Screening of autosomal gene deletions in patients with hypogonadotropic hypogonadism using multiplex ligation-dependent probe amplification: detection of a hemizygosis for the fibroblast growth factor receptor 1

P>Objective Congenital hypogonadotropic hypogonadism with anosmia (Kallmann syndrome) or with normal sense of smell is a heterogeneous genetic disorder caused by defects in the synthesis, secretion and action of gonadotrophin-releasing hormone (GnRH). Mutations involving autosomal genes have been identified in approximately 30% of all cases of hypogonadotropic hypogonadism. However, most studies that screened patients with hypogonadotropic hypogonadism for gene mutations did not include gene dosage methodologies. Therefore, it remains to be determined whether patients without detected point mutation carried a heterozygous deletion of one or more exons. Measurements We used the multiplex ligation-dependent probe amplification (MLPA) assay to evaluate the potential contribution of heterozygous deletions of FGFR1, GnRH1, GnRHR, GPR54 and NELF genes in the aetiology of GnRH deficiency. Patients We studied a mutation-negative cohort of 135 patients, 80 with Kallmann syndrome and 55 with normosmic hypogonadotropic hypogonadism. Results One large heterozygous deletion involving all FGFR1 exons was identified in a female patient with sporadic normosmic hypogonadotropic hypogonadism and mild dimorphisms as ogival palate and cavus foot. FGFR1 hemizygosity was confirmed by gene dosage with comparative multiplex and real-time PCRs. Conclusions FGFR1 or other autosomal gene deletion is a possible but very rare event and does not account for a significant number of sporadic or inherited cases of isolated GnRH deficiency.

An atypical 7q11.23 deletion in a normal IQ Williams-Beuren syndrome patient.

Williams-Beuren syndrome (WBS; OMIM no. 194050) is a multisystemic neurodevelopmental disorder caused by a hemizygous deletion of 1.55 Mb on chromosome 7q11.23 spanning 28 genes. Haploinsufficiency of the ELN gene was shown to be responsible for supravalvular aortic stenosis and generalized arteriopathy, whereas LIMK1, CLIP2, GTF2IRD1 and GTF2I genes were suggested to be linked to the specific cognitive profile and craniofacial features. These insights for genotype-phenotype correlations came from the molecular and clinical analysis of patients with atypical deletions and mice models. Here we report a patient showing mild WBS physical phenotype and normal IQ, who carries a shorter 1 Mb atypical deletion. This rearrangement does not include the GTF2IRD1 and GTF2I genes and only partially the BAZ1B gene. Our results are consistent with the hypothesis that hemizygosity of the GTF2IRD1 and GTF2I genes might be involved in the facial dysmorphisms and in the specific motor and cognitive deficits observed in WBS patients.

Mirror extreme BMI phenotypes associated with gene dosage at the chromosome 16p11.2 locus.

Both obesity and being underweight have been associated with increased mortality. Underweight, defined as a body mass index (BMI) ≤ 18.5 kg per m(2) in adults and ≤ -2 standard deviations from the mean in children, is the main sign of a series of heterogeneous clinical conditions including failure to thrive, feeding and eating disorder and/or anorexia nervosa. In contrast to obesity, few genetic variants underlying these clinical conditions have been reported. We previously showed that hemizygosity of a ∼600-kilobase (kb) region on the short arm of chromosome 16 causes a highly penetrant form of obesity that is often associated with hyperphagia and intellectual disabilities. Here we show that the corresponding reciprocal duplication is associated with being underweight. We identified 138 duplication carriers (including 132 novel cases and 108 unrelated carriers) from individuals clinically referred for developmental or intellectual disabilities (DD/ID) or psychiatric disorders, or recruited from population-based cohorts. These carriers show significantly reduced postnatal weight and BMI. Half of the boys younger than five years are underweight with a probable diagnosis of failure to thrive, whereas adult duplication carriers have an 8.3-fold increased risk of being clinically underweight. We observe a trend towards increased severity in males, as well as a depletion of male carriers among non-medically ascertained cases. These features are associated with an unusually high frequency of selective and restrictive eating behaviours and a significant reduction in head circumference. Each of the observed phenotypes is the converse of one reported in carriers of deletions at this locus. The phenotypes correlate with changes in transcript levels for genes mapping within the duplication but not in flanking regions. The reciprocal impact of these 16p11.2 copy-number variants indicates that severe obesity and being underweight could have mirror aetiologies, possibly through contrasting effects on energy balance.

