Non-coding variation in disorders of sex development.

Genetic studies in Disorders of Sex Development (DSD), representing a wide spectrum of developmental or functional conditions of the gonad, have mainly been oriented towards the coding genome. Application of genomic technologies, such as whole-exome sequencing, result in a molecular genetic diagnosis in ∼50% of cases with DSD. Many of the genes mutated in DSD encode transcription factors such as SRY, SOX9, NR5A1, and FOXL2, characterized by a strictly regulated spatiotemporal expression. Hence, it can be hypothesized that at least part of the missing genetic variation in DSD can be explained by non-coding mutations in regulatory elements that alter gene expression, either by reduced, mis- or overexpression of their target genes. In addition, structural variations such as translocations, deletions, duplications or inversions can affect the normal chromatin conformation by different mechanisms. Here, we review non-coding defects in human DSD phenotypes and in animal models. The wide variety of non-coding defects found in DSD emphasizes that the regulatory landscape of known and to be discovered DSD genes has to be taken into consideration when investigating the molecular pathogenesis of DSD.

Calcium and bone homeostasis in heterozygous carriers of CYP24A1 mutations: A cross-sectional study.

BACKGROUND: Bi-allelic CYP24A1 mutations can cause idiopathic infantile hypercalcemia (IIH), adult-onset nephrocalcinosis, and possibly bone metabolism disturbances. It is currently unclear if heterozygous carriers experience clinical problems or biochemical abnormalities. Our objective is to gain insight in the biochemical profile and health problems in CYP24A1 heterozygotes. STUDY DESIGN: Cross-sectional evaluation of participants. Data of previously reported carriers are reviewed. SETTING AND PARTICIPANTS: Outpatient clinic of a tertiary care hospital. Participants were eight family members of an infant with a well-characterized homozygous CYP24A1 mutation c.1186C>T p.(Arg396Trp). OUTCOMES: Serum vitamin D metabolites. Symptoms or biochemical signs of hypercalcemia, hypercalciuria or nephrocalcinosis. Bone health in heterozygous as compared to wild type (WT) subjects. MEASUREMENTS: Genotyping by Sanger sequencing; vitamin D metabolites by liquid chromatography tandem mass spectrometry; renal, calcium and bone markers by biochemical analyses; presence of nephrocalcinosis by renal ultrasound; bone health by dual-energy X-ray absorptiometry and peripheral quantitative computed tomography. RESULTS: Six participants were heterozygous carriers of the mutation. None of the heterozygous subjects had experienced IIH. One had a documented history of nephrolithiasis, two others had complaints compatible with this diagnosis. No major differences between WT and heterozygous subjects were found regarding bone health, serum or urinary calcium or 25OHD/24,25(OH)2D ratio. Literature reports on three out of 33 heterozygous cases suffering from IIH. In all three, the 25OHD/24,25(OH)2D ratio was highly elevated. Nephrocalcinosis was frequently reported in family members of IIH cases. LIMITATIONS: Small sample size, lack of a large control group. CONCLUSIONS: Our and literature data suggest that most heterozygous CYP24A1 mutation carriers have a normal 25OHD/24,25(OH)2D ratio, are usually asymptomatic and have a normal skeletal status but may possibly be at increased risk of nephrocalcinosis. A review of the available literature suggests that an elevated 25OHD/24,25(OH)2D ratio may be associated with symptoms of IHH, irrespective of carrier status.

FOXL2 copy number changes in the molecular pathogenesis of BPES: unique cohort of 17 deletions.

Blepharophimosis Syndrome (BPES) is an autosomal dominant developmental disorder of the eyelids with or without ovarian dysfunction caused by FOXL2 mutations. Overall, FOXL2deletions represent 12% of all genetic defects in BPES. Here, we have identified and characterized 16 new and one known FOXL2 deletion combining multiplex ligation-dependent probe amplification (MLPA), custom-made quantitative PCR (qPCR) and/or microarray-based copy number screening. The deletion breakpoints could be localized for 13 out of 17 deletions. The deletion size is highly variable (29.8 kb - 11.5 Mb), indicating absence of a recombination hotspot. Although the heterogeneity of their size and breakpoints is not reflected in the uniform BPES phenotype, there is considerable phenotypic variability regarding associated clinical findings including psychomotor retardation (8/17), microcephaly (6/17), and subtle skeletal features (2/17). In addition, in all females in whom ovarian function could be assessed, FOXL2 deletions proved to be associated with variable degrees of ovarian dysfunction. In conclusion, we present the largest series of BPES patients with FOXL2 deletions and standardized phenotyping reported so far. Our genotype-phenotype data can be useful for providing a prognosis (i.e. occurrence of associated features) in newborns with BPES carrying a FOXL2 deletion.

A common NYX mutation in Flemish patients with X linked CSNB.

