16p11.2 Locus modulates response to satiety before the onset of obesity.

BACKGROUND: The 600 kb BP4-BP5 copy number variants (CNVs) at the 16p11.2 locus have been associated with a range of neurodevelopmental conditions including autism spectrum disorders and schizophrenia. The number of genomic copies in this region is inversely correlated with body mass index (BMI): the deletion is associated with a highly penetrant form of obesity (present in 50% of carriers by the age of 7 years and in 70% of adults), and the duplication with being underweight. Mechanisms underlying this energy imbalance remain unknown. OBJECTIVE: This study aims to investigate eating behavior, cognitive traits and their relationships with BMI in carriers of 16p11.2 CNVs. METHODS: We assessed individuals carrying a 16p11.2 deletion or duplication and their intrafamilial controls using food-related behavior questionnaires and cognitive measures. We also compared these carriers with cohorts of individuals presenting with obesity, binge eating disorder or bulimia. RESULTS: Response to satiety is gene dosage-dependent in pediatric CNV carriers. Altered satiety response is present in young deletion carriers before the onset of obesity. It remains altered in adolescent carriers and correlates with obesity. Adult deletion carriers exhibit eating behavior similar to that seen in a cohort of obesity without eating disorders such as bulimia or binge eating. None of the cognitive measures are associated with eating behavior or BMI. CONCLUSIONS: These findings suggest that abnormal satiety response is a strong contributor to the energy imbalance in 16p11.2 CNV carriers, and, akin to other genetic forms of obesity, altered satiety responsiveness in children precedes the increase in BMI observed later in adolescence.

A new highly penetrant form of obesity due to deletions on chromosome 16p11.2.

Obesity has become a major worldwide challenge to public health, owing to an interaction between the Western 'obesogenic' environment and a strong genetic contribution. Recent extensive genome-wide association studies (GWASs) have identified numerous single nucleotide polymorphisms associated with obesity, but these loci together account for only a small fraction of the known heritable component. Thus, the 'common disease, common variant' hypothesis is increasingly coming under challenge. Here we report a highly penetrant form of obesity, initially observed in 31 subjects who were heterozygous for deletions of at least 593 kilobases at 16p11.2 and whose ascertainment included cognitive deficits. Nineteen similar deletions were identified from GWAS data in 16,053 individuals from eight European cohorts. These deletions were absent from healthy non-obese controls and accounted for 0.7% of our morbid obesity cases (body mass index (BMI) >or= 40 kg m(-2) or BMI standard deviation score >or= 4; P = 6.4 x 10(-8), odds ratio 43.0), demonstrating the potential importance in common disease of rare variants with strong effects. This highlights a promising strategy for identifying missing heritability in obesity and other complex traits: cohorts with extreme phenotypes are likely to be enriched for rare variants, thereby improving power for their discovery. Subsequent analysis of the loci so identified may well reveal additional rare variants that further contribute to the missing heritability, as recently reported for SIM1 (ref. 3). The most productive approach may therefore be to combine the 'power of the extreme' in small, well-phenotyped cohorts, with targeted follow-up in case-control and population cohorts.

Split hand/foot malformation with long-bone deficiency and BHLHA9 duplication: report of 13 new families.

Split hand/foot malformation (SHFM) with long-bone deficiency (SHFLD, MIM#119100) is a rare condition characterized by SHFM associated with long-bone malformation usually involving the tibia. Previous published data reported several unrelated patients with 17p13.3 duplication and SHFLD. Recently, the minimal critical region had been reduced, suggesting that BHLHA9 copy number gains are associated with this limb defect. Here, we report on 13 new families presenting with ectrodactyly and harboring a BHLHA9 duplication.

A novel microdeletion syndrome at 3q13.31 characterised by developmental delay, postnatal overgrowth, hypoplastic male genitals, and characteristic facial features.

