Carla S. D'Angelo

Extending the phenotype of monosomy 1p36 syndrome and mapping of a critical region for obesity and hyperphagia.

Rearrangements of 1p36 are the most frequently detected abnormalities in diagnostic testing for chromosomal cryptic imbalances and include variably sized simple terminal deletions, derivative chromosomes, interstitial deletions, and complex rearrangements. These rearrangements result in the specific pattern of malformation and neurodevelopmental disabilities that characterizes monosomy 1p36 syndrome. Thus far, no individual gene within this region has been conclusively determined to be causative of any component of the phenotype. Nor is it known if the rearrangements convey phenotypes via a haploinsufficiency mechanism or through a position effect. We have used multiplex ligation‐dependent probe amplification to screen for deletions of 1p36 in a group of 154 hyperphagic and overweight/obese, PWS negative individuals, and in a separate group of 83 patients initially sent to investigate a variety of other conditions. The strategy allowed the identification and delineation of rearrangements in nine subjects with a wide spectrum of clinical presentations. Our work reinforces the association of monosomy 1p36 and obesity and hyperphagia, and further suggests that these features may be associated with non‐classical manifestations of this disorder in addition to a submicroscopic deletion of ∼2–3 Mb in size. Multiplex ligation probe amplification using the monosomy 1p36 syndrome‐specific kit coupled to the subtelomeric kit is an effective approach to identify and delineate rearrangements at 1p36.

Further delineation of nonhomologous-based recombination and evidence for subtelomeric segmental duplications in 1p36 rearrangements

The mechanisms involved in the formation of subtelomeric rearrangements are now beginning to be elucidated. Breakpoint sequencing analysis of 1p36 rearrangements has made important contributions to this line of inquiry. Despite the unique architecture of segmental duplications inherent to human subtelomeres, no common mechanism has been identified thus far and different nonexclusive recombination–repair mechanisms seem to predominate. In order to gain further insights into the mechanisms of chromosome breakage, repair, and stabilization mediating subtelomeric rearrangements in humans, we investigated the constitutional rearrangements of 1p36. Cloning of the breakpoint junctions in a complex rearrangement and three non-reciprocal translocations revealed similarities at the junctions, such as microhomology of up to three nucleotides, along with no significant sequence identity in close proximity to the breakpoint regions. All the breakpoints appeared to be unique and their occurrence was limited to non-repetitive, unique DNA sequences. Several recombination- or cleavage-associated motifs that may promote non-homologous recombination were observed in close proximity to the junctions. We conclude that NHEJ is likely the mechanism of DNA repair that generates these rearrangements. Additionally, two apparently pure terminal deletions were also investigated, and the refinement of the breakpoint regions identified two distinct genomic intervals ~25-kb apart, each containing a series of 1p36 specific segmental duplications with 90–98% identity. Segmental duplications can serve as substrates for ectopic homologous recombination or stimulate genomic rearrangements.

An inherited atypical 1 Mb 22q11.2 deletion within the DGS/VCFS 3 Mb region in a child with obesity and aggressive behavior

