Molecular characterisation of t(17;22)(q11.2;q11.2) is not consistent with NF1 gene duplication

A tandem duplication of the NF1 gene in 17q11.2 has recently been detected by high-resolution fluorescence in situ hybridisation (FISH) on stretched chromosomes and DNA fibres. These findings suggest not only that, in the 17q11.2 region, the NF1 gene is surrounded by NF1 low-copy repeats on each side of the gene, but also that the NF1 gene and its directly flanking regions are duplicated structures. However, if the NF1 gene is duplicated at 17q11.2, this should be observed by FISH analysis on metaphase chromosomes of relevant translocation carriers with the probes originally used to identify the duplication, since hybridisation signals of some of the probes would be expected on both derivative chromosomes, the der(17) and the der(22). We have only been able to obtain signals on the one or the other derivative of a female translocation carrier. Therefore, our results do not support the hypothesis of a duplication of the NF1 gene and its immediately flanking regions at 17q11.2 as had been previously postulated. Rather, our findings suggest that there is one NF1 gene in the 17q11.2 region.     

Tandem duplication of the NF1 gene detected by high-resolution FISH in the 17q11.2 region

The gene for neurofibromatosis type 1 (NF1), mapping to 17q11.2, has one of the highest observed mutation rates, partially because of its large size and gene conversion primed by NF1 pseudogenes. We have previously shown by means of high resolution fluorescence in situ hybridization (FISH) that a number of the loci flanking the NF1 gene are duplicated, in agreement with the reported presence of NF1 repetitive sequences (REPs). We report a direct tandem duplication of the NF1 gene identified in 17q11.2 by high-resolution FISH. FISH on stretched chromosomes with locus-specific probes revealed the duplication of the NF1 gene from the promoter to 3'UTR, but with at least the absence of exon 22. Fiber FISH with P1 artificial and bacterial artifical chromosomes, including the NF1 5'UTR and 3'UTR and flanking regions, visualized the direct tandem duplication with a similar, but not identical, genomic organization of the NF1 duplicon copies. Duplication was probably present in the human-chimpanzee-gorilla common ancestor, as demonstrated here by the finding of the duplicated NF1 gene at orthologous chromosome loci. The NF1 intrachromosomal duplication may contribute to the high whole-gene mutation rate by gene conversion, although the functional activity of the NF1 copy remains to be investigated. Detection of the NF1 duplicon by high-resolution FISH may pave the way to filling the gaps in the human genomic sequence of the pericentromeric 17q11.2 region.     

Precise localization of NF1 to 17q11.2 by balanced translocation.

A female patient is described with von Recklinghausen neurofibromatosis (NF1) in association with a balanced translocation between chromosome 17 and 22 [46,XX,t(17;22)(q11.2;q11.2)]. The breakpoint in chromosome 17 is cytogenetically identical to a previously reported case of NF1 associated with a 1;17 balanced translocation and suggests that the translocation events disrupt the NF1 gene. This precisely maps the NF1 gene to 17q11.2 and provides a physical reference point for strategies to clone the breakpoint and therefore the NF1 gene. A human-mouse somatic cell hybrid was constructed from patient lymphoblasts which retained the derivative chromosome 22 (22pter----22q11.2::17q11.2----17qter) but not the derivative 17q or normal 17. Southern blot analysis with genes and anonymous probes known to be in proximal 17q showed ErbA1, ErbB2, and granulocyte colony-stimulating factor (CSF3) to be present in the hybrid and therefore distal to the breakpoint, while pHHH202 (D17S33) and beta crystallin (CRYB1) were absent in the hybrid and therefore proximal to the breakpoint. The gene cluster including ErbA1 is known to be flanked by the constitutional 15;17 translocation breakpoint in hybrid SP3 and by the acute promyelocytic leukemia (APL) breakpoint, which provides the following gene and breakpoint order: cen-SP3-(D17S33,CRYB1)-NF1-(CSF3,ERBA1, ERBB2)-APL-tel. The flanking breakpoints of SP3 and API are therefore useful for rapidly localizing new markers to the neurofibromatosis critical region, while the breakpoints of the two translocation patients provide unique opportunities for reverse genetic strategies to clone the NF1 gene.     