BACKGROUND: Congenital deletions affecting 3q11q23 have rarely been reported and only five cases have been molecularly characterised. Genotype-phenotype correlation has been hampered by the variable sizes and breakpoints of the deletions. In this study, 14 novel patients with deletions in 3q11q23 were investigated and compared with 13 previously reported patients. METHODS: Clinical data were collected from 14 novel patients that had been investigated by high resolution microarray techniques. Molecular investigation and updated clinical information of one cytogenetically previously reported patient were also included. RESULTS: The molecular investigation identified deletions in the region 3q12.3q21.3 with different boundaries and variable sizes. The smallest studied deletion was 580 kb, located in 3q13.31. Genotype-phenotype comparison in 24 patients sharing this shortest region of overlapping deletion revealed several common major characteristics including significant developmental delay, muscular hypotonia, a high arched palate, and recognisable facial features including a short philtrum and protruding lips. Abnormal genitalia were found in the majority of males, several having micropenis. Finally, a postnatal growth pattern above the mean was apparent. The 580 kb deleted region includes five RefSeq genes and two of them are strong candidate genes for the developmental delay: DRD3 and ZBTB20. CONCLUSION: A newly recognised 3q13.31 microdeletion syndrome is delineated which is of diagnostic and prognostic value. Furthermore, two genes are suggested to be responsible for the main phenotype.

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

Corpus callosum abnormalities, intellectual disability, speech impairment, and autism in patients with haploinsufficiency of ARID1B. Corpus callosum abnormalities are common brain malformations with a wide clinical spectrum ranging from severe intellectual disability to normal cognitive function. The etiology is expected to be genetic in as much as 30-50% of the cases, but the underlying genetic cause remains unknown in the majority of cases. By next-generation mate-pair sequencing we mapped the chromosomal breakpoints of a patient with a de novo balanced translocation, t(1;6)(p31;q25), agenesis of corpus callosum (CC), intellectual disability, severe speech impairment, and autism. The chromosome 6 breakpoint truncated ARID1B which was also truncated in a recently published translocation patient with a similar phenotype. Quantitative polymerase chain reaction (Q-PCR) data showed that a primer set proximal to the translocation showed increased expression of ARID1B, whereas primer sets spanning or distal to the translocation showed decreased expression in the patient relative to a non-related control set. Phenotype-genotype comparison of the translocation patient to seven unpublished patients with various sized deletions encompassing ARID1B confirms that haploinsufficiency of ARID1B is associated with CC abnormalities, intellectual disability, severe speech impairment, and autism. Our findings emphasize that ARID1B is important in human brain development and function in general, and in the development of CC and in speech development in particular.

Identification of two novel mutations in Shh long-range regulator associated with familial pre-axial polydactyly.

Pre-axial polydactyly type II (PPDII, MIM #174500), Werner mesomelic syndrome (MIM %188770) and Haas polysyndactyly (MIM #186200) are a group of closely related conditions caused by mutations in a long-range Sonic hedgehog (SHH, MIM *600725) regulator called ZRS. To date, 19 point mutations, 10 duplications and 1 triplication of the ZRS associated with those pre-axial polydactylies have been reported in humans, mice, cats and chickens. Some of these have been shown to cause ectopic expression of Shh in the limb bud in mice, leading to a polydactylous phenotype, but the precise mechanism by which ZRS mutations generate this phenotype remains unknown. We present two PPDII families with fully penetrant point mutations in ultra-conserved predicted binding sites for transcription factors SOX9 and PAX3, two possible candidates for regulating SHH expression. Screening for point mutations or copy-number variation of the ZRS, high-resolution array-CGH, and screening of other conserved non-coding sequences (CNS) surrounding SHH in a third family are negative. This is the sixth PPDII pedigree with possible linkage to 7q36 that presents with no detectable ZRS mutation. We hypothesize that another nearby regulatory sequence, or an undetected position effect between ZRS and SHH, could be responsible for negative familial cases linked to 7q36.

