Hans G. Dauwerse

Diagnostic analysis of the Rubinstein-Taybi syndrome: five cosmids should be used for microdeletion detection and low number of protein truncating mutations

Rubinstein-Taybi syndrome (RTS) is a malformation syndrome characterised by facial abnormalities, broad thumbs, broad big toes, and mental retardation. In a subset of RTS patients, microdeletions, translocations, and inversions involving chromosome band 16p13.3 can be detected. We have previously shown that disruption of the human CREB binding protein (CREBBP orCBP) gene, either by these gross chromosomal rearrangements or by point mutations, leads to RTS. CBP is a large nuclear protein involved in transcription regulation, chromatin remodelling, and the integration of several different signal transduction pathways. Here we report diagnostic analysis of CBP in 194 RTS patients, divided into several subsets. In one case the mother is also suspect of having RTS. Analyses of the entireCBP gene by the protein truncation test showed 4/37 truncating mutations. Two point mutations, one 11 bp deletion, and one mutation affecting the splicing of the second exon were detected by subsequent sequencing. Screening theCBP gene for larger deletions, by using different cosmid probes in FISH, showed 14/171 microdeletions. Using five cosmid probes that contain the entire gene, we found 8/89 microdeletions of which 4/8 were 5′ or interstitial. This last subset of microdeletions would not have been detected using the commonly used 3′ probe RT1, showing the necessity of using all five probes.

FRG1P-mediated aggregation of proteins involved in pre-mRNA processing.

FRG1 is considered a candidate gene for facioscapulohumeral muscular dystrophy (FSHD) based on its location at chromosome 4qter and its upregulation in FSHD muscle. The FRG1 protein (FRG1P) localizes to nucleoli, Cajal bodies (and speckles), and has been suggested to be a component of the human spliceosome but its exact function is unknown. Recently, transgenic mice overexpressing high levels of FRG1P in skeletal muscle were described to present with muscular dystrophy. Moreover, upregulation of FRG1P was demonstrated to correlate with missplicing of specific pre-mRNAs. In this study, we have combined colocalization studies with yeast two-hybrid screens to identify proteins that associate with FRG1P. We demonstrate that artificially induced nucleolar aggregates of VSV-FRG1P specifically sequester proteins involved in pre-mRNA processing. In addition, we have identified SMN, PABPN1, and FAM71B, a novel speckle and Cajal body protein, as binding partners of FRG1P. All these proteins are, or seem to be, involved in RNA biogenesis. Our data confirm the presence of FRG1P in protein complexes containing human spliceosomes and support a potential role of FRG1P in either splicing or another step in nuclear RNA biogenesis. Intriguingly, among FRG1P-associated proteins are SMN and PABPN1, both being involved in neuromuscular disorders, possibly through RNA biogenesis-related processes.

A duplication at chromosome 11q12.2–11q12.3 is associated with spinocerebellar ataxia type 20

Spinocerebellar ataxia type 20 (SCA20) has been linked to chromosome 11q12, but the underlying genetic defect has yet to be identified. We applied single-nucleotide polymorphism genotyping to detect structural alterations in the genomic DNA of patients with SCA20. We found a 260 kb duplication within the previously linked SCA20 region, which was confirmed by quantitative polymerase chain reaction and fiber fluorescence in situ hybridization, the latter also showing its direct orientation. The duplication spans 10 known and 2 unknown genes, and is present in all affected individuals in the single reported SCA20 pedigree. While the mechanism whereby this duplication may be pathogenic remains to be established, we speculate that the critical gene within the duplicated segment may be DAGLA, the product of which is normally present at the base of Purkinje cell dendritic spines and contributes to the modulation of parallel fiber-Purkinje cell synapses.