The DiGeorge, Velocardiofacial, and Conotruncal Anomaly Face syndromes (DGS/VCFS/CTAF) are knowncollectively as the 22q11.2 deletion syndrome (22q11 DS) [Shaikh et al., 2000; Swillen et al., 2000]. Although most individuals have the same large 3 Mb 22q11.2 de novo deletion, a recurrent 1.5–2 Mb proximally nested deletion is more common in familial cases of 22q11.2 DS [Iascone et al., 2002; Adeyinka et al., 2004; Fernandez et al., 2005]. Atypical smaller or uniquely placed deletions have been described in only a few patients [Kurahashi et al., 1996, 1997; Amati et al., 1999; Shaikh et al., 2000; Garcia-Minaur et al., 2002; Rauch et al., 2005]. We report on a rare 1 Mb 22q11.2 deletion in a female patient with obesity, hyperphagia, and aggressive behavior, and in her mother who had a major depressive disorder. The deletion was identified serendipitously in the proband during a microdeletion screening for syndromic obesity and was limited to the most telomeric region of the 3 Mb typically deleted region. The patient (Fig. 1) was the first child of nonconsanguinous parents with an unremarkable family history. Decreased fetal movement was reported. She was delivered at term by caesarean. Apgar scores were 8 and 9 at 1 and 5 min, respectively. The birth weight was 2.9 kg ( 25th centile), length 48 cm ( 10th centile), and the OFC 34 cm ( 25th centile). Neonatal jaundice was treated with phototherapy. The sucking reflex was present, but she fed with difficulty until 1 year of age when gastroesophageal reflux was diagnosed. Delayed closure of anterior fontanel was reported. She walked independently at 1 year and 4 months. On examination at 4 years of age, the OFC was 52 cm (50–98th centile), height was 101 cm (25–50th centile), and weight was 27 kg (>97th centile). She had a narrow forehead, synophrys, upslanted palpebral fissures, deep-set eyes, divergent strabismus of the right eye, small mouth and thin lips, high-arched palate, short philtrum, retrognathia, small hands, and pes planus. Her problems included speech articulation, sleep difficulties, hyperphagia, decreased sensitivity to pain, hyperactivity, and aggressive behavior, such as beating strangers, and self-injurious behaviors including head banging, biting, hair pulling, and skin picking. When re-evaluated at age 8 years, there were no significant changes. Compulsive food eating and behavioral problems had worsened. She weighed 67 kg (>97th centile) with truncal obesity, and height was 130 cm (50–75th centile). She took antipsychotic medication to control aggressiveness and attended a special education school. The patient’s mother has had a major depressive disorder and anxiety for 10 years, for which she has taken several psychiatric medications. She had similar facial features to her daughter including divergent strabismus on the right eye, and a hypernasal speech. She weighed 71 kg and was 160 cm tall (BMI 27.7, overweight). She was otherwise

Copy number variants in obesity-related syndromes: review and perspectives on novel molecular approaches.

In recent decades, obesity has reached epidemic proportions worldwide and became a major concern in public health. Despite heritability estimates of 40 to 70% and the long-recognized genetic basis of obesity in a number of rare cases, the list of common obesity susceptibility variants by the currently published genome-wide association studies (GWASs) only explain a small proportion of the individual variation in risk of obesity. It was not until very recently that GWASs of copy number variants (CNVs) in individuals with extreme phenotypes reported a number of large and rare CNVs conferring high risk to obesity, and specifically deletions on chromosome 16p11.2. In this paper, we comment on the recent advances in the field of genetics of obesity with an emphasis on the genes and genomic regions implicated in highly penetrant forms of obesity associated with developmental disorders. Array genomic hybridization in this patient population has afforded discovery opportunities for CNVs that have not previously been detectable. This information can be used to generate new diagnostic arrays and sequencing platforms, which will likely enhance detection of known genetic conditions with the potential to elucidate new disease genes and ultimately help in developing a next-generation sequencing protocol relevant to clinical practice.

Obesity with associated developmental delay and/or learning disability in patients exhibiting additional features: Report of novel pathogenic copy number variants

Obesity is a major threat to public health worldwide, and there is now mounting evidence favoring a role for the central nervous system (CNS) in weight control. A causal relationship has been recognized in both monogenic (e.g., BDNF, TRKB, and SIM1 deficiencies) and syndromic forms of obesity [e.g., Prader–Willi syndrome (PWS)]. Syndromic obesity arising from chromosomal abnormalities, that typically also affect learning and development, are often associated with congenital malformations and behavioral characteristics. We report on nine unrelated patients with a diagnosis of learning disability and/or developmental delay (DD) in addition to obesity that were found to have copy number variants (CNVs) by single nucleotide polymorphism array‐based analysis. Each patient also had a distinct and complex phenotype, and most had hypotonia and other neuroendocrine issues, such as hyperphagia and hypogonadism. Molecular and clinical characterization of these patients enabled us to determine with confidence that the CNVs we observed were pathogenic or likely to be pathogenic. Overall, the CNVs reported here encompassed a candidate gene or region (e.g., SIM1) that has been reported in patients associating obesity and DD and/or intellectual disability (ID) and novel candidate genes and regions.

