Genome-wide equine preimplantation genetic testing enabled by simultaneous haplotyping and copy number detection.

In different species, embryonic aneuploidies and genome-wide errors are a major cause of developmental failure. The increasing number of equine embryos being produced worldwide provides the opportunity to characterize and rank or select embryos based on their genetic profile prior to transfer. Here, we explored the possibility of generic, genome-wide preimplantation genetic testing concurrently for aneuploidies (PGT-A) and monogenic (PGT-M) traits and diseases in the horse, meanwhile assessing the incidence and spectrum of chromosomal and genome-wide errors in in vitro-produced equine embryos. To this end, over 70,000 single nucleotide polymorphism (SNP) positions were genotyped in 14 trophectoderm biopsies and corresponding biopsied blastocysts, and in 26 individual blastomeres from six arrested cleavage-stage embryos. Subsequently, concurrent genome-wide copy number detection and haplotyping by haplarithmisis was performed and the presence of aneuploidies and genome-wide errors and the inherited parental haplotypes for four common disease-associated genes with high carrier frequency in different horse breeds (GBE1, PLOD1, B3GALNT2, MUTYH), and for one color coat-associated gene (STX17) were compared in biopsy-blastocyst combinations. The euploid (n = 12) or fully aneuploid (n = 2) state and the inherited parental haplotypes for 42/45 loci of interest of the biopsied blastocysts were predicted by the biopsy samples in all successfully analyzed biopsy-blastocyst combinations (n = 9). Two biopsies showed a loss of maternal chromosome 28 and 31, respectively, which were confirmed in the corresponding blastocysts. In one of those biopsies, additional complex aneuploidies not present in the blastocyst were found. Five out of six arrested embryos contained chromosomal and/or genome-wide errors in most of their blastomeres, demonstrating their contribution to equine embryonic arrest in vitro. The application of the described PGT strategy would allow to select equine embryos devoid of genetic errors and pathogenetic variants, and with the variants of interest, which will improve foaling rate and horse quality. We believe this approach will be a gamechanger in horse breeding.

Single closed-tube quantitative real-time PCR assay with dual-labelled probes for improved sex determination of equine embryos.

In addition to fulfilling many breeders' curiosity, equine embryonic sex determination can have a profound commercial impact. However, the application of currently described assays for equine embryonic sexing has rendered variable diagnosis and validation rates, with sensitivity being the main problem. In addition, while pregnancy results of in vivo-flushed equine embryos following a needle aspiration biopsy equal those of non-biopsied embryos, the effect on in vitro-produced embryos is unknown. Here, we aimed to develop a highly sensitive and specific assay for equine sex determination that can be directly performed on few embryonic cells, and to test the effect of a needle aspiration biopsy on the viability of the in vitro-produced embryo. To this end, a multiplex quantitative real-time PCR (qPCR) assay with dual-labelled probes was designed to allow the simultaneous generation of both male-specific and control fragments in a single closed-tube reaction, avoiding potential sample loss or contamination. To improve sensitivity, multicopy and polymeric genes were chosen to be specifically amplified, i.e., eight copies of Y-chromosomal ETSTY5 as male-specific and four autosomal UBC monomers as control fragment. Specificity was enhanced by the equine-specific character of ETSTY5 and by using dual-labelled probes. The assay was optimised with equine male and female genomic DNA and demonstrated a 100% accuracy and a >95% qPCR efficiency down to 10 pg of DNA. The assay was subsequently applied to determine the sex of 44 in vitro-produced embryos, collecting trophectoderm biopsies by means of a needle aspiration biopsy and herniating cells. Of all trophectoderm biopsies and herniating cell samples (n = 54), 87% could be diagnosed. Assay results were validated on a second sample obtained from the biopsied embryo (n = 18) or, by ultrasound-based sex determination of the foetus (n = 7) following the transfer of the biopsied embryo to a recipient mare, with about half of the embryos being fillies and colts. The needle aspiration biopsy procedure did not impair initial pregnancy rate or early pregnancy losses as compared to non-biopsied embryos. In conclusion, we report a safe, reliable, fast, and cost-effective assay for equine sex determination which was validated for the sex determination of in vitro-produced embryos based on few embryonic cells, and needle aspiration biopsy did not impair the embryo's viability. The assay and safe biopsy strategy hold potential for other applications.

