research investigation extended to other genes, indicated causal and candidate genes around 40% and 10%. Nonconclusive results are frequent in such ultrarare conditions that recurrence and genotype-phenotype correlations are limited. International data-sharing permits the gathering of additional patients carrying variants in the same gene to draw definitive conclusions on their implication as disease causing. Several web-based tools have been developed and grouped in Matchmaker Exchange. In this study, we report our current experience as a regional center that has implemented ES as a first-line diagnostic test since 2013, working with a research laboratory devoted to disease gene identification. METHODS: We used GeneMatcher over 2.5 years to share 71 novel candidate genes identified by ES. RESULTS: Matches occurred in 60/71 candidate genes allowing to confirm the implication of 39% of matched genes as causal and to rule out 6% of them. CONCLUSION: The introduction of user-friendly gene-matching tools, such as GeneMatcher, appeared to be an essential step for the rapid identification of novel disease genes responsible for ID/MCA.

Constitutional chromoanasynthesis: description of a rare chromosomal event in a patient.

Structural alterations in chromosomes are a frequent cause of cancers and congenital diseases. Recently, the phenomenon of chromosome crisis, consisting of a set of tens to hundreds of clustered genomic rearrangements, localized in one or a few chromosomes, was described in cancer cells under the term chromothripsis. Better knowledge and recognition of this catastrophic chromosome event has brought to light two distinct entities, chromothripsis and chromoanasynthesis. The complexity of these rearrangements and the original descriptions in tumor cells initially led to the thought that it was an acquired anomaly. In fact, a few patients have been reported with constitutional chromothripsis or chromoanasynthesis. Using microarray we identified a very complex chromosomal rearrangement in a patient who had a cytogenetically visible rearrangement of chromosome 18. The rearrangement contained more than 15 breakpoints localized on a single chromosome. Our patient displayed intellectual disability, behavioral troubles and craniofacial dysmorphism. Interestingly, the succession of duplications and triplications identified in our patient was not clustered on a single chromosomal region but spread over the entire chromosome 18. In the light of this new spectrum of chromosomal rearrangements, this report outlines the main features of these catastrophic events and discusses the underlying mechanism of the complex chromosomal rearrangement identified in our patient, which is strongly evocative of a chromoanasynthesis.

KIAA1797/FOCAD encodes a novel focal adhesion protein with tumour suppressor function in gliomas.

In a strategy to identify novel genes involved in glioma pathogenesis by molecular characterization of chromosomal translocation breakpoints, we identified the KIAA1797 gene, encoding a protein with an as yet undefined function, to be disrupted by a 7;9 translocation in a primary glioblastoma culture. Array-based comparative genomic hybridization detected deletions involving KIAA1797 in around half of glioblastoma cell lines and glioblastomas investigated. Quantification of messenger RNA levels in human tissues demonstrated highest KIAA1797 expression in brain, reduced levels in all glioblastoma cell lines and most glioblastomas and similar levels in glial and neuronal cells by analysis of different hippocampal regions from murine brain. Antibodies against KIAA1797 were generated and showed similar protein levels in cortex and subcortical white matter of human brain, while levels were significantly reduced in glioblastomas with KIAA1797 deletion. By immunofluorescence of astrocytoma cells, KIAA1797 co-localized with vinculin in focal adhesions. Physical interaction between KIAA1797 and vinculin was demonstrated via co-immunoprecipitation. Functional in vitro assays demonstrated a significant decrease in colony formation, migration and invasion capacity of LN18 and U87MG glioma cells carrying a homozygous KIAA1797 deletion ectopically expressing KIAA1797 compared with mock-transduced cells. In an in vivo orthotopic xenograft mouse model, U87MG tumour lesions expressing KIAA1797 had a significantly reduced volume compared to tumours not expressing KIAA1797. In summary, the frequently deleted KIAA1797 gene encodes a novel focal adhesion complex protein with tumour suppressor function in gliomas, which we name 'focadhesin'. Since KIAA1797 genetic variation has been implicated in Alzheimer's disease, our data are also relevant for neurodegeneration.