Does germ-line deletion of the PIP gene constitute a widespread risk for cancer?

We wish to draw the attention of cancer geneticists to a particular genetic variant of the Prolactin-Induced Protein (PIP) gene that may be an important predisposing factor to cancer because of its high frequency and significant association with cancer, as determined in this study. In an initial copy number variation (CNV) screen for germ-line deletions in 123 Brazilian cancer patients selected as high risk either because of early age of onset (pediatric cancer) or a positive family history (TP53-negative Li–Fraumeni and APC/MUTYH-negative Familial Adenomatous Polyposis patients), a previously undescribed microdeletion was discovered in four patients. The deletion carried in these patients was apparently identical (Figure 1), with a size of 69 kb and similar base-pair position in chromosome region 7q34. All four deletions were validated by qPCR and found to harbor only the PIP and TAS2R39 (taste receptor type 2 member 39) genes. TAS2R39 is a member of ∼30 TAS2R bitter taste receptors,1 several of which are known to exhibit variation in copy number. It is unlikely that TAS2R39 has a role in tumorigenesis, and will not be considered further in this submission. We here refer to the deletion encompassing the PIP gene as PIP-Sao Paulo, following the convention of adding the place of discovery to the gene name. This deletion was not detected in a control group of 260 non-related individuals from the Sao Paulo urban area that had attended our genetic service for reasons other than cancer (normal relatives of patients with intellectual disabilities). The difference in deletion frequency between patients and controls was significant at the 0.01 level (Fishers exact test). Another example of the PIP-Sao Paulo deletion was serendipitously detected in a TP53-mutated patient who was not part of these cohorts, but had been investigated by aCGH for presenting with an atypically severe course of cancer that commenced at 4 years of age with the diagnosis of a rhabdomyosarcoma, followed by choroid plexus tumor (7 years of age), liposarcoma and osteochondroma (10 years of age), and finally passed away at 11 years. This patient was not included in the statistics of the patient–control comparison; however, a possible interaction between the TP53 mutation and PIP-Sao Paulo deletion may have contributed to the severity of the cancer progression. Figure 1 PIP-Sao Paulo microdeletion at 7q34, detected by array-CGH in cancer patients. Array-CGH profile of a chromosome region at 7q34 (microarray platform Agilent 180K—Agilent Technologies, Santa Clara, CA, USA), showing heterozygous losses ... Following our initial observations, we interrogated the 1000 Genomes Project database and found a comparable deletion to be present in 13 out of 1097 individuals: 9 of European and 4 of Latin-American origin (see Supplementary Table 1). Manual review of the sequence data in this region indicated that 7 out of the 13 individuals had sufficient sequence depth to determine that they shared identical breakpoints. From this information, we designed PCR primers and were able to amplify across the breakpoints in all the five Brazilian deletion cases. Subsequent sequencing of the PCR fragments (see Supplementary Figure 1) demonstrated that all identified PIP-Sao Paulo deletions were identical to the deletions present in the 1000 Genomes Project data set. In a replicate study, we determined by breakpoint PCR the frequency of PIP deletions in an independent Brazilian cancer group (219 individuals) that had either presented with more than one primary cancer prior to 60 years of age (166 individuals) or were probands of hereditary melanoma families (53 individuals with no CDKN2A or CDK4 mutations). This identified a further 6 individuals, who had had cancer and carried the PIP deletion (2.6%), as opposed to 10 out of 847 individuals (1.2%) from a second control group. As in the initial study, the deletion frequency in the replicate study in cancer patients was higher than that in controls, although this was not significant at the 5% level (P=0.11). However, when cancer and control samples from both studies were pooled, statistical significance was attained (P=0.04, Fishers exact test). Importantly, heterogeneity tests demonstrated that no significant differences existed at the 0.05 level between the two control and two patient samples, respectively. We conclude that the relatively frequent germ-line deletion of the PIP gene has more than a two-fold increase in frequency in the Brazilian cancer patients compared with the Brazilian controls. The clinical phenotype, mutation status, type of tumor and age of diagnosis of all patients carrying the