A novel intellectual disability syndrome caused by GPI anchor deficiency due to homozygous mutations in PIGT.

PURPOSE: To delineate the molecular basis for a novel autosomal recessive syndrome, characterised by distinct facial features, intellectual disability, hypotonia and seizures, in combination with abnormal skeletal, endocrine, and ophthalmologic findings. METHODS: We examined four patients from a consanguineous kindred with a strikingly similar phenotype, by using whole exome sequencing (WES). Functional validation of the initial results were performed by flow cytometry determining surface expression of glycosylphosphatidylinositol (GPI) and GPI anchored proteins and, in addition, by in vivo assays on zebrafish embryos. RESULTS: The results from WES identified a homozygous mutation, c.547A>C (p.Thr183Pro), in PIGT; Sanger sequencing of additional family members confirmed segregation with the disease. PIGT encodes phosphatidylinositol-glycan biosynthesis class T (PIG-T) protein, which is a subunit of the transamidase complex that catalyses the attachment of proteins to GPI. By flow cytometry, we found that granulocytes from the patients had reduced levels of the GPI anchored protein CD16b, supporting pathogenicity of the mutation. Further functional in vivo validation via morpholino mediated knockdown of the PIGT ortholog in zebrafish (pigt) showed that, unlike human wild-type PIGT mRNA, the p.Thr183Pro encoding mRNA failed to rescue gastrulation defects induced by the suppression of pigt. CONCLUSIONS: We identified mutations in PIGT as the cause of a novel autosomal recessive intellectual disability syndrome. Our results demonstrate a new pathogenic mechanism in the GPI anchor pathway and expand the clinical spectrum of disorders belonging to the group of GPI anchor deficiencies.

Inherited mosaicism for the supernumerary marker chromosome in cat eye syndrome: inter- and intra-individual variation and correlation to the phenotype.

We have studied a family with repeated transmission of mosaicism for a supernumerary marker chromosome (SMC), giving rise to varying symptoms of the cat eye syndrome (CES) in the offspring. The frequency of the SMC was investigated using FISH with probes from the CES critical region on lymphocytes as well as buccal cells. The same probes were used to study the frequency of the SMC in spermatozoa from the father. The SMC was characterized in detail using array-CGH and was found to correspond to a symmetrical cat eye SMC type I, with two extra copies of the most proximal part of 22q11, not extending into the classical 22q11.2 deletion region. Mosaicism for the SMC was detected in 4 out of 7 family members, the father and all his three children. The degree of mosaicism varied greatly between individuals as well as between tissues, with twice as many cells with the SMC in epithelial cells compared to blood. The highest frequency (almost 50%) was found in spermatozoa from the father. There was a direct correlation between the degree of mosaicism and the symptoms, varying from no obvious symptoms to classical CES. The study confirms the occurrence of direct transmission of SMC-mosaicism in CES. The results indicate that examination of parental epithelial cells should be preferred compared to blood cells in order to exclude a recurrence risk in parents of a child with CES. Interphase FISH analysis of spermatozoa is the most sensitive method to exclude paternal germ line mosaicsm.

Interphase fluorescent in situ hybridization deletion analysis of the 9p21 region and prognosis in childhood acute lymphoblastic leukaemia (ALL): results from a prospective analysis of 519 Nordic patients treated according to the NOPHO-ALL 2000 protocol.

Interphase fluorescent in situ hybridization (FISH) was applied on diagnostic BM smears from 519 children with acute lymphoblastic leukaemia (ALL) in order to establish the frequency and prognostic importance of 9p21 deletion in children enrolled in the Nordic Society of Paediatric Haematology and Oncology (NOPHO) - 2000 treatment protocol. Among the patients, 452 were diagnosed with B-cell precursor (BCP)-ALL and 66 with T-ALL. A higher incidence of 9p21 deletions was found in T-ALL (38%) compared to BCP-ALL (15·7%). Homozygous deletions were found in 19·7% of T-ALL and 4·0% of BCP-ALL; hemizygous deletions were found in 18·2% and 11·7% respectively. In our series, 9p21 deletions were detected in all age groups with a steady rise in the frequency with age. There was no significant difference in outcome between cases with or without 9p21 deletion or between cases with hemi- or homozygous deletions of 9p21. In conclusion, in this large series of childhood ALL deletion of 9p21 was not associated with worse prognosis. However, interphase FISH deletion analysis of 9p21 could be used as a first step to detect unfavourable subtle cytogenetic aberrations such as the dic(9;20) rearrangement.