Williams-Beuren syndrome TRIM50 encodes an E3 ubiquitin ligase.

Williams-Beuren syndrome (WBS) is a neurodevelopmental and multisystemic disease that results from hemizygosity of approximately 25 genes mapping to chromosomal region 7q11.23. We report here the preliminary description of eight novel genes mapping within the WBS critical region and/or its syntenic mouse region. Three of these genes, TRIM50, TRIM73 and TRIM74, belong to the TRIpartite motif gene family, members of which were shown to be associated to several human genetic diseases. We describe the preliminary functional characterization of these genes and show that Trim50 encodes an E3 ubiquitin ligase, opening the interesting hypothesis that the ubiquitin-mediated proteasome pathway might be involved in the WBS phenotype.

No complementation between TP53 or RB-1 and v-src in astrocytomas of GFAP-v-src transgenic mice.

Human low-grade astrocytomas frequently recur and progress to states of higher malignancy. During tumor progression TP53 alterations are among the first genetic changes, while derangement of the p16/p14ARF/RB-1 system occurs later. To probe the pathogenetic significance of TP53 and RB-1 alterations, we introduced a v-src transgene driven by glial fibrillary acidic protein (GFAP) regulatory elements (which causes preneoplastic astrocytic lesions and stochastically astrocytomas of varying degrees of malignancy) into TP53+/- or RB-1+/- mice. Hemizygosity for TP53 or RB-1 did not increase the incidence or shorten the latency of astrocytic tumors in GFAP-v-src mice over a period of up to 76 weeks. Single strand conformation analysis of exons 5 to 8 of non-ablated TP53 alleles revealed altered migration patterns in only 3/16 tumors analyzed. Wild-type RB-1 alleles were retained in all RB-1+/-GFAP-v-src mice-derived astrocytic tumors analyzed, and pRb immunostaining revealed protein expression in all tumors. Conversely, the GFAP-v-src transgene did not influence the development of extraneural tumors related to TP53 or RB-1 hemizygosity. Therefore, the present study indicates that neither loss of RB-1 nor of TP53 confer a growth advantage in vivo to preneoplastic astrocytes expressing v-src, and suggests that RB-1 and TP53 belong to one single complementation group along with v-src in this transgenic model of astrocytoma development. The stochastic development of astrocytic tumors in GFAP-v-src, TP53+/- GFAP-v-src, and RB-1+/- GFAP-v-src transgenic mice indicates that additional hitherto unknown genetic lesions of astrocytes contribute to tumorigenesis, whose elucidation may prove important for our understanding of astrocytoma initiation and progression.

Gene copy number and differential gene expression in haploid and diploid males of the stingless bee, Melipona quadrifasciata

Complementary sex determination in Hymenoptera implies that heterozygosity at the sex locus leads to the development of diploid females, whereas hemizygosity results in haploid males. Diploid males can arise through inbreeding. In social species, these pose a double burden on colony fitness, from significant reduction in its worker force and through being less viable and fertile than haploid males. Apart from being "misfits", diploid males are of interest to assess molecular correlates for possibly ploidy-related bionomic differences. Herein, we generated suppression subtractive cDNA libraries from newly emerged haploid and diploid males of the stingless bee Melipona quadrifasciata to enrich for differentially expressed genes. Gene Ontology classification revealed that in haploid males more DEGs were related to stress responsiveness, biosynthetic processes, reproductive processes and spermatogenesis, whereas in diploid ones differentially expressed genes were associated with cellular organization, nervous system development and amino acid transport were prevalent. Furthermore, both libraries contained over 40 % ESTs representing possibly novel transcripts. Quantitative RT-PCR analyses confirmed the differential expression of a representative DEG set in newly emerged males. Several muscle formation and energy metabolism-related genes were under-expressed in diploid males. On including 5-day-old males in the analysis, changes in transcript abundance during sexual maturation were revealed.

