Stefania Gimelli

Cryptic deletions are a common finding in “balanced” reciprocal and complex chromosome rearrangements: a study of 59 patients

Using array comparative genome hybridisation (CGH) 41 de novo reciprocal translocations and 18 de novo complex chromosome rearrangements (CCRs) were screened. All cases had been interpreted as “balanced” by conventional cytogenetics. In all, 27 cases of reciprocal translocations were detected in patients with an abnormal phenotype, and after array CGH analysis, 11 were found to be unbalanced. Thus 40% (11 of 27) of patients with a “chromosomal phenotype” and an apparently balanced translocation were in fact unbalanced, and 18% (5 of 27) of the reciprocal translocations were instead complex rearrangements with >3 breakpoints. Fourteen fetuses with de novo, apparently balanced translocations, all but two with normal ultrasound findings, were also analysed and all were found to be normal using array CGH. Thirteen CCRs were detected in patients with abnormal phenotypes, two in women who had experienced repeated spontaneous abortions and three in fetuses. Sixteen patients were found to have unbalanced mutations, with up to 4 deletions. These results suggest that genome-wide array CGH may be advisable in all carriers of “balanced” CCRs. The parental origin of the deletions was investigated in 5 reciprocal translocations and 11 CCRs; all were found to be paternal. Using customised platforms in seven cases of CCRs, the deletion breakpoints were narrowed down to regions of a few hundred base pairs in length. No susceptibility motifs were associated with the imbalances. These results show that the phenotypic abnormalities of apparently balanced de novo CCRs are mainly due to cryptic deletions and that spermatogenesis is more prone to generate multiple chaotic chromosome imbalances and reciprocal translocations than oogenesis.

13q Deletion and central nervous system anomalies: further insights from karyotype–phenotype analyses of 14 patients

Background: Chromosome 13q deletion is associated with varying phenotypes, which seem to depend on the location of the deleted segment. Although various attempts have been made to link the 13q deletion intervals to distinct phenotypes, there is still no acknowledged consensus correlation between the monosomy of distinct 13q regions and specific clinical features. Methods: 14 Italian patients carrying partial de novo 13q deletions were studied. Molecular–cytogenetic characterisation was carried out by means of array-comparative genomic hybridisation (array-CGH) or fluorescent in situ hybridisation (FISH). Results: Our 14 patients showed mental retardation ranging from profound–severe to moderate–mild: eight had central nervous system (CNS) anomalies, including neural tube defects (NTDs), six had eye abnormalities, nine had facial dysmorphisms and 10 had hand or feet anomalies. The size of the deleted regions varied from 4.2 to 75.7 Mb. Conclusion: This study is the first systematic molecular characterisation of de novo 13q deletions, and offers a karyotype–phenotype correlation based on detailed clinical studies and molecular determinations of the deleted regions. Analyses confirm that patients lacking the 13q32 band are the most seriously affected, and critical intervals have been preliminarily assigned for CNS malformations. Dose-sensitive genes proximal to q33.2 may be involved in NTDs. The minimal deletion interval associated with the Dandy–Walker malformation (DWM) was narrowed to the 13q32.2–33.2 region, in which the ZIC2 and ZIC5 genes proposed as underlying various CNS malformations are mapped.

Duplications in addition to terminal deletions are present in a proportion of ring chromosomes. Clues to the mechanisms of formation

Background and methods: Ring chromosomes are often associated with abnormal phenotypes because of loss of genomic material at one or both ends. In some cases no deletion has been detected and the abnormal phenotype has been attributed to mitotic ring instability. We investigated 33 different ring chromosomes in patients with phenotypic abnormalities by array based comparative genomic hybridisation (CGH) and fluorescence in situ hybridisation (FISH). Results: In seven cases we found not only the expected terminal deletion but also a contiguous duplication. FISH analysis in some of these cases demonstrated that the duplication was inverted. Thus these ring chromosomes derived through a classical inv dup del rearrangement consisting of a deletion and an inverted duplication. Discussion: Inv dup del rearrangements have been reported for several chromosomes, but hardly ever in ring chromosomes. Our findings highlight a new mechanism for the formation of some ring chromosomes and show that inv dup del rearrangements may be stabilised not only through telomere healing and telomere capture but also through circularisation. This type of mechanism must be kept in mind when evaluating possible genotype–phenotype correlations in ring chromosomes since in these cases: (1) the deletion may be larger or smaller than first estimated based on the size of the ring, with a different impact on the phenotype; and (2) the associated duplication will in general cause further phenotypic anomalies and might confuse the genotype–phenotype correlation. Moreover, these findings explain some phenotypic peculiarities which previously were attributed to a wide phenotypic variation or hidden mosaicism related to the instability of the ring.