Molecular and clinical characterization of patients with overlapping 10p deletions.

Chromosome 10p terminal deletions have been associated with DiGeorge phenotype, and within the same genomic region haploinsufficiency of GATA3 causes the HDR syndrome (hypoparathyroidism, sensorineural deafness, renal dysplasia). We have performed detailed molecular analysis of four patients with partial overlapping 10p deletions by using FISH-mapping, array-CGH, and custom-designed high-resolution oligonucleotide array. All four patients had mental retardation and speech impairment and three of them showed variable signs of HDR syndrome. In addition, two patients had autistic behaviors and had similar dysmorphic features giving them a striking physical resemblance. A review of the literature identified 10 previously published cases with similar 10p deletions and reliable molecular or molecular cytogenetic mapping data. The combined information of present and previous cases suggests that partial deletions of 10p14-p15 represent a syndrome with a distinct and more severe phenotype than previously assumed. The main characteristics include severe mental retardation, language impairment, autistic behavior, and characteristic clinical features. A critical region involved in mental retardation and speech impairment is defined within 1.6 Mb in 10p15.3. In addition, deletion of 4.3 Mb within 10p14 is associated with autism and characteristic clinical findings.

Chimerism resulting from parthenogenetic activation and dispermic fertilization.

Whole-body human chimerism is the result of two zygotes giving rise to one individual, and is a rarely detected condition. We have studied the molecular background and discuss the likely mechanism for the chimerism in a patient with a 46,XX/47,XY,+14 karyotype and ambiguous genitalia, cryptorchidism, pigment anomalies, and normal psychomotor development. We have used karyotyping, interphase-FISH and array-CGH analysis as well as molecular analysis of polymorphic markers from 48 loci in order to define the origin and percentage of 47,XY,+14 cells in different tissues. Based on the findings of two paternal alleles and the detection of homozygous maternal alleles without evidence of crossing-over, and the fact that four alleles were never detected, our results indicate that the chimerism in our patient is the result of dispermic fertilization of a parthenogenetically activated oocyte. Our report underlines that cytogenetic findings suggesting mosaicism might actually indicate chimerism as an underlying mechanism in patients. It also highlights the difficulties in predicting the clinical outcome in patients with genetic aberrations in mosaic or chimeric form.

Clinical and cytogenetic features of a population-based consecutive series of 285 pediatric T-cell acute lymphoblastic leukemias: rare T-cell receptor gene rearrangements are associated with poor outcome.

Clinical characteristics and cytogenetic aberrations were ascertained and reviewed in a population-based consecutive series of 285 pediatric T-cell acute lymphoblastic leukemias (T-ALLs) diagnosed between 1992 and 2006 in the Nordic countries. Informative karyotypic results were obtained in 249 (87%) cases, of which 119 (48%) were cytogenetically abnormal. Most (62%) of the aberrant T-ALLs were pseudodiploid. Structural changes were more common than numerical ones; 86% displayed at least one structural abnormality and 41% at least one numerical anomaly. The most frequent abnormalities were T-cell receptor (TCR) gene rearrangements (20%) [TCR;11p13 (10%), TCR;10q24 (3%), TCR;other (8%)], del(9p) (17%), +8 (14%), del(6q) (12%), and 11q23 rearrangements (6%). The TCR;other group comprised the rare rearrangements t(X;14)(p11;q11), t(X;7)(q22;q34), t(1;14)(p32;q11), ins(14;5)(q11;q?q?), inv(7)(p15q34), t(8;14)(q24;q11), t(7;11)(q34;p15), and t(12;14)(p13;q11). The clinical characteristics of this Nordic patient cohort agreed well with previous larger series, with a median age of 9.0 years, male predominance (male/female ratio 3.1), median white blood cell (WBC) count of 66.5 x 10(9)/l, and a high incidence of mediastinal mass and central nervous system involvement (59% and 9.5%, respectively). These features did not differ significantly among the various genetic subgroups. 5-year event-free survival (EFS) and overall survival for all patients were 0.61 (+/-0.03) and 0.67 (+/-0.03), respectively. In a multivariate analysis, two factors affected negatively the EFS, namely a WBC count of > or =200 x 10(9)/l (P < 0.001) and the presence of rare TCR rearrangements (P = 0.001). In conclusion, in this large series of childhood T-ALLs from the Nordic countries, the cytogenetic findings were not associated with risk of therapy failure with the exception of the TCR;other group. However, further prospective and collaborative investigations of this genetically heterogeneous entity are needed to confirm these results.