A Turkish patient with large 17p11.2 deletion presenting with Smith Magenis syndrome

Smith-Magenis syndrome (SMS), which occurs as a result of an interstitial deletion within chromosome 17p11.2-p12, is a disorder that presents itself with minor dysmorphic features, brachydactyly, short stature, hypotonia, delayed speech, cognitive deficits and neurobehavioral problems including sleep disturbances and maladaptive repetitive and self-injurious behavior. We present a girl with full SMS phenotype. G-banding cytogenetic analysis showed normal 46,XX karyotype. Whole-genome array comparative genomic hybridization (CGH) was performed due to the severity of the phenotype and the unusual features present in the patient. An interstitial deletion in 17p11.2-p12, approximately 4.73 Mb in size was determined. Characteristic physical and behavioral phenotype strongly suggested SMS. This, to the best of our knowledge is the first patient with SMS reported in Turkey. We emphasize the need for whole genome analysis in multiple congenital abnormalities/mental retardation disorders with unusual and severe phenotypes.     

Parathyroid gland dysfunction in 22q11.2 deletion syndrome

BACKGROUND: 22q11 deletion syndrome (22q11DS) is characterized by conotruncal cardiac defects and hypoplasia of parathyroid glands and thymus, which result in variable hypoparathyroidism (HPT) and immune deficiency. METHODS: To study the course of HPT and the spectrum of other associated manifestations we evaluated all patients with 22q11DS, confirmed by fluorescence in situ hybridization, and HPT who were under follow-up at the Calcium-bone clinic, The Hospital for Sick Children, Toronto. Patients were clinically assessed and their hospital records were reviewed. RESULTS: Eighteen patients were included. At follow-up assessment at median age of 7.3 years HPT was judged complete in 11 (61%) and partial in 7 patients (39%). Patients with complete HPT presented with hypocalcemia later (median age at diagnosis 2.4 vs. 0.0 years) and more often with a hypocalcemic seizure than patients with partial HPT (73 vs. 29%). The spectrum of other associated manifestations did not differ between the groups. CONCLUSIONS: HPT in patients with 22q11DS is often partial. Many of the patients present with a hypocalcemic seizure which is predictive of complete HPT. Patients with complete and partial HPT do not differ in respect to their other associated features. Patients with features of 22q11DS should be actively screened for hypocalcemia to prevent development of symptomatic hypocalcemia.     

Genomic findings in patients with clinical suspicion of 22q11.2 deletion syndrome

Chromosome 22q11.2 deletion syndrome, one of the most common human genomic syndromes, has highly heterogeneous clinical presentation. Patients usually harbor a 1.5 to 3 Mb hemizygous deletion at chromosome 22q11.2, resulting in pathognomic TBX1, CRKL and/or MAPK1 haploinsufficiency. However, there are some individuals with clinical features resembling the syndrome who are eventually diagnosed with genomic disorders affecting other chromosomal regions. The objective of this study was to evaluate the additive value of high-resolution array-CGH testing in the cohort of 41 patients with clinical features of 22q11.2 deletion syndrome and negative results of standard cytogenetic diagnostic testing (karyotype and FISH for 22q11.2 locus). Array-CGH analysis revealed no aberrations at chromosomes 22 or 10 allegedly related to the syndrome. Five (12.2 %) patients were found to have other genomic imbalances, namely 17q21.31 microdeletion syndrome (MIM#610443), 1p36 deletion syndrome (MIM#607872), NF1 microduplication syndrome (MIM#613675), chromosome 6pter-p24 deletion syndrome (MIM#612582) and a novel interstitial deletion at 3q26.31 of 0.65 Mb encompassing a dosage-dependent gene NAALADL2. Our study demonstrates that the implementation of array-CGH into the panel of classic diagnostic procedures adds significantly to their efficacy. It allows for detection of constitutional genomic imbalances in 12 % of subjects with negative result of karyotype and FISH targeted for 22q11.2 region. Moreover, if used as first-tier genetic test, the method would provide immediate diagnosis in ～40 % phenotypic 22q11.2 deletion subjects.     

Geneticl and clinical characteristics of 22q11.2 deletion syndrome

In a group of 140 patients with typical phenotype, the 22q11.2 microdeletion was detected in 43 patients (32%) using FISH and MLPA methods. There were no deletions of other chromosomal loci causing to phenotypes similar to the 22q11.2 deletion syndrome (22q11.2DS). Sequencing of the TBX1 gene did not detect any mutations, except for some common neutral polymorphisms. For the first time in the Russian Federation, the diagnostic efficiency of 22q11.2DS appeared to be 32%, as a result of the application of a combination of genetic approaches for a large group of patients with suspected 22q11.2DS.     