Detailed phenotype-genotype study in five patients with chromosome 6q16 deletion : narrowing the critical region for Prader-Willi-like phenotype

Most patients with an interstitial deletion of 6q16 have Prader–Willi-like phenotype, featuring obesity, hypotonia, short hands and feet, and developmental delay. In all reported studies, the chromosome rearrangement was detected by karyotype analysis, which provides an overview of the entire genome but has limited resolution. Here we describe a detailed clinical presentation of five patients, two of whom were previously reported, with overlapping interstitial 6q16 deletions and Prader–Willi-like phenotype. Our patients share the following main features with previously reported cases: global developmental delay, hypotonia, obesity, hyperphagia, and eye/vision anomalies. All rearrangement breakpoints have been accurately defined through array-CGH at about 100u2009Kb resolution. We were able to narrow the shortest region of deletion overlap for the presumed gene(s) involved in the Prader–Willi-like syndrome to 4.1u2009Mb located at 6q16.1q16.2. Our results support the evidence that haploinsufficiency of the SIM1 gene is responsible for obesity in these patients. A possible involvement of the GRIK2 gene in autistic-like behaviour, of POPDC3 in heart development, and of MCHR2 in the control of feeding behaviour and energy metabolism is also hypothesized.

Mutations in SOX17 are Associated with Congenital Anomalies of the Kidney and the Urinary Tract

Congenital anomalies of the kidney and the urinary tract (CAKUT) represent a major source of morbidity and mortality in children. Several factors (PAX, SOX,WNT, RET, GDFN, and others) play critical roles during the differentiation process that leads to the formation of nephron epithelia. We have identified mutations in SOX17, an HMG‐box transcription factor and Wnt signaling antagonist, in eight patients with CAKUT (seven vesico‐ureteric reflux, one pelvic obstruction). One mutation, c.775T>A (p.Y259N), recurred in six patients. Four cases derived from two small families; renal scars with urinary infection represented the main symptom at presentation in all but two patients. Transfection studies indicated a 5–10‐fold increase in the levels of the mutant protein relative to wild‐type SOX17 in transfected kidney cells. Moreover we observed a corresponding increase in the ability of SOX17 p.Y259N to inhibit Wnt/β‐catenin transcriptional activity, which is known to regulate multiple stages of kidney and urinary tract development. In conclusion, SOX17 p.Y259N mutation is recurrent in patients with CAKUT. Our data shows that this mutation correlates with an inappropriate accumulation of SOX17‐p.Y259N protein and inhibition of the β‐catenin/Wnt signaling pathway. These data indicate a role of SOX17 in human kidney and urinary tract development and implicate the SOX17–p.Y259N mutation as a causative factor in CAKUT.Hum Mutat 31:1352–1359, 2010.

Complex pathogenesis of Hirschsprung's disease in a patient with hydrocephalus, vesico-ureteral reflux and a balanced translocation t(3;17)(p12;q11)

Hirschsprungs disease (HSCR), a congenital complex disorder of intestinal innervation, is often associated with other inherited syndromes. Identifying genes involved in syndromic HSCR cases will not only help understanding the specific underlying diseases, but it will also give an insight into the development of the most frequent isolated HSCR. The association between hydrocephalus and HSCR is not surprising as a large number of patients have been reported to show the same clinical association, most of them showing mutations in the L1CAM gene, encoding a neural adhesion molecule often involved in isolated X-linked hydrocephalus. L1 defects are believed to be necessary but not sufficient for the occurrence of the intestinal phenotype in syndromic cases. In this paper, we have carried out the molecular characterization of a patient affected with Hirschsprungs disease and X-linked hydrocephalus, with a de novo reciprocal balanced translocation t(3;17)(p12;q21). In particular, we have taken advantage of this chromosomal defect to gain access to the predisposing background possibly leading to Hirschsprungs disease. Detailed analysis of the RET and L1CAM genes, and molecular characterization of MYO18A and TIAF1, the genes involved in the balanced translocation, allowed us to identify, besides the L1 mutation c.2265delC, different additional factors related to RET-dependent and -independent pathways which may have contributed to the genesis of enteric phenotype in the present patient.

Identification and molecular modelling of a novel familial mutation in the SRY gene implicated in the pure gonadal dysgenesis

SRY gene is responsible for initiating male sexual differentiation. The protein encoded by SRY contains a homeobox (HMG) domain, which is a DNA-binding domain. Mutations of the SRY gene are reported to be associated with XY pure gonadal dysgenesis. The majority of these are de novo mutations affecting only one individual in a family. Only a small subset of mutations is shared between the father and one or more of his children. Most of these familial mutations are localized within the HMG box and only two are at the N-terminal domain of the SRY protein. Herein, we describe a young girl with pure gonadal dysgenesis and her father carrying a novel familial mutation in the SRY gene at codon number 3. This mutation is resulting in a serine (S) to leucine (L) substitution. The secondary structure of the SRY protein was carried out by protein modelling studies. This analysis suggests, with high possibility, that the N-terminal domain of the SRY protein, where we found the mutation, could form an α-helix from amino acid in position 2 to amino acid in position 13. The secondary structure prediction and the chemical properties of serine to leucine substitution stands for a potential disruption of this N-terminal α-helix in the SRY protein. This mutation could have some role in impeding the normal function of the SRY protein.