Genomewide copy number alteration screening of circulating plasma DNA: potential for the detection of incipient tumors.

Background: Early cancer diagnosis might improve survival rates. As circulating tumor DNA (ctDNA) carries cancer-specific modifications, it has great potential as a noninvasive biomarker for detection of incipient tumors. Patients and methods: We collected cell-free DNA (cfDNA) samples of 1002 elderly without a prior malignancy, carried out whole-genome massive parallel sequencing and scrutinized the mapped sequences for the presence of (sub)chromosomal copy number alterations (CNAs) predictive for a malignancy. When imbalances were detected, 6-monthly clinical follow-up was carried out. Results: In 3% of participants chromosomal imbalances were detected. Follow-up analyses, including whole-body MRI screening, confirmed the presence of five hematologic malignancies: one Hodgkin lymphoma (HL), stage II; three non-HL (type chronic lymphocytic leukemia, Rai I-Binet A; type SLL, stage III; type mucosa-associated lymphoid tissue, stage I) and one myelodysplastic syndrome with excess blasts, stage II. The CNAs detected in cfDNA were tumor-specific. Furthermore, one case was identified with monoclonal B-cell lymphocytosis, a potential precursor of B-cell malignancy. In 24 additional individuals, CNAs were identified but no cancer diagnosis was made. For 9 of them, the aberrant cfDNA profile originated from peripheral blood cells. For 15 others the origin of aberrations in cfDNA remains undetermined. Conclusion(s): Genomewide profiling of cfDNA in apparently healthy individuals enables the detection of incipient hematologic malignancies as well as clonal mosaicism with unknown clinical significance. CNA screening of cellular DNA of peripheral blood in elderly has established that clonal mosaicism for these chromosomal anomalies predicts a 5- to 10-fold enhanced risk of a subsequent cancer. We demonstrate that cfDNA screening detects CNAs, which are not only derived from peripheral blood, but even more from other tissues. Since the clinical relevance of clonal mosaics in other tissues remains unknown, long-term follow-up is warranted. Taken together, this study demonstrates that genomewide cfDNA analysis has potential as an unbiased screening approach for hematological malignancies and premalignant conditions.

Recent developments in genetics and medically assisted reproduction: from research to clinical applications.

Two leading European professional societies, the European Society of Human Genetics and the European Society for Human Reproduction and Embryology, have worked together since 2004 to evaluate the impact of fast research advances at the interface of assisted reproduction and genetics, including their application into clinical practice. In September 2016, the expert panel met for the third time. The topics discussed highlighted important issues covering the impacts of expanded carrier screening, direct-to-consumer genetic testing, voiding of the presumed anonymity of gamete donors by advanced genetic testing, advances in the research of genetic causes underlying male and female infertility, utilisation of massively parallel sequencing in preimplantation genetic testing and non-invasive prenatal screening, mitochondrial replacement in human oocytes, and additionally, issues related to cross-generational epigenetic inheritance following IVF and germline genome editing. The resulting paper represents a consensus of both professional societies involved.

Modulation of Aneuploidy in Leishmania donovani during Adaptation to Different In Vitro and In Vivo Environments and Its Impact on Gene Expression.