Characterization of C1q found in a patient with hypocomplementemic vasculitis-urticaria syndrome

C1q, a subcomponent of the first complement component, of a 60-year-old female patient with hypocomplementemic vasculitis-urticaria syndrome (HVUS) was characterized. The C1q-precipitin activity (C1q-p) could not be detected by a routine method with 0.6% agarose in 10 mM Na-phosphate buffer containing 10 mM EDTA (pH 7.2). Hemolytic activity of her serum complement (CH50) and levels of C1 and C4 were significantly reduced at the exacerbation stage, but levels of other complement components were almost within the normal range throughout her clinical course. The specific activity of C1q at her exacerbation stage was significantly low, and its elution position on Sephacryl S-300 column was spread toward the low molecular weight in comparison with that of normal plasma. Molecular weights of the delayed fraction of C1q were estimated to be approximately 300,000 on the Sephacryl and 440,000 by the polyacrylamide gel electrophoresis (PAGE) containing sodium dodecyl sulfate (SDS) followed by immunoblotting, respectively. On reduction of her plasma, two bands with molecular weights equivalent to those of B and C chains of the normal C1q in an approximate molar ratio of 2:1 were immunostained. Plasma at her exacerbation stage showed only one precipitation line against anti-human C1q-antiserum which was completely fused with that formed between purified normal human C1q and the same antiserum. The probable structural change of the hypofunctional C1q in the case of this HVUS is discussed.     

13q deletion syndrome in an adult mentally retarded patient.

Clinical features of the 13q deletion syndrome are difficult to define and include retinoblastoma, mental and growth retardation, craniofacial abnormalities, brain, gastrointestinal, renal and heart malformations, anal atresia and limb and digit malformations. The critical region for development of major organ systems has been defined in 13q32 between the proximal marker 13S132 and distal marker D13S147. We report a severely mentally retarded male patient with a deletion of the distal part of chromosome 13 (13q32.3-->qter) without major organ malformations.     

Visual perception skills: a comparison between patients with Noonan syndrome and 22q11.2 deletion syndrome

Ventral and dorsal streams are visual pathways deputed to transmit information from the photoreceptors of the retina to the lateral geniculate nucleus and then to the primary visual cortex (V1). Several studies investigated whether one pathway is more vulnerable than the other during development, and whether these streams develop at different rates. The results are still discordant. The aim of the present study was to understand the functionality of the dorsal and the ventral streams in two populations affected by different genetic disorders, Noonan syndrome (NS) and 22q11.2 deletion syndrome (22q11.2DS), and explore the possible genotype–phenotype relationships. ‘Form coherence’ abilities for the ventral stream and ‘motion coherence’ abilities for the dorsal stream were evaluated in 19 participants with NS and 20 participants with 22q11.2DS. Collected data were compared with 55 age‐matched controls. Participants with NS and 22q11.2DS did not differ in the form coherence task, and their performance was significantly lower than that of controls. However, in the motion coherence task, the group with NS and controls did not differ, and both obtained significantly higher scores than the group with 22q11.2DS. Our findings indicate that deficits in the dorsal stream are related to the specific genotype, and that in our syndromic groups the ventral stream is more vulnerable than the dorsal stream.     

Extensively high load of internal tumors determined by whole body MRI scanning in a patient with neurofibromatosis type 1 and a non-LCR-mediated 2-Mb deletion in 17q11.2

Deletions in 17q11.2 affecting the NF1 gene and surrounding regions occur in 5% of patients with NF1. The two major types of NF1 deletions encompass 1.4-Mb and 1.2-Mb, respectively, and have breakpoints in the NF1 low-copy repeats or in the JJAZ gene and its pseudogene. Deletions larger than 1.4-Mb are rare, and only seven cases have been reported so far. Here, we describe a 26-year-old NF1 patient with an atypical NF1 deletion of 2-Mb. In contrast to the 1.4-Mb deletions, which preferentially occur by interchromosomal recombination during maternal meiosis, the deletion described here occurred intrachromosomally on the paternal chromosome. The centromeric deletion breakpoint lies in an L1-element located 1.3-Mb proximal to the NF1 gene. The telomeric deletion boundary is located in a single copy segment between an AT-rich segment and an AluSx-element in intron 15 of the JJAZ1 gene. Structural analysis implies that non-B DNA conformations at the breakpoints destabilized the duplex DNA and caused double-strand breaks. Although the breakpoints of this 2-Mb deletion are not recurrent, it is conspicuous that one breakpoint is located in the JJAZ1 gene. Paralogous recombination between the JJAZ1 gene and its pseudogene causes the recurrent 1.2 Mb deletions. The genomic architecture of the NF1 gene region, influenced by paralogous sequences such as the JJAZ1 gene and its pseudogene, seems also to stimulate the occurrence of non-recurrent deletions mediated by non-homologous end joining. Patient 442 described here suffers from a very high burden of subdermal neurofibromas. Magnetic resonance imaging of the whole body revealed numerous internal tumors, mainly plexiform neurofibromas and spinal tumors. This demonstrates the value of whole-body MRI scanning in determining the total tumor load, which is an important aspect in genotype/phenotype correlations with regard to large NF1 deletions.     