Genomic imbalances in mental retardation

Introduction: It has been estimated that cytogenetically visible rearrangements are present in ~1% of newborns. These chromosomal changes can cause a wide range of deleterious developmental effects, including mental retardation (MR). It is assumed that many other cases exist where the cause is a submicroscopic deletion or duplication. To facilitate the detection of such cases, different techniques have been developed, which have differing efficiency as to the number of loci and patients that can be tested. Methods: We implemented multiplex amplifiable probe hybridisation (MAPH) to test areas known to be rearranged in MR patients (for example, subtelomeric/pericentromeric regions and those affected in microdeletion syndromes) and to look for new regions that might be related to MR. Results: In this study, over 30 000 screens for duplications and deletions were carried out; 162 different loci tested in each of 188 developmentally delayed patients. The analysis resulted in the detection of 19 rearrangements, of which ~65% would not have been detected by conventional cytogenetic analysis. A significant fraction (46%) of the rearrangements found were interstitial, despite the fact that only a limited number of these loci have so far been tested. Discussion: Our results strengthen the arguments for whole genome screening within this population, as it can be assumed that many more interstitial rearrangements would be detected. The strengths of MAPH for this analysis are the simplicity, the high throughput potential, and the high resolution of analysis. This combination should help in the future identification of the specific genes that are responsible for MR.

Genotype-Phenotype Correlation in Patients Suspected of Having Sotos Syndrome

Background: Deletions and mutations in the NSD1 gene are the major cause of Sotos syndrome. We wanted to evaluate the genotype-phenotype correlation in patients suspected of having Sotos syndrome and determine the best discriminating parameters for the presence of a NSD1 gene alteration. Methods: Mutation and fluorescence in situ hybridization analysis was performed on blood samples of 59 patients who were clinically scored into 3 groups. Clinical data were compared between patients with and without NSD1 alterations. With logistic regression analysis the best combination of predictive variables was obtained. Results: In the groups of typical, dubious and atypical Sotos syndrome, 81, 36 and 0% of the patients, respectively, showed NSD1 gene alterations. Four deletions were detected. In 23 patients (2 families) 19 mutations were detected (1 splicing defect, 3 non-sense, 7 frameshift and 8 missense mutations). The best predictive parameters for a NSD1 gene alteration were frontal bossing, down-slanted palpebral fissures, pointed chin and overgrowth. Higher incidences of feeding problems and cardiac anomalies were found. The parameters, delayed development and advanced bone age, did not differ between the 2 subgroups. Conclusions: In our patients suspected of having Sotos syndrome, facial features and overgrowth were highly predictive of a NSD1 gene aberration, whereas developmental delay and advanced bone age were not.

Common regulatory elements in the polycystic kidney disease 1 and 2 promoter regions

The PKD1 and PKD2 genes are mutated in patients with autosomal dominant polycystic kidney disease (ADPKD), a systemic disease, with the formation of renal cysts as main clinical feature. The genes are developmentally regulated and aberrant expression of PKD1 or PKD2 leads to cystogenesis. To date, however, the transcription factors regulating expression of these genes have hardly been studied. To identify conserved putative transcription factor-binding sites, we cloned and characterized the 5′-flanking regions of the murine and canine Pkd1 genes and performed a multispecies comparison by including sequences from the human and Fugu rubripes orthologues as well as the Pkd2 promoters from mouse and human. Sequence analysis revealed a variety of conserved putative binding sites for transcription factors and no TATA-box element. Nine elements were conserved in the mammalian Pkd1 promoters: AP2, E2F, E-Box, EGRF, ETS, MINI, MZF1, SP1, and ZBP-89. Interestingly, six of these elements were also found in the mammalian Pkd2 promoters. Deletion studies with the mouse Pkd1 promoter showed that a ∼280 bp fragment is capable of driving luciferase reporter gene expression, whereas reporter constructs containing larger fragments of the Pkd1 promoter showed a lower activity. Furthermore, mutating a potential E2F-binding site within this 280 bp fragment diminished the reporter construct activity, suggesting a role for E2F in regulating cell cycle-dependent expression of the Pkd1 gene. Our data define a functional promoter region for Pkd1 and imply that E2F, EGRF, Ets, MZF1, Sp1, and ZBP-89 are potential key regulators of PKD1 and PKD2 in mammals.European Journal of Human Genetics advance online publication, 16 March 2005; doi:10.1038/sj.ejhg.5201392

Chromosomal localization of the 5-HT1F receptor gene: no evidence for involvement in response to sumatriptan in migraine patients.