Overexpression of CD123 correlates with the hyperdiploid genotype in acute lymphoblastic leukemia.

We evaluated CD123 expression in 95 pediatric and 24 adult ALL patients and compared the results with the CD123 expression in normal B-cell precursors. Early B-cell precursors were negative while intermediate precursors and mature B cells showed weak CD123 expression. Leukemic blasts in 31% of precursor-B ALL samples exhibited strong expression of CD123, 61% had moderate CD123 expression and 8% were negative; 81.5% of ALL with hyperdiploid karyotype (>/= 52 chromosomes) showed strong CD123 overexpression. In contrast, cases with ETV6/RUNX1 rearrangement had weak CD123 expression. Our study suggests that overexpression of CD123 is an aberrant phenotype present in a subset of precursor-B ALL with hyperdiploid genotype, and represents an additional marker of good prognosis in pediatric precursor-B ALL. Moreover, aberrant CD123 expression in ALL is a good marker for monitoring of minimal residual disease.

Array-CGH reveals hidden gene dose changes in children with acute lymphoblastic leukaemia and a normal or failed karyotype by G-banding.

A tiling path 33K BAC array was used to study 28 children with acute lymphoblastic leukaemia (ALL) who had normal or failed G-banded karyotypes. Twenty-two patients (79%) had a total of 135 copy number alterations (CNA) (69 gains and 66 losses); most of these patients showed CNA that were below the resolution of G-banding. Molecular cytogenetic and array comparative genomic hybridization results enabled the division of B-precursor ALL patients into five groups: high hyperdiploidy, intrachromosomal amplification of 21q, ETV6/RUNX1 rearrangement, others and no CNA. Apart from a shared deletion of 9p21.3, T-ALL patients had additional small CNA, with no region in common.

Outcome of ETV6/RUNX1-positive childhood acute lymphoblastic leukaemia in the NOPHO-ALL-1992 protocol: frequent late relapses but good overall survival.

The prognostic impact of t(12;21)(p13;q22) [ETV6/RUNX1 fusion] in paediatric acute lymphoblastic leukaemia (ALL) has been extensively debated, particularly with regard to the frequency of late relapses and appropriate treatment regimens. We have retrospectively collected 679 ALLs with known ETV6/RUNX1 status, as ascertained by fluorescence in situ hybridization or reverse-transcription polymerase chain reaction, treated according to the Nordic Society of Paediatric Haematology and Oncology -ALL-1992 protocol. The assigned risk groups/treatment modalities for the 171 (25%) patients with t(12;21)-positive ALLs were 74 (43%) standard risk, 71 (42%) intermediate risk and 26 (15%) high risk. The 5- and 10-year event-free survival (EFS) of the 171 patients was 80% and 75% respectively, with no significant differences among the three risk groups. Most of the relapses occurred in boys and were late, with almost 50% of all relapses occurring > or = 5 years after diagnosis. Of all relapses after 6 years, 80% occurred in the t(12;21)-positive group. The overall survival was 94% at 5 years and 88% at 10 years; thus, the treatment of patients in second or later remission is usually successful. As yet, there is no reliable plateau in the EFS curve, a fact that raises the question as to when the prognostic ramifications of ALLs harbouring ETV6/RUNX1 should be evaluated.

Combined spectral karyotyping, comparative genomic hybridization, and in vitro apoptyping of a panel of Burkitt's lymphoma-derived B cell lines reveals an unexpected complexity of chromosomal aberrations and a recurrence of specific abnormalities in chemoresistant cell lines.