Array-CGH in unclear syndromic nephropathies identifies a microdeletion in Xq22.3-q23.

To investigate whether submicroscopic chromosomal deletions or duplications can be causative of unclear syndromic nephropathies, we analyzed ten patients with congenital abnormalities of the kidney and urinary tract or glomerulopathies combined with important extrarenal anomalies by whole-genome array-based comparative genomic hybridization. In a 14-year-old girl presenting with hematuria, proteinuria, mental retardation (MR), sensorineural hearing loss, dysmorphisms, and epilepsy, we detected a microdeletion in chromosome Xq22.3-q23. This deletion was verified and characterized by fluorescence in situ hybridization and multiplex ligation-dependent probe amplification analyses, found to be de novo, uniallelic and 3.3 Mb in size. Electron microscopy of a kidney biopsy showed glomerular basement membrane thinning and segmental splitting of the lamina densa compatible with Alport syndrome. Cranial magnetic resonance and diffusion tensor imaging detected a severe neuronal migration disorder with double cortex formation and pronounced reduction of the fronto-occipital tract system. Thus, in one of ten patients with unclear syndromic nephropathies we identified a previously undescribed contiguous gene syndrome at Xq22.3-q23. The microdeletion contains the X-linked Alport syndrome gene COL4A5, the MR genes FACL4 and PAK3, and parts of the X-chromosomal lissencephaly gene DCX associated with double cortex formation in girls, MR, and epilepsy. The phenotype in our patient combines features of the Alport-MR contiguous gene syndrome with lissencephaly.

Identification of genomic aberrations associated with shorter overall survival in patients with oligodendroglial tumors.

Deletions on chromosomes 1p and 19q are associated with favorable prognosis in patients with oligodendroglial tumors. The aim of our study was to identify additional genomic aberrations linked to patient survival. We performed a genome-wide screen for genomic imbalances by comparative genomic hybridization on tumors from 70 patients, including 40 oligodendrogliomas, 30 oligoastrocytomas (21 WHO grade II tumors, 49 WHO grade III tumors). Data were correlated with overall patient survival (OS, median follow-up: 5.8 years). The most frequent aberrations were losses on chromosome 19q (64%), 1p (59%), 9p (26%), 4q (21%), 10q (19%), 18q (17%); gains on 7q (24%), 19p (19%), 7p (17%). In univariate analyses, combined 1p/19q and 19q loss were significantly associated with longer OS, and gains on 7, 8q, 19q, 20, losses on 9p, 10, 18q, Xp with shorter OS. Multivariate analyses showed the most significant prognostic factors for OS of patients with any oligodendroglial tumor to be WHO grade [odds ratio (OR) 8], 7p gain (OR 6), 9p loss (OR 3); for OS of patients with anaplastic tumors to be 7p gain (OR 10), 8q gain (OR 5), 18q loss (OR 3). Patients with anaplastic oligodendroglial tumors containing one or more prognostically unfavorable genomic aberration had a poor outcome independent of the 1p/19q status. In summary, we identified several independent genomic markers of shorter survival in patients with oligodendroglial tumors. Thus, molecular diagnostic testing, which is usually restricted to 1p/19q deletion analysis, may need to be refined by additionally assessing the prognostically unfavorable genomic aberrations identified.

Towards mapping phenotypical traits in 18p- syndrome by array-based comparative genomic hybridisation and fluorescent in situ hybridisation.