The Effects of Oxybutynin on Urinary Symptoms in Children with Williams-Beuren Syndrome

Purpose: Williams-Beuren syndrome is a genomic disorder caused by a hemizygous contiguous gene deletion on chromosome 7q11.23. Lower urinary tract symptoms are common in children with Williams-Beuren syndrome. However, there are few data on the management of voiding symptoms in this population. We report our experience using oxybutynin to treat urinary symptoms in children with Williams-Beuren syndrome. Materials and Methods: We prospectively analyzed 42 patients with Williams-Beuren syndrome and significant lower urinary tract symptoms due to detrusor overactivity diagnosed on urodynamics in a 12-week, open-label study. Urological assessment included symptomatic evaluation, the impact of lower urinary tract symptoms on quality of life, frequency-volume chart, urodynamics and urinary tract sonography. After 12 weeks of treatment with 0.6 mg/kg oxybutynin per day given in 3 daily doses, patients were assessed for treatment efficacy and side effects. Results: A total of 17 girls and 19 boys completed medical therapy and were assessed at 12 weeks. Mean +/- SD patient age was 9.2 +/- 4.3 years (range 3 to 18). The most common urinary complaint was urgency, which occurred in 31 patients (86.1%), followed by urge incontinence, which was seen in 29 (80.5%). Compared to baseline, urinary symptoms were substantially improved. The negative impact of storage symptoms on quality of life was significantly decreased from a mean +/- SD of 3.3 +/- 1.7 to 0.5 +/- 0.9 (p <0.001). Mean +/- SD maximum urinary flow improved from 14.2 +/- 15.0 to 20.5 +/- 6.4 ml per second (p <0.001). Conclusions: A total of 12 weeks of therapy with 0.6 mg/kg oxybutynin daily resulted in improvement of lower urinary tract symptoms, quality of life and maximum flow rate in most patients with Williams-Beuren syndrome.

Physical and functional mapping of a novel tumor suppressor locus for prostate cancer within chromosome 10p12.31-q11

Prostate cancer remains the second leading cause of male cancer deaths in the United States, yet the molecular mechanisms underlying this disease remain largely unknown. Cytogenetic and molecular analyses of prostate tumors suggest a consistent association with the loss of chromosome 10. Previously, we have defined a novel tumor suppressor locus PAC-1 within chromosome 10pter-q11. Introduction of the short arm of chromosome 10 into a prostatic adenocarcinoma cell line PC-3H resulted in dramatic tumor suppression and restoration of a programmed cell death pathway. Using a combined approach of comparative genomic hybridization and microsatellite analysis of PC-3H, I have identified a region of hemizygosity within 10p12-p15. This region has been shown to be involved in frequent loss of heterozygosity in gliomas and melanoma. To functionally dissect the region within chromosome 10p containing PAC-1, we developed a strategy of serial microcell fusion, a technique that allows the transfer of defined fragments of chromosome 10p into PC-3H. Serial microcell fusion was used to transfer defined 10p fragments into a mouse A9 fibrosarcoma cell line. Once characterized by FISH and microsatellite analyses, the 10p fragments were subsequently transferred into PC-3H to generate a panel of microcell hybrid clones containing overlapping deletions of chromosome 10p. In vivo and microsatellite analyses of these PC hybrids identified a small chromosome 10p fragment (an estimated 31 Mb in size inclusive of the centromere) that when transferred into the PC-3H background, resulted in significant tumor suppression and limited a region of functional tumor suppressor activity to chromosome 10p12.31-q11. This region coincides with a region of LOH demonstrated in prostate cancer. These studies demonstrate the utility of this approach as a powerful tool to limit regions of functional tumor suppressor activity. Furthermore, these data used in conjunction with data generated by the Human Genome Project lent a focused approach to identify candidate tumor suppressor genes involved in prostate cancer. ^