Aneuploidy is usually deleterious in multicellular organisms but appears to be tolerated and potentially beneficial in unicellular organisms, including pathogens. Leishmania, a major protozoan parasite, is emerging as a new model for aneuploidy, since in vitro-cultivated strains are highly aneuploid, with interstrain diversity and intrastrain mosaicism. The alternation of two life stages in different environments (extracellular promastigotes and intracellular amastigotes) offers a unique opportunity to study the impact of environment on aneuploidy and gene expression. We sequenced the whole genomes and transcriptomes of Leishmania donovani strains throughout their adaptation to in vivo conditions mimicking natural vertebrate and invertebrate host environments. The nucleotide sequences were almost unchanged within a strain, in contrast to highly variable aneuploidy. Although high in promastigotes in vitro, aneuploidy dropped significantly in hamster amastigotes, in a progressive and strain-specific manner, accompanied by the emergence of new polysomies. After a passage through a sand fly, smaller yet consistent karyotype changes were detected. Changes in chromosome copy numbers were correlated with the corresponding transcript levels, but additional aneuploidy-independent regulation of gene expression was observed. This affected stage-specific gene expression, downregulation of the entire chromosome 31, and upregulation of gene arrays on chromosomes 5 and 8. Aneuploidy changes in Leishmania are probably adaptive and exploited to modulate the dosage and expression of specific genes; they are well tolerated, but additional mechanisms may exist to regulate the transcript levels of other genes located on aneuploid chromosomes. Our model should allow studies of the impact of aneuploidy on molecular adaptations and cellular fitness.IMPORTANCE Aneuploidy is usually detrimental in multicellular organisms, but in several microorganisms, it can be tolerated and even beneficial. Leishmania-a protozoan parasite that kills more than 30,000 people each year-is emerging as a new model for aneuploidy studies, as unexpectedly high levels of aneuploidy are found in clinical isolates. Leishmania lacks classical regulation of transcription at initiation through promoters, so aneuploidy could represent a major adaptive strategy of this parasite to modulate gene dosage in response to stressful environments. For the first time, we document the dynamics of aneuploidy throughout the life cycle of the parasite, in vitro and in vivo We show its adaptive impact on transcription and its interaction with regulation. Besides offering a new model for aneuploidy studies, we show that further genomic studies should be done directly in clinical samples without parasite isolation and that adequate methods should be developed for this.

Recent developments in genetics and medically-assisted reproduction: from research to clinical applications†‡.

Two leading European professional societies, the European Society of Human Genetics and the European Society for Human Reproduction and Embryology, have worked together since 2004 to evaluate the impact of fast research advances at the interface of assisted reproduction and genetics, including their application into clinical practice. In September 2016, the expert panel met for the third time. The topics discussed highlighted important issues covering the impacts of expanded carrier screening, direct-to-consumer genetic testing, voiding of the presumed anonymity of gamete donors by advanced genetic testing, advances in the research of genetic causes underlying male and female infertility, utilisation of massively-parallel sequencing in preimplantation genetic testing and non-invasive prenatal screening, mitochondrial replacement in human oocytes, and additionally, issues related to cross-generational epigenetic inheritance following IVF and germline genome editing. The resulting paper represents a consensus of both professional societies involved.

Clinical implementation of NIPT - technical and biological challenges.

Non-invasive prenatal testing (NIPT) for fetal aneuploidy detection is increasingly being offered in the clinical setting. Whereas the majority of tests only report fetal trisomies 21, 18 and 13, genome-wide analyses have the potential to detect other fetal, as well as maternal, aneuploidies. In this review, we discuss the technical and clinical advantages and challenges associated with genome-wide cell-free fetal DNA profiling.

Real-time PCR evaluation of cell-free DNA subjected to various storage and shipping conditions.

In this study, we attempted to explore the factors affecting the yield of cell-free fetal DNA (cffDNA) obtained from maternal blood samples, including the use of different types of collection tubes, the interval between sample processing, and sample shipping under extreme weather conditions. Blood samples were drawn into K3EDTA tubes and cell-stabilizing tubes (Streck blood collection tube, BCT) from women pregnant with male fetuses. Real time PCR was used to amplify a ß-actin gene fragment to measure the total plasma cell-free DNA concentration, while an SRY gene fragment was used to quantify the cffDNA. The samples in the K3EDTA tubes revealed a decreased quantity of SRY after 5 days of transportation, with a median of 25.9 copies/mL (P < 0.01); however, the value remained stable at 33.4 copies/mL in the BCT tubes. We observed a statistically significant increase in stability of the amount of total DNA in the blood samples stored in K3EDTA tubes (P < 0.01) and transportated under extreme outdoor temperatures (-20°-0°C) than that of the control values. These results indicate that it could be possible to avoid the presence of excess maternal DNA in samples shipped under extreme weather conditions for no more than 2 days, by collecting the blood samples in BCT tubes.

