Maria Kirchhoff

Enrichment of autologous fat grafts with ex-vivo expanded adipose tissue-derived stem cells for graft survival: a randomised placebo-controlled trial

BACKGROUND Autologous fat grafting is increasingly used in reconstructive surgery. However, resorption rates ranging from 25% to 80% have been reported. Therefore, methods to increase graft viability are needed. Here, we report the results of a triple-blind, placebo-controlled trial to compare the survival of fat grafts enriched with autologous adipose-derived stem cells (ASCs) versus non-enriched fat grafts. METHODS Healthy participants underwent two liposuctions taken 14 days apart: one for ASC isolation and ex-vivo expansion, and another for the preparation of fat grafts. Two purified fat grafts (30 mL each) taken from the second liposuction were prepared for each participant. One graft was enriched with ASCs (20 × 10(6) cells per mL fat), and another graft without ASC enrichment served as a control. The fat grafts were injected subcutaneously as a bolus to the posterior part of the right and left upper arm according to the randomisation sequence. The volumes of injected fat grafts were measured by MRI immediately after injection and after 121 days before surgical removal. The primary goal was to compare the residual graft volumes of ASC-enriched grafts with those of control grafts. This study is registered at www.clinicaltrialsregister.eu, number 2010-023006-12. FINDINGS 13 participants were enrolled, three of whom were excluded. Compared with the control grafts, the ASC-enriched fat grafts had significantly higher residual volumes: 23·00 (95% CI 20·57-25·43) cm(3) versus 4·66 (3·16-6·16) cm(3) for the controls, corresponding to 80·9% (76·6-85·2) versus 16·3% (11·1-21·4) of the initial volumes, respectively (p<0·0001). The difference between the groups was 18·34 (95% CI 15·70-20·98) cm(3), equivalent to 64·6% (57·1-72·1; p<0·0001). No serious adverse events were noted. INTERPRETATION The procedure of ASC-enriched fat grafting had excellent feasibility and safety. These promising results add significantly to the prospect of stem cell use in clinical settings, and indicate that ASC graft enrichment could render lipofilling a reliable alternative to major tissue augmentation, such as breast surgery, with allogeneic material or major flap surgery. FUNDING Danish Cancer Society, Centre of Head and Orthopaedics Rigshospitalet, and Moalem Weitemeyer Bendtsen.

DETECTION OF CHROMOSOMAL GAINS AND LOSSES IN COMPARATIVE GENOMIC HYBRIDIZATION ANALYSIS BASED ON STANDARD REFERENCE INTERVALS

Criteria for detection of chromosome aberrations by Comparative Genomic Hybridization (CGH) are not standardized and improvement of this part of the analysis is of paramount importance to the applicability of the technique. The aim of this work was to suggest CGH detection criteria that increase the specificity and sensitivity and at the same time include chromosome regions previously excluded from CGH analysis. We analyzed 33 hybridizations with normal DNA and modified our CGH software in order to use a selection of these normal analyses as a model for interpretation of analyses of unknown samples. This approach was successfully tested on 14 samples with known aberrations.

Deletions below 10 megabasepairs are detected in comparative genomic hybridization by standard reference intervals

Comparative genomic hybridization (CGH) is a widely used technique for studying chromosomal imbalances. The sensitivity of the technique is, however, relatively low. Deletions down to a size of 10–12 Mbp have been detected by the use of fixed diagnostic thresholds. In this study, we applied standard reference intervals as detection criteria on a number of deletions in the range of 3 Mbp to 14–18 Mbp. All deletions were detected. Thus, detection by standard reference intervals confers a considerably higher sensitivity to CGH analysis compared to fixed diagnostic thresholds. Genes Chromosomes Cancer 25:410–413, 1999.