AIMS: The Schubert-Bornschein type of complete congenital stationary night blindness (CSNB) is a genetically heterogeneous retinal disorder. It is characterised by a non-progressive disease course, often associated with high myopia and nystagmus. So far, mutations in two genes, NYX (nyctalopin) and GRM6 (metabotropic glutamate receptor 6) have been associated with this form of CSNB. The purpose of this study was to identify the genetic defect in affected male patients from Flemish families with complete CSNB. METHODS: Probands with CSNB from three large Flemish families underwent ophthalmological examination. DNA was extracted from peripheral blood, and the coding region of NYX along with parts of the 5'UTR and 3'UTR and intronic regions covering the splice sites were PCR amplified and sequenced. RESULTS: In the affected individuals of three Flemish families with the complete form of CSNB a novel NYX mutation, c.855delG was identified. This deletion is predicted to lead to a frameshift mutation, p.Asp286ThrfsX62 causing a premature stop codon. CONCLUSION: Previously, both single families with different mutations in NYX as well as different families with an identical mutation, suggestive of a founder mutation, have been described. The c.855delG deletion in NYX seems to be a common mutation associated with CSNB in the Flemish population from Belgium. Thus, we suggest performing diagnostic testing for CSNB in the Flemish population initially directed towards the identification of this mutation. Subsequent screening for other mutations in NYX or GRM6 could be performed as a second step.

A structured simple form for ordering genetic tests is needed to ensure coupling of clinical detail (phenotype) with DNA variants (genotype) to ensure utility in publication and databases.

Researchers and clinicians ideally need instant access to all the variation in their gene/locus of interest to efficiently conduct their research and genetic healthcare to the highest standards. Currently much key data resides in the laboratory books or patient records around the world, as there are many impediments to submitting this data. It would be ideal therefore if a semiautomated pathway was available, with a minimum of effort, to make the deidentified data publicly available for others to use. The Human Variome Project (HVP) meeting listed 96 recommendations to work toward this situation. This article is planned to initiate a strategy to enhance the collection of phenotype and genotype data from the clinician/diagnostic laboratory nexus. Thus, the aim is to develop universally applicable forms that people can use when investigating patients for each inherited disease, to assist in satisfying many of the recommendations of the HVP Meeting [Cotton et al., 2007]. We call for comment and collaboration in this article.

Deletions involving long-range conserved nongenic sequences upstream and downstream of FOXL2 as a novel disease-causing mechanism in blepharophimosis syndrome.

The expression of a gene requires not only a normal coding sequence but also intact regulatory regions, which can be located at large distances from the target genes, as demonstrated for an increasing number of developmental genes. In previous mutation studies of the role of FOXL2 in blepharophimosis syndrome (BPES), we identified intragenic mutations in 70% of our patients. Three translocation breakpoints upstream of FOXL2 in patients with BPES suggested a position effect. Here, we identified novel microdeletions outside of FOXL2 in cases of sporadic and familial BPES. Specifically, four rearrangements, with an overlap of 126 kb, are located 230 kb upstream of FOXL2, telomeric to the reported translocation breakpoints. Moreover, the shortest region of deletion overlap (SRO) contains several conserved nongenic sequences (CNGs) harboring putative transcription-factor binding sites and representing potential long-range cis-regulatory elements. Interestingly, the human region orthologous to the 12-kb sequence deleted in the polled intersex syndrome in goat, which is an animal model for BPES, is contained in this SRO, providing evidence of human-goat conservation of FOXL2 expression and of the mutational mechanism. Surprisingly, in a fifth family with BPES, one rearrangement was found downstream of FOXL2. In addition, we report nine novel rearrangements encompassing FOXL2 that range from partial gene deletions to submicroscopic deletions. Overall, genomic rearrangements encompassing or outside of FOXL2 account for 16% of all molecular defects found in our families with BPES. In summary, this is the first report of extragenic deletions in BPES, providing further evidence of potential long-range cis-regulatory elements regulating FOXL2 expression. It contributes to the enlarging group of developmental diseases caused by defective distant regulation of gene expression. Finally, we demonstrate that CNGs are candidate regions for genomic rearrangements in developmental genes.

A recurrent polyalanine expansion in the transcription factor FOXL2 induces extensive nuclear and cytoplasmic protein aggregation.