Exome sequencing identifies a recessive PIGN splice site mutation as a cause of syndromic congenital diaphragmatic hernia.

Using exome sequencing we identify a homozygous splice site mutation in the PIGN gene in a foetus with multiple congenital anomalies including bilateral diaphragmatic hernia, cardiovascular anomalies, segmental renal dysplasia, facial dysmorphism, cleft palate, and oligodactyly. This finding expands the phenotypic spectrum associated with homozygous loss of function mutations in PIGN, and adds further support for defective GPI anchor biosynthesis as a cause of developmental abnormalities. We demonstrate that exome sequencing is a valuable approach for the identification of a genetic cause in sporadic cases of multiple congenital anomalies (MCA) due to inherited mutations.

Identification of dosage-sensitive genes in fetuses referred with severe isolated congenital diaphragmatic hernia.

OBJECTIVE: Congenital diaphragmatic hernia (CDH) is a fetal abnormality affecting diaphragm and lung development with a high mortality rate despite advances in fetal and neonatal therapy. CDH may occur either as an isolated defect or in syndromic form for which the prognosis is worse. Although conventional karyotyping and, more recently, chromosomal microarrays support a substantial role for genetic factors, causal genes responsible for isolated CDH remain elusive. We propose that chromosomal microarray analysis will identify copy number variations (CNVs) associated with isolated CDH. METHODS: We perform a prospective genome-wide screen for CNVs using chromosomal microarrays on 75 fetuses referred with apparently isolated CDH, six of which were later reclassified as non-isolated CDH. RESULTS: The results pinpoint haploinsufficiency of NR2F2 as a cause of CDH and cardiovascular malformations. In addition, the 15q25.2 and 16p11.2 recurrent microdeletions are associated with isolated CDH. By using gene prioritisation and network analysis, we provide strong evidence for several novel dosage-sensitive candidate genes associated with CDH. CONCLUSIONS: Chromosomal microarray analysis detects submicroscopic CNVs associated with isolated CDH or CDH with cardiovascular malformations.

Microarray analysis reveals abnormal chromosomal complements in over 70% of 14 normally developing human embryos.

STUDY QUESTION: What are the aneuploidy rates and incidence of mosaicism in good-quality human preimplantation embryos. SUMMARY ANSWER: High-level mosaicism and structural aberrations are not restricted to arrested or poorly developing embryos but are also common in good-quality IVF embryos. WHAT IS KNOWN ALREADY: Humans, compared with other mammals, have a poor fertility rate, and even IVF treatments have a relatively low success rate. It is known that human gametes and early preimplantation embryos carry chromosomal abnormalities that are thought to lower their developmental potential. STUDY DESIGN, SIZE AND DURATION: The embryos studied came from nine young (age <35 years old) IVF patients and were part of a cohort of embryos that all resulted in healthy births. These 14 embryos inseminated by ICSI and cryopreserved on Day 2 of development were thawed, cultured overnight and allowed to succumb by being left at room temperature for 24 h. Following removal of the zona pellucida, blastomeres were disaggregated and collected. PARTICIPANTS/MATERIALS, SETTING AND METHODS: There were 91 single blastomeres collected and amplified by multiple displacement amplification. Array-comparative genomic hybridization was performed on the amplified DNA. Array-data were normalized and aneuploidy was detected by the circular binary segmentation method. MAIN RESULTS AND THE ROLE OF CHANCE: The good-quality embryos exhibited high rates of aneuploidy, 10 of 14 (71.4%) of the embryos being mosaic. While none of the embryos had the same aneuploidy pattern in all cells, 4 of 14 (28.6%) were uniformly diploid. Of the 70 analysed blastomeres, 55.7% were diploid and 44.3% had chromosomal abnormalities, while 29% of the abnormal cells carried structural aberrations. WIDER IMPLICATIONS OF THE FINDINGS: Finding such a high rate of aneuploidy and mosaicism in excellent quality embryos from cycles with a high implantation rate warrants further research on the origin and significance of chromosomal abnormalities in human preimplantation embryos. STUDY FUNDING/COMPETING INTEREST(S): This research was supported by the Instituut voor de aanmoediging van innovatie door Wetenschap en Technologie in Vlaanderen (IWT-Vlaanderen). A.M. is a PhD student at the IWT-Vlaanderen. C.S. is a postdoctoral fellow at the FWO Vlaanderen. There are no competing interests.