Proline and COMT status affect visual connectivity in children with 22q11.2 deletion syndrome

Background

Individuals with the 22q11.2 deletion syndrome (22q11DS) are at increased risk for schizophrenia and Autism Spectrum Disorders (ASDs). Given the prevalence of visual processing deficits in these three disorders, a causal relationship between genes in the deleted region of chromosome 22 and visual processing is likely. Therefore, 22q11DS may represent a unique model to understand the neurobiology of visual processing deficits related with ASD and psychosis.

Methodology

We measured Event-Related Potentials (ERPs) during a texture segregation task in 58 children with 22q11DS and 100 age-matched controls. The C1 component was used to index afferent activity of visual cortex area V1; the texture negativity wave provided a measure for the integrity of recurrent connections in the visual cortical system. COMT genotype and plasma proline levels were assessed in 22q11DS individuals.

Principal Findings

Children with 22q11DS showed enhanced feedforward activity starting from 70 ms after visual presentation. ERP activity related to visual feedback activity was reduced in the 22q11DS group, which was seen as less texture negativity around 150 ms post presentation. Within the 22q11DS group we further demonstrated an association between high plasma proline levels and aberrant feedback/feedforward ratios, which was moderated by the COMT ¹⁵⁸ genotype.

Conclusions

These findings confirm the presence of early visual processing deficits in 22q11DS. We discuss these in terms of dysfunctional synaptic plasticity in early visual processing areas, possibly associated with deviant dopaminergic and glutamatergic transmission. As such, our findings may serve as a promising biomarker related to the development of schizophrenia among 22q11DS individuals.     

Molecular characterization of a deletion in the HPRT1 gene in a patient with Lesch-Nyhan syndrome

Lesch-Nyhan syndrome is caused by a deficiency of hypoxanthine phosphoribosyltransferase (HPRT) encoded by HPRT1. About 20% of patients have a deletion of HPRT1 and large deletions of HPRT1 are not always fully characterized at the molecular level. Here, we report on a case of Lesch-Nyhan syndrome with a 33-kb deletion involving exon 1 of HPRT1. This novel mutation is caused by a nonhomologous recombination between different classes of interspersed repetitive DNA.     

Molecular characterization and gene content of breakpoint boundaries in patients with neurofibromatosis type 1 with 17q11.2 microdeletions

Homologous recombination between poorly characterized regions flanking the NF1 locus causes the constitutional loss of approximately 1.5 Mb from 17q11.2 covering > or =11 genes in 5%-20% of patients with neurofibromatosis type 1 (NF1). To elucidate the extent of microheterogeneity at the deletion boundaries, we used single-copy DNA fragments from the extreme ends of the deleted segment to perform FISH on metaphase chromosomes from eight patients with NF1 who had large deletions. In six patients, these probes were deleted, suggesting that breakage and fusions occurred within the adjacent highly homologous sequences. Reexamination of the deleted region revealed two novel functional genes FLJ12735 (AK022797) and KIAA0653-related (WI-12393 and AJ314647), the latter of which is located closest to the distal boundary and is partially duplicated. We defined the complete reading frames for these genes and two expressed-sequence tag (EST) clusters that were reported elsewhere and are associated with the markers SHGC-2390 and WI-9521. Hybrid cell lines carrying only the deleted chromosome 17 were generated from two patients and used to identify the fusion sequences by junction-specific PCRs. The proximal breakpoints were found between positions 125279 and 125479 in one patient and within 4 kb of position 143000 on BAC R-271K11 (AC005562) in three patients, and the distal breakpoints were found at the precise homologous position on R-640N20 (AC023278). The interstitial 17q11.2 microdeletion arises from unequal crossover between two highly homologous WI-12393-derived 60-kb duplicons separated by approximately 1.5 Mb. Since patients with the NF1 large-deletion syndrome have a significantly increased risk of neurofibroma development and mental retardation, hemizygosity for genes from the deleted region around the neurofibromin locus (CYTOR4, FLJ12735, FLJ22729, HSA272195 (centaurin-alpha2), NF1, OMGP, EVI2A, EVI2B, WI-9521, HSA272196, HCA66, KIAA0160, and WI-12393) may contribute to the severe phenotype of these patients.     