Microdeletion at chromosome 4q21 defines a new emerging syndrome with marked growth restriction, mental retardation and absent or severely delayed speech.

BACKGROUND Genome-wide screening of large patient cohorts with mental retardation using microarray-based comparative genomic hybridisation (array-CGH) has recently led to identification several novel microdeletion and microduplication syndromes. METHODS Owing to the national array-CGH network funded by the French Ministry of Health, shared information about patients with rare disease helped to define critical intervals and evaluate their gene content, and finally determine the phenotypic consequences of genomic array findings. RESULTS In this study, nine unrelated patients with overlapping de novo interstitial microdeletions involving 4q21 are reported. Several major features are common to all patients, including neonatal muscular hypotonia, severe psychomotor retardation, marked progressive growth restriction, distinctive facial features and absent or severely delayed speech. The boundaries and the sizes of the nine deletions are different, but an overlapping region of 1.37 Mb is defined; this region contains five RefSeq genes: PRKG2, RASGEF1B, HNRNPD, HNRPDL and ENOPH1. DISCUSSION Adding new individuals with similar clinical features and 4q21 deletion allowed us to reduce the critical genomic region encompassing two genes, PRKG2 and RASGEF1B. PRKG2 encodes cGMP-dependent protein kinase type II, which is expressed in brain and in cartilage. Information from genetically modified animal models is pertinent to the clinical phenotype. RASGEF1B is a guanine nucleotide exchange factor for Ras family proteins, and several members have been reported as key regulators of actin and microtubule dynamics during both dendrite and spine structural plasticity. CONCLUSION Clinical and molecular delineation of 4q21 deletion supports a novel microdeletion syndrome and suggests a major contribution of PRKG2 and RASGEF1B haploinsufficiency to the core phenotype.

Delineation of 15q13.3 microdeletions.

The increasing use of array-comparative genomic hybridization (array-CGH) to identify copy number variations (CNVs) in patients with developmental delay (DD), mental retardation and/or dysmorphic features has allowed the recent recognition of numerous genomic imbalances, including the 15q13.3 microdeletion. Patients with this microdeletion generally present with relatively consistent breakpoints at BP4 and BP5, which include the CHRNA7 gene. About 100 index cases have been reported since the first publication in 2008. This large number of patients ascertained through highly variable samples has been necessary to describe the full phenotypic spectrum of this microdeletion, ranging from mental retardation with dysmorphic features, epilepsy, neuropsychiatric disturbances with or without cognitive impairment to complete absence of anomalies. Here, we describe a collaborative study reporting a new cohort of 12 index patients and 13 relatives carrying a heterozygous BP4-BP5 microdeletion out of a series of 4625 patients screened by array-CGH for DD. We confirm the clinical expressivity of the disease as well as the incomplete penetrance in seven families. We showed through a review of the literature that males are more likely to be symptomatic. Sequence analysis of CHRNA7 yielded no data to support the unmasking of recessive variants as a cause of phenotypic variability. We also report the first patient carrying a 15q13.3 homozygous microdeletion inherited from both parents. He had severe epileptic encephalopathy with retinopathy, autistic features and choreoathetosis. Besides the classical approximately 1.5 Mb BP4-BP5 microdeletion, we also describe three index patients and two relatives with a smaller 500 kb microdeletion, including the CHRNA7 gene.

Tibial developmental field defect in valproic acid embryopathy: Report on three cases.

Prenatal exposure to valproic acid (VA) is associated with an increased risk of congenital malformations, especially limb defects like radial ray defects. Tibial developmental field defect in VA embryopathy remains exceptional. We report on three patients presenting with tibial hypo/aplasia associated with either femoral bifurcation or radial ray defect following prenatal exposure to VA. Femoral bifurcation and tibial agenesis has been described in the Gollop-Wolfgang complex and in the tibial agenesis-ectrodactyly syndrome. Tibial agenesis has also been reported in VACTERL association. The relation between prenatal exposure to VA and tibial agenesis is discussed.