Sporadic male patients with intellectual disability: contribution of X-chromosome copy number variants.

Genome-wide array comparative genome hybridization has become the first in line diagnostic tool in the clinical work-up of patients presenting with intellectual disability. As a result, chromosome X-copy number variations are frequently being detected in routine diagnostics. We retrospectively reviewed genome wide array-CGH data in order to determine the frequency and nature of chromosome X-copy number variations (X-CNV) in a cohort of 2222 sporadic male patients with intellectual disability (ID) referred to us for diagnosis. In this cohort, 68 males were found to have at least one X-CNV (3.1%). However, correct interpretation of causality remains a challenging task, and is essential for proper counseling, especially when the CNV is inherited. On the basis of these data, earlier experience and literature data we designed and propose an algorithm that can be used to evaluate the clinical relevance of X-CNVs detected in sporadic male ID patients. Applied to our cohort, 19 male ID patients (0.85%) were found to carry a (likely) pathogenic X-CNV.

Differences in Copy Number Variation between Discordant Monozygotic Twins as a Model for Exploring Chromosomal Mosaicism in Congenital Heart Defects.

Studies addressing the role of somatic copy number variation (CNV) in the genesis of congenital heart defects (CHDs) are scarce, as cardiac tissue is difficult to obtain, especially in non-affected individuals. We explored the occurrence of copy number differences in monozygotic (MZ) twins discordant for the presence of a CHD, as an illustrative model for chromosomal mosaicism in CHDs. Array comparative genomic hybridization was performed on peripheral blood-derived DNA obtained from 6 discordant MZ twin pairs and on sex-matched reference samples. To identify CNV differences between both twin members as well as potential CNVs in both twins contributing to the phenotype, DNA from each twin was hybridized against its co-twin, and against a normal control. Three copy number differences in 1 out of 6 MZ twin pairs were detected, confirming the occurrence of somatic CNV events in MZ twins. Further investigation by copy number and (epi)genome sequencing analyses in MZ twins, discordant for the presence of CHDs, is required to improve our knowledge on how postzygotic genetic, environmental and stochastic factors can affect human heart development.

Holoprosencephaly and ZIC2 microdeletions: novel clinical and epidemiological specificities delineated.

Holoprosencephaly (HPE), the most common malformation of the human brain results from abnormal cleavage of the forebrain during the early embryonic developmental stages. The spectrum of malformations in HPE is wide, ranging from the classical cyclopia/proboscis to fairly asymptomatic forms [i.e. a single maxillary central incisor (SMCI)]. HPE may be caused by environmental or genetic factors. ZIC2 (13q32) was the second gene identified in which mutations cause HPE and recently a specific phenotype was ascribed to ZIC2-mutation HPE. Earlier, we reported a boy presenting HPE and deafness. Cytogenetic analyses were normal. Using array-comparative genomic hybridization (aCGH), we found a de novo 129 kb del(13)(q32) encompassing ZIC2 and ZIC5. There is no evidence for the involvement of ZIC5 in human diseases. We reviewed the literature for ZIC2-ZIC5 deletions and their involvement in neural tube defects (NTDs). Interestingly, we found evidence for a specific facial phenotype for ZIC2 gene deletion patients distinct from those with point mutations. In addition, based on the clinical data together with pathology, imaging and functional studies, we suggest an outline for a model explaining the genetic heterogeneity of ZIC2-ZIC5-associated NTDs and propose further studies for validation.