Blepharophimosis syndrome is an autosomal dominant disease characterised by eyelid malformations, associated or not with premature ovarian failure. It is caused by mutations in the FOXL2 gene, which encodes a forkhead transcription factor containing a polyalanine (polyAla) domain of 14 alanines. Expansions of the polyAla tract from 14 to 24 residues account for 30% of the reported mutations and lead mainly to isolated palpebral defects. We have transfected COS-7 cells with DNA constructs driving the expression of the wildtype and mutant FOXL2 proteins fused to the green fluorescent protein. The polyAla expansion was found to induce the formation of intranuclear aggregates and a mislocalisation of the protein due to extensive cytoplasmic aggregation. These findings were confirmed by immunofluorescence. Co-transfection experiments suggest that the wildtype and mutant proteins can co-aggregate. We propose that the mechanism for the molecular pathogenesis of the polyAla expansions of FOXL2 may be its mislocalisation concomitant with its inclusion into nuclear aggregates. This may diminish the pool of active protein. Potential effects of aggregation on cell viability are under study.

Structure, evolution and expression of the FOXL2 transcription unit.

FOXL2 is a putative transcription factor involved in ovarian development and function. Its mutations in humans are responsible for the blepharophimosis syndrome, characterized by eyelid malformations and premature ovarian failure (POF). Here we have performed a comparative sequence analysis of FOXL2 sequences of ten vertebrate species. We demonstrate that the entire open reading frame (ORF) is under purifying selection leading to strong protein conservation. We also review recent data on FOXL2 transcript and protein expression. FOXL2 has been shown 1) to be the earliest known sex dimorphic marker of ovarian determination/differentiation in vertebrates, 2) to have, at least in mammals, an ovarian expression persisting until adulthood. The conservation of its sequence and pattern of expression suggests that FOXL2 might be a key factor in the early development of the vertebrate female gonad and involved later in adult ovarian function. Finally, we provide arguments for the existence of an alternative transcript in rodents, that may arise from a differential polyadenylation. Although it has only been demonstrated in rodents, its presence/absence in other species deserves further investigation.

Spectrum of FOXL2 gene mutations in blepharophimosis-ptosis-epicanthus inversus (BPES) families demonstrates a genotype--phenotype correlation.

Mutations in FOXL2, a forkhead transcription factor gene, have recently been shown to cause blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) types I and II, a rare genetic disorder. In BPES type I a complex eyelid malformation is associated with premature ovarian failure (POF), whereas in BPES type II the eyelid defect occurs as an isolated entity. In this study, we describe the identification of novel mutations in the FOXL2 gene in BPES types I and II families, in sporadic BPES patients, and in BPES families where the type could not be established. In 67% of the patients studied, we identified a mutation in the FOXL2 gene. In total, 21 mutations (17 of which are novel) and one microdeletion were identified. Thirteen of these FOXL2 mutations are unique. In this study, we demonstrate that there is a genotype--phenotype correlation for either types of BPES by the finding that mutations predicted to result in a truncated protein either lacking or containing the forkhead domain lead to BPES type I. In contrast, duplications within or downstream of the forkhead domain, and a frameshift downstream of them, all predicted to result in an extended protein, cause BPES type II. In addition, in 30 unrelated patients with isolated POF no causal mutations were identified in FOXL2. Our study provides further evidence that FOXL2 haploinsufficiency may cause BPES types I and II by the effect of a null allele and a hypomorphic allele, respectively. Furthermore, we propose that in a fraction of the BPES patients the genetic defect does not reside within the coding region of the FOXL2 gene and may be caused by a position effect.

Cloning and characterization of human WDR10, a novel gene located at 3q21 encoding a WD-repeat protein that is highly expressed in pituitary and testis.

Members of the steroid-thyroid-retinoid receptor superfamily regulate a spectrum of cellular functions, including metabolism and growth and differentiation. We sought to isolate novel members of this family by using degenerate oligonucleotide primers directed to sequences encoding the AF-2 domain of these molecules in a PCR-based approach. The AF-2 domain serves a critical function in recruiting coregulatory molecules and in transcriptional activation. We report the cloning and initial characterization of a novel gene, WDR10, which encodes a 140-kD protein that is highly expressed in pituitary and testis. This protein, WDR10p, contains an AF-2 domain as well as seven N-terminal WD repeats and is highly conserved through evolution. Chromosomal localization studies placed WDR10 at 3q21, near a locus for the Moebius syndrome, Hailey-Hailey disease, and rhodopsin, which is involved in several forms of retinitis pigmentosa. The expression pattern of WDR10 and its chromosomal location makes this novel gene a candidate gene for the hypogonadism associated with some forms of retinitis pigmentosa and the Moebius syndrome.

Identification of BPESC1, a novel gene disrupted by a balanced chromosomal translocation, t(3;4)(q23;p15.2), in a patient with BPES.