PIP-Sao Paulo deletion are summarized in Table 1, demonstrating that several types of cancer are involved in this study and that no single type or group of cancers predominates. Presence of the PIP-Sao Paulo deletion was investigated in paraffin blocks from several different primary tumors taken from four of the deleted patients. In all samples, except for one from patient 7, the PIP-Sao Paulo deletion was detected (Supplementary Figure 1); additionally, at least one exon of the PIP gene was amplified from each of the tested samples, indicating that one allele was retained, similar to blood. Unfortunately, DNA quality did not allow sequence screening for point mutations or other deletions that may have occurred in the remaining allele. We conclude that if the PIP-Sao Paulo deletion is directly responsible for tumor induction, this has not occurred by induction of homozygosity of the deletion itself in the cases studied. Table 1 Characteristics of the PIP-Sao Paulo deletion patients: mutation status, clinical phenotype, type of tumor and age at diagnosis To date, several publications have reported association between PIP expression and tumor progression, particularly for prostate and breast cancers.2, 3 The gene has been implicated in multiple functions, including apoptosis, cell proliferation and migration,4 but mutations have only been studied in tumor cell lines and never investigated as germ-line mutations. According to the TCGA database (http://cancergenome.nih.gov/), PIP gains in tumors are more frequent than deletions; however, data in different type of tumors are very heterogeneous and difficult to interpret. We have no specific insights into the mechanisms through which the PIP-Sao Paulo deletion could be oncogenic. It is evident from the foregoing that the PIP-Sao Paulo deletion is not specific to Brazil. Inspection of the HapMap3 database shows that the haplotype adjacent to the deletion (Table 2) is either absent or extremely rare in African populations. Interestingly, although about ∼25% of the 1094 individuals sequenced in data set 1 of the 1000 Genome Project were of African origin, none of the 13 individuals carrying the deletion were African and, except for one Colombian and two Mexican individuals from Los Angeles, CA, USA, all others were of European origin. Ethnic certainty of Brazilian cancer patients and controls is obscured by miscegenation and ethnic diversity; however, they are most likely to be mainly Caucasians, given the relatively high Caucasian composition of Sao Paulo, and through hospital attendance preferences and other socioeconomic differences. It is probable that PIP-Sao Paulo arose in an European population as a founder mutation that was subsequently exported to the New World. Table 2 SNPs, genomic positions (Hg19) and the common haplotype deduced from the proximal region of the patients with 7q34 deletion The crucial question of whether the PIP-Sao Paulo deletion constitutes a widespread cancer risk can only be answered by similar investigations in other countries, which we hope will be stimulated by this report. However, maintenance of a deletion variant associated with cancer at such high frequency is surprising and probably implies that, if the cancer association is confirmed by independent studies, PIP-Sao Paulo appears to have a relatively low cancer penetrance rate so that many carriers either do not manifest cancer or do so at later age, following transmission of the deletion to their offspring, similar to the well-documented Brazilian TP53 variant R337H4. Whereas R337H has an estimated population frequency of ∼0.3% in Southern Brazil and exhibits an ∼8% penetrance rate in families with adrenocortical cancer,5 PIP-Sao Paulo is practically an order of magnitude more frequent (estimated at ∼2% in European populations from the 1000 Genome Project data set) and appears as a very frequent factor associated with increased cancer risk. Further, if the PIP-Sao Paulo deletion frequency is at least two-fold higher in European cancer patients than in controls, as in the Sao Paulo study, then even allowing for a low penetrance rate, there would be an increased cancer risk for Europeans. In the case of PIP-Sao Paulo, it is the high frequency and not the penetrance that constitutes the main risk parameter. This completely unanticipated aspect deserves a rapid, coordinated response to confirm whether indeed such an increased risk exists, and if so, to establish its magnitude and specificity. Rapid and cost-effective detection of the deletion can be performed using either PCR across the breakpoints, or copy number estimates by RT-PCR, MLPA and so on. The exact genome location of PIP-Sao Paulo is chr7: 142824847-142893913 (Genome build GRCh37/hg19).