A 7 Mb Duplication at 22q13 in a Girl With Bipolar Disorder and Hippocampal Malformation

We identified a duplication of 22q13.1‐q13.2 in a 10‐year‐old girl and demonstrated that this duplication was the recombinant product of a maternal intrachromosomal insertion. Phenotypic characteristics included prominent forehead, small low‐set ears, hypertelorism, epicanthal folds, small palpebral fissures, short philtrum, and syndactyly. MRI of the brain revealed high signal abnormalities in the periventricular white matter, a hypoplastic corpus callosum, under‐rotated hippocampus on the left and atrophic hippocampus on the right. Since age 5, the childs behavior has shown cyclic maniacal episodes with severely disorganized mood and behavior. Psychiatric and cognitive assessment led to a diagnosis of bipolar disorder not otherwise specified, manic episodes, attention deficit hyperactivity disorder and moderate mental retardation. Array‐CGH revealed an interstitial duplication of 6.9 Mb at chromosome 22q: dup(22)(q13.1q13.2). FISH using BAC clones confirmed the array‐CGH results and demonstrated that the duplication was inverted. G‐banding analysis in the propositas mother revealed a banding pattern suggestive of an intrachromosomal insertion, as demonstrated by dual‐color FISH with BACs that were duplicated in the proposita and multicolor‐banding (MCB) based on microdissection derived region‐specific libraries for chromosome 22. Our findings suggest that in both seemingly de novo deletions and duplications, the parent transmitting the imbalance should be investigated for possible balanced rearrangements. This report reinforces previous evidence that chromosome imbalances, and thus gene dosage effects, may be at the basis of some psychiatric disorders. Stringent correlations between submicroscopic imbalances, specific behavioral phenotypes and brain imaging will possibly help in dissecting complex behavioral traits.

Presence of 1q gain and absence of 7p gain are new predictors of local or metastatic relapse in localized resectable neuroblastoma

We have addressed the search of novel genetic prognostic markers in a selected cohort of patients with stroma-poor localized resectable neuroblastoma (NB) who underwent relapse or progression (group 1) or complete remission (group 2) over a minimum follow-up of 32 months from diagnosis. Twenty-three Italian patients with localized resectable NB (stages 1 and 2) diagnosed from 1994 through 2005 were studied. All patients received surgical treatment. Chemotherapy was administered only to the three stage 2 patients who had MYCN-amplified tumors. High-resolution array-comparative genomic hybridization (CGH) DNA copy-number analysis technology was used to identify novel prognostic markers. Chromosome 1p36.22p36.32 loss and 1q22qter gain, detected almost exclusively in group 1 patients, were significantly associated with poor event-free survival (EFS) (p = 0.0024 and p = 0.024, respectively). In contrast, patients with 7p11.2p22 gain, who belonged predominantly to group 2, had a significantly better EFS (p = 0.015). The frequency of 17q gain or 3p and 11q losses did not differ significantly in group 1 versus group 2 NBs. The sensitive technique allowed us to define the smallest region of 1p deletion. In conclusion, 1q22qter gain and 7p11.2p22 gain might represent new prognostic markers in localized resectable NB, but the small study size and the retrospective nature of the findings warrant further validation of the results in larger studies.

A 46,X,inv(Y) young woman with gonadal dysgenesis and gonadoblastoma: Cytogenetics, molecular, and methylation studies

Cytogenetic analysis of a young woman with gonadal dysgenesis and bilateral gonadoblastoma shared a male karyotype with a rearranged Y chromosome, interpreted as a pericentric inversion. The breakpoints, defined by fluorescent in situ hybridization (FISH), were located on the very distal short arm on band Yp11.31 and in the middle of the Yq12 long arm heterochromatic region. FISH analysis documented that the short arm breakpoint was 93 Kb distal to SRY and disrupted the CD99 gene, which was transposed to the distal portion of Yq12. The propositas phenotype was similar to that of XY individuals with gonadal dysgenesis but without signs of Ullrich—Turner syndrome. There were no mutations in the SRY gene. Cytogenetic analysis in the propositas father showed mosaicism of a normal Y chromosome and several different rearrangements, such as deletion of a heterochromatin portion at band Yq12.2, a fragile site at the same band, structural rearrangements between the Y‐chromosome and other autosomes, Y‐chromosome aneuploidies, and “Premature Centromere Division” (PCD) anomaly. The propositas inverted Y chromosome appears to have originated from paternal Y chromosome instability. The patients female phenotype could be due to SRY CpG methylation‐mediated positional effects (PEV).