The blepharophimosis syndrome (BPES) is a rare genetic disorder characterized by blepharophimosis, ptosis, epicanthus inversus, and telecanthus. In type I, BPES is associated with female infertility, while in type II, the eyelid defect occurs by itself. The BPES syndrome has been mapped to 3q23. Previously, we constructed a YAC-, PAC-, and cosmid-based physical map surrounding the 3q23 translocation breakpoint of a t(3;4)(q23;p15.2) BPES patient, containing a 110-kb PAC (169-C 10) and a 43-kb cosmid (11-L 10) spanning the breakpoint. In this report, we present the identification of BPESC1 (BPES candidate 1), a novel candidate gene that is disrupted by the translocation on chromosome 3. Cloning of the cDNA has been performed starting from a testis-specific EST, AI032396, found in cosmid 11-L 10. The cDNA sequence of BPESC1 is 3518 bp in size and contains an open reading frame of 351 bp. No significant similarities with known proteins have been found in the sequence databases. BPESC1 contains three exons and spans a genomic fragment of 17.5 kb. Expression of BPESC1 was observed in adult testis tissue. We performed mutation analysis in 28 unrelated familial and sporadic BPES patients, but, apart from the disruption by the translocation, found no other disease-causing mutations. These data make it unlikely that BPESC1 plays a major role in the pathogenesis of BPES.

Closing in on the BPES gene on 3q23: mapping of a de Novo reciprocal translocation t(3;4)(q23;p15.2) breakpoint within a 45-kb cosmid and mapping of three candidate genes, RBP1, RBP2, and beta'-COP, distal to the breakpoint.

BPES is a genetic disorder presenting with blepharophimosis, ptosis of the eyelids, epicanthus inversus, and telecanthus. BPES type I is associated with female infertility, whereas type II presents without additional symptoms. Hitherto, it remains unknown whether BPES type I results from a defect in a single gene or from a contiguous gene syndrome. Previous cytogenetic and linkage analyses have assigned a BPES locus to 3q23, in a 5-cM interval between D3S1615 and D3S1316. In this report, we describe the molecular and physical characterization of the 3q23 breakpoint in a BPES patient with a t(3;4)(q23;p15.2) translocation. Eight YACs located around and within the D3S1615-D3S1316 interval were mapped relative to the 3q23 breakpoint; 5 YACs spanning the 3q23 breakpoint were identified. Thirteen STSs and ESTs were localized on the YAC map. Subsequent hybridization of 2 YACs spanning the breakpoint to the Human RPCI1 PAC Library and the Human Chromosome 3 LLNL Cosmid Library resulted in the identification of 12 PACs and 50 cosmids respectively, allowing the construction of a detailed PAC and cosmid physical map. A refined position-telomeric to the breakpoint-of 3 candidate genes, cellular retinol-binding proteins 1 and 2 (RBP1, RBP2) and the coatomer beta' subunit (beta'-COP), was obtained on this physical map. Furthermore, a PAC and cosmid contig encompassing the breakpoint was constructed. PAC 169-C 10 and cosmid 11-L 10 crossing the breakpoint have sizes of 110 and 45 kb, respectively. The isolation of coding sequences in these clones and in the rest of the contig will greatly facilitate further efforts toward positional cloning of the gene(s) involved in BPES.

Real-life radiation burden to relatives of patients treated with iodine-131: a study in eight centres in Flanders (Belgium).

In view of the EURATOM 96/29 [1] regulations, a prospective multicentre study was performed to evaluate the present guidelines given to relatives of patients treated with iodine-131 for both thyroid carcinoma and thyrotoxicosis, based on the real-life radiation burden. This study comprised 166 measurements carried out on a group of 94 relatives of 65 patients. All relatives wore a thermoluminescent dosemeter (TLD) on the wrist for 7 days. Sixty-one relatives agreed to wear another TLD for an additional 7 days. TLD were placed on nine patients' bedside tables. The eight participating centres were arbitrarily divided into three groups according to the period of time they advised their patients to sleep separately. Groups I, II and III respectively advised their patients to sleep separately for 0, 7-10 and 14-21 days. The median dose received by in-living relatives of thyroid carcinoma patients during the 14 days following hospital discharge was 281 microSv (doses to infinity not calculated); the median dose to infinity received by in-living relatives of ambulatory treated thyrotoxicosis patients was 596 microSv, as compared with 802 microSv for in-living relatives of hospitalised thyrotoxicosis patients. In general the children of patients received a significantly (P < 0.1) lower mean dose than their partners. For thyroid carcinoma patients, only two relatives out of 19 (10%) exceeded the EURATOM 96/29 limit of 1 mSv/year. For thyrotoxic patients, 28% of relatives exceeded the EURATOM 96/29 limit, but none of them were relatives of patients who followed guidelines for 21 days. The results of this study indicate that sleeping separately for 7 days, after a period of hospitalisation of 2-3 days, will usually be sufficient for thyroid carcinoma patients. For thyrotoxicosis patients, up to 21 days of sleeping separately could be necessary in order to strictly abide by EURATOM 96/29. Therefore, the authors propose the implementation of a non-rigid dose constraint for people who "knowingly and willingly" help patients treated with 131I, while still following the ALARA principle.