The 5-HT1F receptor, which is present in both human vascular and neuronal tissue, may mediate the therapeutic effect and/or side-effects of sumatriptan. We investigated the chromosomal localization of the 5-HT1F receptor gene and the relation between eventually existing polymorphisms and the clinical response to sumatriptan in migraine patients. The 5-HT1F receptor gene was localized using a monochromosomal mapping panel, followed by a radiation-reduced hybrid mapping and fluorescent in situ hybridization. The results of these techniques show that the 5-HT1F receptor gene is localized at 3p12. We investigated the presence of polymorphisms by single strand conformation polymorphism analysis in 14 migraine patients who consistently responded well to sumatriptan, 12 patients who consistently experienced recurrence of the headache after initial relief, 12 patients with no response to sumatriptan, and in 13 patients who consistently experienced chest symptoms after use of sumatriptan. No polymorphisms were detected in any of the patients. We therefore conclude that genetic diversity of the 5-HT1F receptor gene is most probably not responsible for the variable clinical response to sumatriptan.

Genomic acute myeloid leukemia-associated inv(16)(p13q22) breakpoints are tightly clustered

The inv(16) and related t(16;16) are found in 10% of all cases with de novo acute myeloid leukemia. In these rearrangements the core binding factor β (CBFB) gene on 16q22 is fused to the smooth muscle myosin heavy chain gene (MYH11) on 16p13. To gain insight into the mechanisms causing the inv(16) we have analysed 24 genomic CBFB-MYH11 breakpoints. All breakpoints in CBFB are located in a 15-Kb intron. More than 50% of the sequenced 6.2 Kb of this intron consists of human repetitive elements. Twenty-one of the 24 breakpoints in MYH11 are located in a 370-bp intron. The remaining three breakpoints in MYH11 are located more upstream. The localization of three breakpoints adjacent to a V(D)J recombinase signal sequence in MYH11 suggests a V(D)J recombinase-mediated rearrangement in these cases. V(D)J recombinase-associated characteristics (small nucleotide deletions and insertions of random nucleotides) were detected in six other cases. CBFB and MYH11 duplications were detected in four of six cases tested.

Possible phenotypic dosage effect in patients compound heterozygous for FSHD-sized 4q35 alleles.

Objective: Autosomal dominant facioscapulohumeral muscular dystrophy (FSHD) is associated with a contraction of the D4Z4 repeat array on chromosome 4. So far, homozygosity or compound heterozygosity for FSHD alleles has not been described, and it has been debated whether the absence of such subjects is because of the rarity or the lethality of the disorder. Methods: Two unrelated families in which the probands are compound heterozygous for two FSHD-sized alleles were studied. Clinical examination, pulsed-field gel electrophoresis (PFGE) studies of DNA with probes proximal and distal to D4Z4, and cytogenetic analysis of metaphase chromosomes by FISH were performed. Results: Complementary molecular and cytogenetic approaches confirmed the chromosome 4qA origin of all FSHD-sized repeat arrays that segregate in the families. Conclusions: Heterozygosity for FSHD-sized alleles is compatible with life in men and women. A possible dosage effect was observed in both probands in whom each 4qA allele contributed to the FSHD phenotype. Because at least one of the FSHD alleles in both families showed an unusual low penetrance, the authors propose that susceptibility for FSHD is partly determined by intrinsic properties of the disease allele other than the residual D4Z4 repeat size alone.

CBFB/MYH11 fusion in a patient with AML‐M4Eo and cytogenetically normal chromosomes 16

We present a unique case of acute myeloid leukemia M4Eo (AML‐M4Eo) with a CBFB/MYH11 fusion transcript and a trisomy 22, but in whom cytogenetic analyses did not disclose an inv(16). Fluorescence in situ hybridization (FISH) analysis with chromosome arm‐specific painting probes as well as with the c40 and c36 cosmids also revealed no evidence for an inv(16), whereas the application of locus‐specific probes confirmed the presence of a masked inv(16). The results of our comprehensive FISH investigations indicate that the events leading to this masked inv(16) were complex and concurred with deletions on both the long and short arms. The most likely explanation for the formation of the relevant CBFB/MYH11 fusion is an insertion of parts of the MYH11 into the CBFB gene, although it is also possible that it was formed by a double inversion.