Molecular analysis of the Retinoic Acid Induced 1 gene (RAI1) in patients with suspected Smith-Magenis syndrome without the 17p11.2 deletion

Smith-Magenis syndrome (SMS) is a complex neurobehavioral disorder characterized by multiple congenital anomalies. The syndrome is primarily ascribed to a ～3.7 Mb de novo deletion on chromosome 17p11.2. Haploinsufficiency of multiple genes likely underlies the complex clinical phenotype. RAI1 (Retinoic Acid Induced 1) is recognized as a major gene involved in the SMS phenotype. Extensive genetic and clinical analyses of 36 patients with SMS-like features, but without the 17p11.2 microdeletion, yielded 10 patients with RAI1 variants, including 4 with de novo deleterious mutations, and 6 with novel missense variants, 5 of which were familial. Haplotype analysis showed two major RAI1 haplotypes in our primarily Caucasian cohort; the novel RAI1 variants did not occur in a preferred haplotype. RNA analysis revealed that RAI1 mRNA expression was significantly decreased in cells of patients with the common 17p11.2 deletion, as well as in those with de novo RAI1 variants. Expression levels varied in patients with familial RAI1 variants and in non-17p11.2 deleted patients without identified RAI1 defects. No correlation between SNP haplotype and RAI1 expression was found. Two clinical features, ocular abnormalities and polyembolokoilomania (object insertion), were significantly correlated with decreased RAI1 expression. While not significantly correlated, the presence of hearing loss, seizures, hoarse voice, childhood onset of obesity and specific behavioral aspects and the absence of immunologic abnormalities and cardiovascular or renal structural anomalies, appeared to be specific for the de novo RAI1 subgroup. Recognition of the combination of these features will assist in referral for RAI1 analysis of patients with SMS-like features without detectable microdeletion of 17p11.2. Moreover, RAI1 expression emerged as a genetic target for development of therapeutic interventions for SMS.     

Choroideremia and deafness with stapes fixation: a contiguous gene deletion syndrome in Xq21.

The study of contiguous gene deletion syndromes by using reverse genetic techniques provides a powerful tool for precisely defining the map location of the genes involved. We have made use of individuals with overlapping deletions producing choroideremia as part of a complex phenotype, to define the boundaries on the X chromosome for this gene, as well as for X-linked mixed deafness with perilymphatic gusher (DFN3). Two patients with deletions and choroideremia are affected by an X-linked mixed conductive/sensorineural deafness; one patient, XL-62, was confirmed at surgery to have DFN3, while the other patient, XL-45, is suspected clinically to have the same disorder. A third choroideremia deletion patient, MBU, has normal hearing. Patient XL-62 has a cytogenetically detectable deletion that was measured to be 7.7% of the X chromosome by dual laser flow cytometry; the other patient, XL-45, has a cytogenetically undetectable deletion that measures only 3.3% of the X chromosome. We have produced a physical map of the X-chromosome region containing choroideremia and DFN3 by using routine Southern blotting, chromosome walking and jumping techniques, and long-range restriction mapping to generate and link anonymous DNA sequences in this region. DXS232 and DXS233 are located within 450 kb of each other on the same SfiI and MluI fragments and share partial SalI fragments of 750 and greater than 1,000 kb but are separated by at least one SalI site. In addition, DXS232, which lies outside the MBU deletion, detects the proximal breakpoint of this deletion. We have isolated two new anonymous DNA sequences by chromosome jumping from DXS233; one of these detects a new SfiI fragment distal to DXS233 in the direction of the choroideremia gene, while the other jump clone is proximal to DXS233 and detects a new polymorphism. These data refine the map around the loci for choroideremia and for mixed deafness with stapes fixation and will provide points from which to isolate candidate gene sequences for these disorders.