Congenital skin pedicles with or without amniotic band sequence: Extending the human phenotype resembling mouse disorganization.

Congenital skin pedicles are very rare and usually described in association with multiple congenital anomalies. Here, we report on six patients with congenital pedicle skin hamartomatous lesions. Two patients showed a single skin pedicle lesion, one of whom was also shown to have 22q11.2 microdeletion syndrome, and four patients also had severe limb anomalies for which they were originally diagnosed with amniotic band sequence (ABS). We propose that all these infants instead show various forms of the phenotype resembling disorganization in the mouse. This article supports previous reports suggesting that "Disorganization-like" mutations may cause cases with apparent ABS. Owing to these reports, we propose the hypothesis that hamartomatous skin pedicles and "ABS plus" are different phenotypes of the human disorder resembling disorganization.

Autosomal dominant spondylocarpotarsal synostosis syndrome: phenotypic homogeneity and genetic heterogeneity.

Spondylocarpotarsal synostosis syndrome (SCT) (OMIM 272460), originally thought to be a failure of normal spine segmentation, is characterized by progressive fusion of vertebras and associates unsegmented bars, scoliosis, short stature, carpal and tarsal synostosis. Cleft palate, sensorineural or mixed hearing loss, joint limitation, clinodactyly, and dental enamel hypoplasia are variable manifestations. Twenty-five patients have been reported. Thirteen affected individuals were siblings from six families and four of these families were consanguineous. In four of those families, Krakow et al. [Krakow et al. (2004) Nat Genet 36:405-410] found homozygosity or compound heterozygosity for mutations in the gene encoding FLNB. This confirmed autosomal recessive inheritance of the disorder. We report on two new patients (a mother and her son) representing the first case of autosomal dominant inheritance. These patients met the clinical and radiological criteria for SCT and did not present any features which could exclude this diagnosis. Molecular analysis failed to identify mutations in NOG and FLNB. SCT is therefore, genetically heterogeneous. Both dominant and autosomal recessive forms of inheritance should be considered during genetic counseling.

Optimization and evaluation of single-cell whole-genome multiple displacement amplification.

The scarcity of genomic DNA can be a limiting factor in some fields of genetic research. One of the methods developed to overcome this difficulty is whole genome amplification (WGA). Recently, multiple displacement amplification (MDA) has proved very efficient in the WGA of small DNA samples and pools of cells, the reaction being catalyzed by the phi29 or the Bst DNA polymerases. The aim of the present study was to develop a reliable, efficient, and fast protocol for MDA at the single-cell level. We first compared the efficiency of phi29 and Bst polymerases on DNA samples and single cells. The phi29 polymerase generated accurately, in a short time and from a single cell, sufficient DNA for a large set of tests, whereas the Bst enzyme showed a low efficiency and a high error rate. A single-cell protocol was optimized using the phi29 polymerase and was evaluated on 60 single cells; the DNA obtained DNA was assessed by 22 locus-specific PCRs. This new protocol can be useful for many applications involving minute quantities of starting material, such as forensic DNA analysis, prenatal and preimplantation genetic diagnosis, or cancer research.

Detection of genomic imbalances by array based comparative genomic hybridisation in fetuses with multiple malformations.