Comparative genomic hybridization reveals a recurrent pattern of chromosomal aberrations in severe dysplasia/carcinoma in situ of the cervix and in advanced‐stage cervical carcinoma

We analyzed 17 cases of dysplasia/carcinoma in situ (CIS) of the cervix and 29 advanced‐stage cervical squamous cell carcinomas by comparative genomic hybridization (CGH). A comparable recurrent pattern of aberrations was detected in both preinvasive and invasive cases, although the total number of aberrations was much higher in the latter category. The most consistent chromosomal gain was mapped to chromosome arm 3q in 35% of preinvasive cases and in 72% of invasive cases. Chromosome aberrations were detected in 13/17 preinvasive cases with a total of 61 involved chromosome arms. In the invasive cases, frequent gains also occurred on 1q (45%), 8q (41%), 15q (41%), 5p (34%), and Xq (34%), and frequent losses were mapped to chromosome arms 3p (52%), 11q (48%), 13q (38%), 6q (38%), and 4p (34%). A recurrent pattern of aberrations has not previously been described in preinvasive lesions of the cervix. Our finding is surprising considering that only few preinvasive lesions are expected to progress to invasive cancer. Genes Chromosomes Cancer 24:144–150, 1999.

High resolution comparative genomic hybridisation in clinical cytogenetics.

High resolution comparative genomic hybridisation (HR-CGH) is a diagnostic tool in our clinical cytogenetics laboratory. The present survey reports the results of 253 clinical cases in which 47 abnormalities were detected. Among 144 dysmorphic and mentally retarded subjects with a normal conventional karyotype, 15 (10%) had small deletions or duplications, of which 11 were interstitial. In addition, a case of mosaic trisomy 9 was detected. Among 25 dysmorphic and mentally retarded subjects carrying apparently balanced de novo translocations, four had deletions at translocation breakpoints and two had deletions elsewhere in the genome. Seventeen of 19 complex rearrangements were clarified by HR-CGH. A small supernumerary marker chromosome occurring with low frequency and the breakpoint of a mosaic r(18) case could not be clarified. Three of 19 other abnormalities could not be confirmed by HR-CGH. One was a Williams syndrome deletion and two were DiGeorge syndrome deletions, which were apparently below the resolution of HR-CGH. However, we were able to confirm Angelman and Prader-Willi syndrome deletions, which are about 3-5 Mb. We conclude that HR-CGH should be used for the evaluation of (1) dysmorphic and mentally retarded subjects where normal karyotyping has failed to show abnormalities, (2) dysmorphic and mentally retarded subjects carrying apparently balanced de novo translocations, (3) apparently balanced de novo translocations detected prenatally, and (4) for clarification of complex structural rearrangements.

Duplication or amplification of chromosome band 11q23, including the unrearranged MLL gene, is a recurrent abnormality in therapy-related MDS and AML, and is closely related to mutation of the TP53 gene and to previous therapy with alkylating agents

Gene amplification is a rare phenomenon in acute leukemia, but recently amplification of specific chromosome bands containing genes rearranged in leukemia‐specific balanced chromosome translocations has been reported in a few cases. We detected duplication or amplification of chromosome band 11q23 with 3–7 copies of the MLL gene by fluorescence in situ hybridization in 12 out of 70 unselected patients with therapy‐related myelodysplasia or acute myeloid leukemia (17%). In all but one case, the supernumerary copies of MLL were located to previously unidentified marker chromosomes or unbalanced translocations. In 4 of the 12 patients, 2–6 copies were located together on the same chromosome arm representing amplification, 7 patients had single, extra duplicated copies of MLL, whereas both amplification and duplication were observed in the same cell in 1 patient. Comparative genomic hybridization demonstrated gain of varying, often large parts of 11q in five patients. The MLL gene was shown to be unrearranged in all 12 patients. Seven out of eight patients with duplication or amplification of MLL had mutations of TP53. Patients with supernumerary copies of MLL were in general older (P = 0.007) and had a shorter survival (P < 0.001) compared to other patients. Duplication or amplification of MLL was significantly associated with a complex karyotype (P = 0.002), with deletion or loss of 5q (P = 0.001), and with prior therapy with alkylating agents. These results support the existence of a specific genetic pathway in t‐MDS and t‐AML with many previously unidentified chromosome aberrations demonstrated to represent extra copies of parts of 11q, including the unrearranged MLL gene.

Corpus callosum abnormalities, intellectual disability, speech impairment, and autism in patients with haploinsufficiency of ARID1B

Halgren C, Kjaergaard S, Bak M, Hansen C, El‐Schich Z, Anderson CM, Henriksen KF, Hjalgrim H, Kirchhoff M, Bijlsma EK, Nielsen M, den Hollander NS, Ruivenkamp CAL, Isidor B, Le Caignec C, Zannolli R, Mucciolo M, Renieri A, Mari F, Anderlid B‐M, Andrieux J, Dieux A, Tommerup N, Bache I. Corpus callosum abnormalities, intellectual disability, speech impairment, and autism in patients with haploinsufficiency of ARID1B.