Challenges of interpreting copy number variation in syndromic and non-syndromic congenital heart defects.

Array comparative genomic hybridization (aCGH) has led to an increased detection of causal chromosomal imbalances in individuals with congenital heart defects (CHD). The introduction of aCGH as a diagnostic tool in a clinical cardiogenetic setting entails numerous challenges. Based on our own experience as well as those of others described in the literature, we outline the state of the art and attempt to answer a number of outstanding questions such as the detection frequency of causal imbalances in different patient populations, the added value of higher-resolution arrays, and the existence of predictive factors in syndromic cases. We introduce a step-by-step approach for clinical interpretation of copy number variants (CNV) detected in CHD, which is primarily based on gene content and overlap with known chromosomal syndromes, rather than on CNV inheritance and size. Based on this algorithm, we have reclassified the detected aberrations in aCGH studies for their causality for syndromic and non-syndromic CHD. From this literature overview, supplemented with own investigations in a cohort of 46 sporadic patients with severe non-syndromic CHD, it seems clear that the frequency of causal CNVs in non-syndromic CHD populations is lower than that in syndromic CNV populations (3.6 vs. 19%). Moreover, causal CNVs in non-syndromic CHD mostly involve imbalances with a moderate effect size and reduced penetrance, whereas the majority of causal imbalances in syndromic CHD consistently affects human development and significantly reduces reproductive fitness.

The human cleavage stage embryo is a cradle of chromosomal rearrangements.

The first cell cycles following in vitro fertilization (IVF) of human gametes are prone to chromosome instability. Many, but often not all, blastomeres of an embryo acquire a genetic makeup during cleavage that is not representative of the original zygotic genome. Whole chromosomes are missegregated, but also structural rearrangements of chromosomes do occur in human cleavage stage embryogenesis following IVF. Analysis of pre- and postnatal DNA samples indicates that the in vivo human conceptions also endure instability of chromosome number and structure during cleavage of the fertilized oocyte. This embryonic chromosome instability not necessarily undermines normal human development, but may lead to a spectrum of conditions, including loss of conception, genetic disease and genetic variation development. In this review, the structural instability of chromosomes during human cleavage stage embryogenesis is catalogued, channeled into etiologic models and linked to genomic profiles of healthy and diseased newborns.

PGD for a complex chromosomal rearrangement by array comparative genomic hybridization.

Patients carrying a chromosomal rearrangement (CR) have an increased risk for chromosomally unbalanced conceptions. Preimplantation genetic diagnosis (PGD) may avoid the transfer of embryos carrying unbalanced rearrangements, therefore increasing the chance of pregnancy. Only 7-12 loci can be screened by fluorescence in situ hybridization whereas microarray technology can detect genome-wide imbalances at the single cell level. We performed PGD for a CR carrier with karyotype 46,XY,ins(3;2)(p23;q23q14.2),t(6;14)(p12.2;q13) using array comparative genomic hybridization. Selection of embryos for transfer was only based on copy number status of the chromosomes involved in both rearrangements. In two ICSI-PGD cycles, nine and seven embryos were analysed by array, leaving three and one embryo(s) suitable for transfer, respectively. The sensitivity and specificity of single cell arrays was 100 and 88.8%, respectively. In both cycles a single embryo was transferred, resulting in pregnancy following the second cycle. The embryo giving rise to the pregnancy was normal/balanced for the insertion and translocation but carried a trisomy 8 and nullisomy 9 in one of the two biopsied blastomeres. After 7 weeks of pregnancy the couple miscarried. Genetic analysis following hystero-embryoscopy showed a diploid (90%)/tetraploid (10%) mosaic chorion, while the gestational sac was empty. No chromosome 8 aneuploidy was detected in the chorion, while 8% of the cells carried a monosomy for chromosome 9. In summary, we demonstrate the feasibility and determine the accuracy of single cell array technology to test against transmission of the unbalanced meiotic products that can derive from CRs. Our findings also demonstrate that the genomic constitution of extra-embryonic tissue cannot necessarily be predicted from the copy number status of a single blastomere.