The comprehensive cytogenetic profiles of a panel of 10 Burkitt's lymphoma (BL)-derived B cell lines, designated Akata, BL-28, BL-41, Daudi, DG-75, Mutu I, Mutu III, Namalwa, Rael, and Ramos, respectively, are reported herein. The unique origin of each cell line was established using multiplex quantitative fluorescence polymerase chain reaction (QF-PCR). Spectral karyotyping (SKY) revealed a large number of structural and numerical chromosomal aberrations, many of which had not been previously identified or resolved by conventional G-banding techniques. Notably, whereas all 10 cell lines harbored the hallmark translocation t(8;14)(q24;q32), no other common structural aberrations were identified, although translocations involving chromosomes 3, 13, and 17 were frequently seen. Moreover, analysis of chromosomal breakpoints by comparative genomic hybridization (CGH) revealed a number of recurring aberrations, such as gain of chromosomes 7 and 20, gains of regions at 2p, 3q, 13q and 16q, and losses at 3p, 4q and 17p. In addition, apoptyping (i.e. determination of in vitro responses to apoptosis stimulation) of the cell lines suggested specific association patterns between karyotypic changes (e.g. translocations involving 17p, and gains of portions of chromosomes 7 and 20) and resistance to the chemotherapeutic agent, etoposide. The current molecular cytogenetic characterization of 10 BL cell lines has thus identified several novel sites of rearrangements; moreover, the combined karyotyping and functional assessment (apoptyping) of these cell lines serves to enhance their utility in future studies aimed at gene discovery and gene function.

Cryptic subtelomeric 6p deletion in a girl with congenital malformations and severe language impairment.

Several cases with microscopically visible, terminal 6p deletions have been described, and a distinct clinical phenotype has emerged, including developmental delay, congenital heart malformations, ocular abnormalities, hearing loss and a characteristic facial appearance. We report a patient with a submicroscopic 6p deletion, detected by subtelomeric screening using fluorescence in situ hybridisation. This girl presented with typical facial dysmorphic features, hearing impairment, malformation of the anterior eye segment, an ASD and severe language impairment. However, her cognitive functions were within the normal range. Detailed FISH analysis with 20 BAC probes covering the distal 6p25 region estimated the size of the terminal deletion to 2.1 Mb, and thus this case narrows down the critical region for the 6p phenotype. The forkhead transcription factor gene FOXC1, involved in a spectrum of anterior eye chamber disorders, is deleted in this patient, together with several characterised and putative genes with yet unknown function.

A comparison of different metaphase CGH methods for the detection of cryptic chromosome aberrations of defined size.

An increasing body of evidence indicates that submicroscopic gene dose alterations may cause mental impairment and malformations. During the last decade, comparative genomic hybridization (CGH) has become a useful tool in the detection and mapping of chromosome aberrations. Modifications of CGH with increased resolution down to 3-5 Mb have been reported and CGH is now offered as a diagnostic procedure in the evaluation of patients with idiopathic mental retardation (MR). In order to increase the resolution, we modified the CGH protocol using freshly prepared high-quality metaphase slides and chemical labeling, and tested the method on a set of patients with well-defined submicroscopic chromosome abnormalities with confirmed size 1.3-20.5 Mb. Subsequently, a completely blinded test was performed to compare the performance of the chemical labeling CGH to the commercially available HR-CGH. Using the two different CGH methods, we were able to detect chromosome imbalances down to 2-3 Mb approximately. The HR-CGH method detected all aberrations >6 Mb and a few smaller, while the modified CGH method was able to detect all but three aberrations >1.8 Mb. The modified CGH method was superior in the detection of terminal imbalances, while the HR-CGH software was more successful in the detection of imbalances located very close to the centromeric regions. In conclusion, the resolution of metaphase CGH may be as high as 2-3 Mb but is most likely depending on the chromosomal region involved, a clear limitation when used as a screening method for chromosome aberrations in patients with idiopathic MR.

An excess of chromosome 1 breakpoints in male infertility.