Molecular Cytogenetic Characterization of an Inherited Maternal Duplication 20p11.21p13 Associated With a Small 20p11.21 Deletion

Partial trisomy for the short arm of chromosome 20 is a relatively rare chromosomal abnormality. Most cases are derived from reciprocal translocations with the presence of partial monosomy of other chromosomes, and thus it has been difficult to delineate a specific phenotype [review in Chaabouni et al., 2007]. The molecular cytogenetic characterization of chromosome 20p rearrangements has been described in a few cases, which has allowed not only a more accurate genotype–phenotype correlation, but also the investigation of its underlying mechanisms. Sidwell et al. [2000] reported on a case of pure trisomy 20p arisen from de novo isochromosome formation, and associated with a non-reciprocal rearrangement involving the translocation of 20q11.1qter sequences onto chromosome 4p. Ravel et al. [2003] described a de novo isolated interstitial tandem duplication of chromosome 20p12 with no evidence of repeated sequences mediating this rearrangement. Ardalan et al. [2005] reported on a derivative with 20pter duplication and 20qter deletion originated from a familial pericentric intrachromosomal insertion following homologous pairing and crossover at meiosis. Chaabouni et al. [2007] described a case of de novo duplication spanning almost the whole short arm of chromosome 20p and associated with a small distal 20q deletion. Recently, Leclercq et al. [2009] reported on the first case of inversion duplication deletion involving the short arm of chromosome 20p (inv dup del 20p), which likely formed through a U-type exchange. High-resolution oligonucleotide CGH array revealed an 18.16 Mb duplication of 20p11.22p13 in association with a 20p13 terminal deletion sizing 2.02 Mb. Herein, we report on a case of trisomy 20p of maternal origin initially detected by conventional cytogenetic investigation. Molecular cytogenetic studies with fluorescent in situ hybridization (FISH) and single-nucleotide polymorphism (SNP) array revealed a direct tandem duplication of 20p11.21p13 associated with a contiguous 20p11.21 proximal microdeletion. The clinically normal mother had the same chromosome abnormality detected in about 15% of her lymphocyte cells. We have compared the phenotype of our patient to similar cases from the literature in order to further delineate trisomy 20p syndrome. The patient, a 21-year-old woman (Fig. 1), was the first child of non-consanguineous normal parents, aged 23 (mother) and 22 years (father). The mother had two miscarriages at 10 weeks of gestation and a normal son was subsequently delivered. Patient’s pregnancy was uncomplicated and she was delivered at term by caesarean. Apgar scores were 6 and 8 at 1 and 5 min, respectively. Birth weight was 2,960 g ( 10th centile) and birth length 49 cm (25th centile). The sucking reflex was present. At 2 months she had surgery for bilateral inguinal and umbilical hernia. By the age of 9 months she had little control over her head and neck and could not sit without support. She started walking at the age of 18 months with frequent falls. Independent walk was achieved at 2 years of age. On examination at 21 years of age, occipital-frontal circumference (OFC) was 54 cm (2–50th centile), height 1.70 m (90th centile) and weight 65 kg (75–90th centile). She had a high forehead, hypertelorism, downslanted palpebral fissures, deep-set eyes, thick