Oculocerebral hypopigmentation syndrome maps to chromosome 3q27.1q29.

BACKGROUND: In 1967, Cross et al. [J Pediatr 1967;70:398-406] reported four siblings with intellectual disability, microcephaly, neurologic and ocular disorders, and hypopigmentation involving skin and hair. This novel entity, known as oculocerebral hypopigmentation syndrome (OCHS) or Cross syndrome (OMIM 257800), is assumed to be autosomal recessive. However, its genetic cause is still unknown. CASE REPORT: A 4-year-old girl is reported with OCHS, a history of recurrent infections and vertebral fusion of L4-L5. Central nervous system and cardiac imaging as well as metabolic screening were normal. Microscopic hair investigations did not show any melanin deposit defects. RESULTS: Using molecular cytogenetics, we detected a de novo interstitial del(3)(q27.1q29) of the paternal chromosome. To our knowledge, this is the first molecular genetics finding in a patient with OCHS. Here we discuss the genotype-phenotype correlations and suggest candidate genes for this disorder. CONCLUSION: Investigating further patients would enable fine-mapping the OCHS locus and identifying its putative gene.

Duplication of the VHL and IRAK2 genes in a patient with mental retardation/multiple congenital anomalies, epilepsy and ectomorphic habitus.

Partial 3p duplications are very rare. Often they are reported in translocations involving other chromosomes, whereas deletions encompassing the VHL gene in 3p25.3 predispose to Van-Hippel Lindau syndrome. We report here a paternally-inherited microduplication of 3p25.3 detected by array comparative genomic hybridisation (aCGH) in a 17 year-old male patient presenting with mental retardation and multiple congenital anomalies (MR/MCA), epilepsy and ectomorphic habitus. He has no tumour and there is no history of familial cancer. We refined the duplication by Multiplex Ligation-dependent Probe Amplification (MLPA) to a 251 kb region encompassing the VHL and IRAK2 genes. The duplication is likely to be causal. Interestingly, duplication of IRAK2 can cause epilepsy. Disruption of the GHRL gene can explain the ectomorphic habitus. To our knowledge, this is the smallest 3p duplication encompassing the VHL region. Its prognosis is unknown and a long-term follow-up is essential for an early diagnosis of malignancy.

FOXD1 Duplication Causes Branchial Defects and Interacts with the TFAP2A Gene Implicated in the Branchio-Oculo-Facial Syndrome in Causing Eye Effects in Zebrafish.

Branchio-oculo-facial syndrome (BOFS) is a rare disorder characterized by maldevelopment of the first and second branchial arches, skin defects, facial dysmorphism, auricular, ophthalmological and oral abnormalities. A high clinical variability has been reported. Recently, mutations in TFAP2A were found to underlie this condition. A small duplication on 5q13 was detected in 2 family members with mild BOFS features. Molecular cytogenetic delineation of the duplication demonstrated that only 7 genes are affected: LOC100289045, RGNEF, UTP15, ANKRA2, FUNDC2P1, BTF3 and FOXD1. The latter is expressed in the developing branchial arches and involved in cranio-facial development. Zebrafish embryos with combined inhibition of the expression of foxd1l and tfap2a show optic axis defects. We identified a novel locus associated with a mild BOFS-like phenotype. The functional in vivo experiments suggest an interaction between FOXD1 and TFAP2A.