BACKGROUND: Malformations are a major cause of morbidity and mortality in full term infants and genomic imbalances are a significant component of their aetiology. However, the causes of defects in many patients with multiple congenital malformations remain unexplained despite thorough clinical examination and laboratory investigations. METHODS: We used a commercially available array based comparative genomic hybridisation method (array CGH), able to screen all subtelomeric regions, main microdeletion syndromes, and 201 other regions covering the genome, to detect submicroscopic chromosomal imbalances in 49 fetuses with three or more significant anomalies and normal karyotype. RESULTS: Array CGH identified eight genomic rearrangements (16.3%), all confirmed by quantitative multiplex PCR of short fluorescent fragments. Subtelomeric and interstitial deletions, submicroscopic duplications, and a complex genomic imbalance were identified. In four de novo cases (15qtel deletion, 16q23.1-q23.3 deletion, 22q11.2 deletion, and mosaicism for a rearranged chromosome 18), the genomic imbalance identified clearly underlay the pathological phenotype. In one case, the relationship between the genotype and phenotype was unclear, since a subtelomeric 6q deletion was detected in a mother and her two fetuses bearing multiple malformations. In three cases, a subtelomeric 10q duplication, probably a genomic polymorphism, was identified. CONCLUSIONS: The detection of 5/49 causative chromosomal imbalances (or 4/49 if the 6qtel deletion is not considered as causative) suggests wide genome screening when standard chromosome analysis is normal and confirms that array CGH will have a major impact on pre and postnatal diagnosis as well as providing information for more accurate genetic counselling.

The SOX10 transcription factor: evaluation as a candidate gene for central and peripheral hereditary myelin disorders.

The SOX10 transcription factor is involved in development of neural crest derivatives and fate determination in glial cells. SOX10 mutations have been found in patients with intestinal aganglionosis and depigmentation with deafness (Waardenburg-Hirschsprung). Associated neurological signs have been reported in some cases, including a patient exhibiting a central and peripheral myelin deficiency. Therefore, we screened for SOX10 mutations in a large cohort of patients with peripheral and central myelin disorders. 56 were affected by classical demyelinating Charcot-Marie-Tooth disease without identified mutations in the genes encoding PNS myelin proteins (PMP22, P0), connexin 32 and the zinc-finger transcription factor, EGR2. 88 patients with undetermined leukodystrophy were selected from a large European prospective study. Associated clinical, magnetic resonance imaging and electrophysiological signs were consistent with a defect in CNS myelination in 83 and with an active degeneration of the CNS myelin in 5. No abnormalities in the proteolipid protein gene (PLP) were found. The absence of SOX100 mutation in this large cohort of patients suggests that this gene is not frequently involved in peripheral or central inherited myelin disorders.

Distinct phenotype of PHF6 deletions in females.

We report on two female patients carrying small overlapping Xq26.2 deletions of 100 kb and 270 kb involving the PHF6 gene. Mutations in PHF6 have been reported in individuals with Borjeson-Forssman-Lehmann syndrome, a condition present almost exclusively in males. Two very recent papers revealed de novo PHF6 defects in seven female patients with intellectual disability and a phenotype resembling Coffin-Siris syndrome (sparse hair, bitemporal narrowing, arched eyebrows, synophrys, high nasal root, bulbous nasal tip, marked clinodactyly with the hypoplastic terminal phalanges of the fifth fingers and cutaneous syndactyly of the toes, Blaschkoid linear skin hyperpigmentation, dental anomalies and occasional major malformations). The clinical presentation of these patients overlaps completely with our first patient, who carries a germline deletion involving PHF6. The second patient has a mosaic deletion and presented with a very mild phenotype of PHF6 loss in females. Our report confirms that PHF6 loss in females results in a recognizable phenotype overlapping with Coffin-Siris syndrome and distinct from Borjeson-Forssman-Lehmann syndrome. We expand the clinical spectrum and provide the first summary of the recommended medical evaluation.

Complex chromosomal rearrangement and intracytoplasmic sperm injection: a case report.