In a search for potential infertility loci, which might be revealed by clustering of chromosomal breakpoints, we compiled 464 infertile males with a balanced rearrangement from Mendelian Cytogenetics Network database (MCNdb) and compared their karyotypes with those of a Danish nation-wide cohort. We excluded Robertsonian translocations, rearrangements involving sex chromosomes and common variants. We identified 10 autosomal bands, five of which were on chromosome 1, with a large excess of breakpoints in the infertility group. Some of these could potentially harbour a male-specific infertility locus. However, a general excess of breakpoints almost everywhere on chromosome 1 was observed among the infertile males: 26.5 versus 14.5% in the cohort. This excess was observed both for translocation and inversion carriers, especially pericentric inversions, both for published and unpublished cases, and was significantly associated with azoospermia. The largest number of breakpoints was reported in 1q21; FISH mapping of four of these breakpoints revealed that they did not involve the same region at the molecular level. We suggest that chromosome 1 harbours a critical domain whose integrity is essential for male fertility.

Simian AIDS-related lymphoma growth in severe combined immunodeficiency mice is independent of karyotypic abnormalities or Bcl-6 mutations.

Simian AIDS-related lymphomas (sARL) of cynomolgus monkeys infected with a simian immunodeficiency virus (SIVsm) were studied in relation to growth in severe combined immunodeficient (SCID) mice, karyotype abnormalities, and DNA sequence of the first noncoding region of the Bcl-6 gene. The tumors were diffuse large B cell lymphomas and expressed a simian homolog to Epstein-Barr virus (HVMF-1) in 12 of 13 primary tumors and corresponding cell lines. A tested cell line was tumorigenic in SCID mice. Tumors in the SCID mice showed cell growth features similar to those in the original lymphoma, suggesting that no subpopulation with growth advantage was selected for in the mice. Spectral karyotype analysis of sARL cell lines showed normal cytogenetic features except for a trisomy of monkey chromosome 2 (corresponding to human chromosomes 7 and 21) in two of five sARL lines, which was not recovered in SCID tumors established from the same cell line. Sequence analysis of a Bcl-6 gene fragment showed sequence variations indicative of population polymorphism(s) in 10 of 13 sARLs, and no evidence of Bcl-6 mutations. Thus Bcl-6 mutations in the first noncoding region are irrelevant for sARL development in cynomolgus monkeys and for tumorigenicity of sARL cell lines. We also demonstrate that no cytogenetic alterations are needed for the development of highly aggressive lymphomas in the SIV-immunosuppressed host.

Subtelomeric rearrangements detected in patients with idiopathic mental retardation.

A screening for submicroscopic rearrangements was performed in 111 patients with idiopathic mental retardation (MR) using fluorescence in situ hybridization (FISH) probes from the subtelomeric regions of all chromosome arms. Ten cryptic rearrangements were found (9%): five de novo deletions; one unbalanced de novo translocation; three unbalanced inherited translocations; and one unbalanced recombinant chromosome, inherited from a parent with a pericentric inversion. In addition, 50 of the patients were screened for interstitial rearrangements with spectral karyotyping (SKY), but no aberrations were found. However, SKY detected the subtelomeric rearrangement in three of the four unbalanced translocations. Dysmorphic features were present in all patients with detected subtelomeric rearrangements.

Combined genetic and transcriptional profiling of acute myeloid leukemia with normal and complex karyotypes.

BACKGROUND AND OBJECTIVES: Acute myeloid leukemia (AML) is a heterogeneous group of diseases. Patients with a normal karyotype constitute the largest single group; multiple chromosome rearrangements involving three or more chromosomes occur in 5 10% of AML patients. The pathophysiologic mechanisms underlying both groups are largely unknown. In the current study, we have systematically combined transcriptional profiles with cytogenetic data from 15 AML patients with either normal or complex karyotypes. DESIGN AND METHODS: The expression profiles were investigated by unsupervised hierarchical clustering, supervised cluster analysis, and comparative genomic microarray analysis. In addition, the samples were analyzed by G-banding and/or spectral karyotyping and comparative genomic hybridization. RESULTS: Our results show that AML with complex karyotypes exhibit a gene expression profile that is specific to this group of patients. The differentially expressed genes included several located on 5q and 7q, as well as genes involved in controlling cell division. We also found that DNA gains and losses caused by multiple chromosome rearrangements result in altered gene expression in a gene-dosage-dependent manner. INTERPRETATION AND CONCLUSIONS: These data provide insight into the mechanisms of multiple chromosome rearrangements and further demonstrate that the expression patterns of AML are strongly linked to the karyotypic status, even for the relatively undefined cytogenetic subgroup AML with complex karyotype.