Molecular karyotyping holds the promise of improving genotype-phenotype correlations for frequent chromosome conditions such as the 18p- syndrome. In spite of more than 150 reported cases with deletions in 18p, no reliable phenotype map for the characteristic clinical findings such as mental retardation, post-natal growth retardation and typical facial features has been established yet. Here, we report on four patients with partial monosomy 18p of different sizes owing to unbalanced translocations that were thoroughly characterised clinically and by molecular karyotyping. One patient had a terminal deletion of 1.6 Mb in 18p and a trisomy of 8q24.23-qter as determined by array-based comparative genomic hybridisation and large insert clone fluorescent in situ hybridisation. In two sibs and a fourth patient, cytogenetic and molecular-cytogenetic analyses showed the terminal deletions in 18p (8.0 and 13.84 Mb, respectively) to be accompanied by partial trisomies of 20p. Literature analyses of typical phenotypic features of 18p-, 8q+ and 20p+ syndromes allowed the attribution of clinical findings in our patients to the respective chromosomal aberration. Based on these data, we propose a phenotype map for several clinical features of the 18p- syndrome: Round face was tentatively mapped to the distal 1.6 Mb of 18p; post-natal growth retardation and seizures to the distal 8 Mb and ptosis and short neck to the proximal half of 18p.

Delineation by molecular cytogenetics of 5q deletion breakpoints in myelodyplastic syndromes and acute myeloid leukemia.

Deletions of 5q in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are of different extents and the majority map to sub-bands 5q13.3 and 5q33.1. To further pinpoint these deletions, we have performed a detailed interphase fluorescence in situ hybridization (I-FISH) analysis with precisely mapped BAC probes. Eleven MDS and two AML patients with a sole cytogenetically visible del(5q) were studied. The proximal deletion endpoints were localized between 75 and 86 megabases (Mb) (5q13, five times), 86 and 96 Mb (5q14 approximately q15, four times), and at various sites in the other four. The distal breakpoints mapped between 153 and 155 Mb (5q33.2, five times), 156 and 158 Mb (5q33.3, three times), 158 and 164 Mb (5q34, two times), and 164 and 181 Mb (telomere) in three. The largest deletion was approximately 70 Mb and the smallest was 43 Mb. These studies show that cytogenetically similar appearing deletions in 5q are highly variable in molecular terms. We also found that in MDS cases with a blast count between 0 and 13%, cells with a del(5q) were present in 24-90% of interphase (nondividing) cells and in 30-100% of metaphase (dividing) cells. In the two AML patients with a blast count of 30 and 80%, del(5q) was found in 35 and 95% interphase cells and 95 and 100% of metaphase cells, respectively. This demonstrates that a low blast count can be associated with a high proportion of 5q- cells in the bone marrow.

Molecular cytogenetic profiling of complex karyotypes in primary myelodysplastic syndromes and acute myeloid leukemia.

Complex chromosomal aberrations are present in < or =30% of patients with primary myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML) and are associated with a poor prognosis. Specific alterations in complex karyotypes are difficult to define by conventional cytogenetics alone. To obtain a more comprehensive view of the recurrent aberrations, we performed spectral karyotyping (SKY) and fluorescence in situ hybridization (FISH) with selected probes on bone marrow samples from 17 patients with primary MDS and 3 with primary AML. All cases had chromosome 5 alterations. Two different types of 5q loss were identified: unbalanced translocations and interstitial deletions, or del(5q), each occurring in 10 patients. The most frequent additional chromosome aberrations were -3/-3p/-3q, -7/7q-, +8, 13q-, -16, 17p-, -18/18p-, -20/20q-, and +21q, each occurring in 25%. In the five cases with gain of 21q, involvement of the AML1 gene was excluded. Unbalanced 5q translocations occurred more often in combination with monosomy 3 and 7 and with gain of 21q, whereas del(5q) was associated more often with -1p and trisomy 8. A detailed analysis of specific breakpoints and deletions revealed recurrent involvement of specific chromosomal bands harboring known tumor suppressor genes or oncogenes. Analysis of large numbers of MDS and AML cases in a similar detailed manner with SKY and FISH will reveal whether new subgroups can be identified according to their genetic alterations. Correlation with clinical parameters may reveal the prognostic significance of these genetic subgroups.