Phenotype and 244k array-CGH characterization of chromosome 13q deletions: An update of the phenotypic map of 13q21.1-qter†

Partial deletions of the long arm of chromosome 13 lead to variable phenotypes dependant on the size and position of the deleted region. In order to update the phenotypic map of chromosome 13q21.1‐qter deletions, we applied 244k Agilent oligonucleotide‐based array‐CGH to determine the exact breakpoints in 14 patients with partial deletions of this region. Subsequently, we linked the genotype to the patients phenotype. Using this approach, we were able to refine the smallest deletion region linked to short stature (13q31.3: 89.5–91.6 Mb), microcephaly (13q33.3–q34), cortical development malformations (13q33.1‐qter), Dandy–Walker malformation (DWM) (13q32.2–q33.1), corpus callosum agenesis (CCA) (13q32.3–q33.1), meningocele/encephalocele (13q31.3‐qter), DWM, CCA, and neural tube defects (NTDs) taken together (13q32.3–q33.1), ano‐/microphthalmia (13q31.3–13qter), cleft lip/palate (13q31.3–13q33.1), lung hypoplasia (13q31.3–13q33.1), and thumb a‐/hypoplasia (13q31.3–q33.1 and 13q33.3–q34). Based on observations of this study and previous reports we suggest a new entity, “distal limb anomalies association,” linked to 13q31.3q33.1 segment. Most of the individuals with deletion of any part of 13q21qter showed surprisingly similar facial dysmorphic features, and thus, a “13q deletion facial appearance” was suggested. Prominent nasal columella was mapped between 13q31.3 and 13q33.3, and micrognathia between 13q21.33 and 13q31.1. The degree of mental delay did not display a particular phenotype–genotype correlation on chromosome 13. In contrast to previous reports of carriers of 13q32 band deletions as the most seriously affected patients, we present two such individuals with long‐term survival, 28 and 2.5 years.

Impact of low copy repeats on the generation of balanced and unbalanced chromosomal aberrations in mental retardation

Low copy repeats (LCRs) are stretches of duplicated DNA that are more than 1 kb in size and share a sequence similarity that exceeds 90%. Non-allelic homologous recombination (NAHR) between highly similar LCRs has been implicated in numerous genomic disorders. This study aimed at defining the impact of LCRs on the generation of balanced and unbalanced chromosomal rearrangements in mentally retarded patients. A cohort of 22 patients, preselected for the presence of submicroscopic imbalances, was analysed using submegabase resolution tiling path array CGH and the results were compared with a set of 41 patients with balanced translocations and breakpoints that were mapped to the BAC level by FISH. Our data indicate an accumulation of LCRs at breakpoints of both balanced and unbalanced rearrangements. LCRs with high sequence similarity in both breakpoint regions, suggesting NAHR as the most likely cause of rearrangement, were observed in 6/22 patients with chromosomal imbalances, but not in any of the balanced translocation cases studied. In case of chromosomal imbalances, the likelihood of NAHR seems to be inversely related to the size of the aberration. Our data also suggest the presence of additional mechanisms coinciding with or dependent on the presence of LCRs that may induce an increased instability at these chromosomal sites.

Disruption of the CNTNAP2 gene in a t(7;15) translocation family without symptoms of Gilles de la Tourette syndrome

Caspr2 is a member of neurexin superfamily, members of which are transmembrane proteins that mediate cellular interactions in the nervous system. Recently, truncation of the CNTNAP2 gene coding for the Caspr2 protein has been suggested to be associated with the Gilles de la Tourette syndrome, a neurological disorder characterized by motor and vocal tics, and behavioral anomalies. In this study, we describe a familial balanced reciprocal translocation t(7;15)(q35;q26.1) in phenotypically normal individuals. The 7q35 breakpoint disrupts the CNTNAP2 gene, indicating that truncation of this gene does not necessarily lead to the symptoms of the complex Gilles de la Tourette syndrome.