Complex chromosomal rearrangements (CCRs) are rare events in human pathology and are usually considered to induce severe reproductive impairment by disturbing the meiotic process and producing unbalanced gametes responsible for high reproductive risk. One-third of all CCRs are familial and tend to implicate fewer breakpoints and fewer chromosomes than de novo cases. CCRs are rarely transmitted through spermatogenesis and are primarily ascertained by male infertility. We report a familial balanced CCR, with seven breakpoints involving three chromosomes, which was detected prenatally in a female fetus conceived after intracytoplasmic sperm injection (ICSI) in a couple initially thought to be a carrier of a paternal reciprocal translocation involving two chromosomal breakpoints. Fluorescent in-situ hybridization (FISH) was used to elucidate the complexity of this CCR. The karyotype of the female CCR carrier was balanced and determined as 46,XX.ish t(1;4)(q42;q32)(WCP1+, D1Z5+, WCP4+, D1S3738-, D4S2930+; WCP4+, D4Z1+, WCP1+, D4S2930-, D1S3738+), ins(1;11)(q41;q23q24)(WCP1+,WCP11+, D11S2071-, MLL+; WCP11+, D11S2071+, WCP1-, MLL-), ins(4;11)(q23;q14q23)(WCP4+,WCP11+; WCP11+,WCP4-). The same balanced CCR was confirmed in her oligozoospermic father. We report, to our knowledge, the first case of ICSI performed in an infertile male with CCR, resulting in a balanced CCR carrier female with a normal clinical follow-up at 4 years of age. This particular case stresses the point of the relevance and feasibility of ICSI procedure in cases of balanced CCRs.

Interaction among SOX10, PAX3 and MITF, three genes altered in Waardenburg syndrome.

Waardenburg syndrome (WS) is an autosomal dominant disorder with an incidence of 1 in 40 000 that manifests with sensorineural deafness and pigmentation defects. It is classified into four types depending on the presence or absence of additional symptoms. WS1 and WS3 are due to mutations in the PAX3 gene whereas some WS2 cases are associated with mutations in the microphthalmia-associated transcription factor (MITF) gene. The WS4 phenotype can result from mutations in the endothelin-B receptor gene (EDNRB), in the gene for its ligand, endothelin-3 (EDN3), or in the SOX10 gene. PAX3 has been shown to regulate MITF gene expression. The recent implication of SOX10 in WS4 prompted us to test whether this transcription factor, known to cooperate in vitro with PAX3, is also able to regulate expression from the MITF promoter. Here we show that SOX10, in synergy with PAX3, strongly activates MITF expression in transfection assays. Analyses revealed that PAX3 and SOX10 interact directly by binding to a proximal region of the MITF promoter containing binding sites for both factors. Moreover, SOX10 or PAX3 mutant proteins fail to transactivate this promoter, providing further evidence that the two genes act in concert to directly regulate expression of MITF. In situ hybridization experiments carried out in the dominant megacolon (DOM:) mouse, confirmed that SOX10 dysfunction impairs MITF: expression as well as melanocytic development and survival. These experiments, which demonstrate an interaction between three of the genes that are altered in WS, could explain the auditory-pigmentary symptoms of this disease.

Familial deafness, congenital heart defects, and posterior embryotoxon caused by cysteine substitution in the first epidermal-growth-factor-like domain of jagged 1.

In the present study, we report a kindred with hearing loss, congenital heart defects, and posterior embryotoxon, segregating as autosomal dominant traits. Six of seven available affected patients manifested mild-to-severe combined hearing loss, predominantly affecting middle frequencies. Two patients were diagnosed with vestibular pathology. All patients had congenital heart defects, including tetralogy of Fallot, ventricular septal defect, or isolated peripheral pulmonic stenosis. No individual in this family met diagnostic criteria for any previously described clinical syndrome. A candidate-gene approach was undertaken and culminated in the identification of a novel Jagged 1 (JAG1) missense mutation (C234Y) in the first cysteine of the first epidermal-growth-factor-like repeat domain of the protein. JAG1 is a cell-surface ligand in the Notch signaling pathway. Mutations in JAG1 have been identified in patients with Alagille syndrome. Our findings revealed a unique phenotype with highly penetrant deafness, posterior embryotoxon, and congenital heart defects but with variable expressivity in a large kindred, which demonstrates that mutation in JAG1 can cause hearing loss.