A region close to Tp53 shows LOH in familial breast cancer.

The proportions of mutation of BRCA1 and BRCA2 detected in familial breast cancer vary in different regions. Most breast cancer families in Sweden cannot be explained by mutations in the known major susceptibility genes. Our previous studies have found a high frequency of LOH in the Tp53 region in familial breast cancer suggesting a putative tumor suppressor gene in this region, and the Tp53 gene was excluded as predisposing gene in these families by mutation screening. In order to identify other candidate tumor suppressor genes responsible for familial breast cancer, we performed LOH analysis in 98 paired tumor and blood samples from 91 breast cancer families using 11 microsatellite markers on chromosome 17p. Two loci with high frequency of LOH were found. One spanned the Tp53 gene, the other was distal to Tp53. Linkage studies were performed on 17p with 11 microsatellite markers in 102 breast cancer families with no detectable mutations in the BRCA1 and BRCA2 gene. The linkage analysis did not further support any of the regions suggested by the LOH study. However, since the Tp53 gene is already known to predispose to breast cancer as well as being involved in tumor progression, it is possible that also this region, close but distal to Tp53 contains a gene involved in familial and/or sporadic breast cancer development similar to Tp53.

DNA copy-number analysis of the 22q11 deletion-syndrome region using array-CGH with genomic and PCR-based targets.

Deletions and duplications of genomic segments commonly cause developmental disorders. The resolution and efficiency in diagnosing such gene-dosage alterations can be drastically increased using microarray-based comparative genomic hybridization (array-CGH). However, array-CGH currently relies on spotting genomic clones as targets, which confers severe limitations to the approach including resolution of analysis and reliable gene-dosage assessment of regions with high content of redundant sequences. To improve the methodology for analysis, we compared the use of genomic clones, repeat-free pools of amplified genomic DNA and cDNAs (single and pooled) as targets on the array. For this purpose, we chose q11.2 locus on chromosome 22 as a testing ground. Microdeletions at 22q11 cause birth defects collectively described as the DiGeorge/velocardiofacial syndrome. The majority of patients present 3 Mb typical deletions. Here, we report the construction of a gene-dosage array, covering 6 Mb of 22q11 and including the typically deleted region. We hybridized DNA from six DiGeorge syndrome patients to the array, and show that as little as 11.5 kb non-redundant, repeat-free PCR-generated sequence can be used for reliable detection of hemizygous deletions. By extrapolation, this would allow analysis of the genome with an average resolution of 25 kb. In the case of cDNAs our results indicate that 3.5 kb sequence is necessary for accurate identification of haploid/diploid dosage alterations. Thus, for regions rich in redundant sequences and repeats, such as 22q11, a specifically tailored array-CGH approach is good for gene copy number profiling.

Prenatal diagnosis: molecular genetics and cytogenetics.

The technologies developed for the Human Genome Project, the recent surge of available DNA sequences resulting from it and the increasing pace of gene discoveries and characterization have all contributed to new technical platforms that have enhanced the spectrum of disorders that can be diagnosed prenatally. The importance of determining the disease-causing mutation or the informativeness of linked genetic markers before embarking upon a DNA-based prenatal diagnosis is, however, still emphasized. Different fluorescence in situ hybridization (FISH) technologies provide increased resolution for the elucidation of structural chromosome abnormalities that cannot be resolved by more conventional cytogenetic analyses, including microdeletion syndromes, cryptic or subtle duplications and translocations, complex rearrangements involving many chromosomes, and marker chromosomes. Interphase FISH and the quantitative fluorescence polymerase chain reaction are efficient tools for the rapid prenatal diagnosis of selected aneuploidies, the latter being considered to be most cost-effective if analyses are performed on a large scale. There is some debate surrounding whether this approach should be employed as an adjunct to karyotyping or whether it should be used as a stand-alone test in selected groups of women.