Biallelic mutations in FLNB cause a skeletal dysplasia with 46,XY gonadal dysgenesis by activating ß-catenin.

Filamin B (FLNB) functions as a switch that can affect chrondrocyte development and endochondral bone formation through a series of signaling molecules and transcription factors that also affect Sertoli cell development. Here, we report a subject with a novel skeletal dysplasia and co-existing 46,XY gonadal dysgenesis and biallelic mutations in FLNB. Whole exome sequencing was performed to identify mutations. Quantitative polymerase chain reaction (qPCR) and flow variant assays were performed to quantify RNA, proteins and phosphorylated proteins. The TOPFLASH reporter was performed to quantify ß-catenin activity. Mutations were identified in the FLNB gene (FLNB:p.F964L, FLNB:p.A1577V). These mutations increased binding of FLNB protein to the MAP3K1 and RAC1 signal transduction complex and activated ß-catenin and had different effects on phosphorylation of MAP kinase pathway intermediates and SOX9 expression. Direct activation of ß-catenin through the FLNB-MAP3K1-RAC1 complex by FLNB mutations is a novel mechanism for causing 46,XY gonadal dysgenesis. The mechanism of action varies from those reported previously for loss of function mutations in SOX9 and gain-of-function mutations in MAP3K1.

Bioinformatics in otolaryngology research. Part two: other high-throughput platforms in genomics and epigenetics.

OBJECTIVES: This second segment of the two-part review summarises several modern high-throughput methods in genomics, epigenetics and molecular biology. Many principles from nucleotide sequencing and transcriptomics can be applied to other high-throughput molecular biology techniques. Specifically, this manuscript reviews: array comparative genome hybridisation; single nucleotide polymorphism arrays; microarray technology, used to study epigenetics; and methodology applied in proteomics. Finally, the review describes current methods for the integration of multiple molecular biology platforms. CONCLUSION: Progress in treating human disease in general will require close collaboration with experts in bioinformatics. Improved understanding, by clinicians and physician-scientists in our field, of the concepts presented in both parts of this review will advance diagnosis and therapy for diseases of the head and neck.

Bioinformatics in otolaryngology research. Part one: concepts in DNA sequencing and gene expression analysis.

BACKGROUND: Advances in high-throughput molecular biology, genomics and epigenetics, coupled with exponential increases in computing power and data storage, have led to a new era in biological research and information. Bioinformatics, the discipline devoted to storing, analysing and interpreting large volumes of biological data, has become a crucial component of modern biomedical research. Research in otolaryngology has evolved along with these advances. OBJECTIVES: This review highlights several modern high-throughput research methods, and focuses on the bioinformatics principles necessary to carry out such studies. Several examples from recent literature pertinent to otolaryngology are provided. The review is divided into two parts; this first part discusses the bioinformatics approaches applied in nucleotide sequencing and gene expression analysis. CONCLUSION: This paper demonstrates how high-throughput nucleotide sequencing and transcriptomics are changing biology and medicine, and describes how these changes are affecting otorhinolaryngology. Sound bioinformatics approaches are required to obtain useful information from the vast new sources of data.

Prostate cancer susceptibility genes on 8p21-23 in a Dutch population.

BACKGROUND: Prostate cancer is the most commonly diagnosed cancer in men in Europe and the United States. Numerous studies have indicated genetics to have a major role in the aetiology of this disease; as much as 42% of the risk may be explained by heritable factors. Genome-wide association studies have detected an association between prostate cancer and chromosome 8p21-23. In this study, we analysed eight microsatellite (MS) markers in that region in order to confirm previous results and narrow down the location of candidate prostate cancer genes. METHODS: 292 cases and 278 controls were selected from the Netherlands Cohort Study (NLCS). The following MSs were used in the analyses: D8S136, D8S1734, D8S1742, D8S261, D8S262, D8S351, D8S511 and D8S520. Associations were evaluated using a χ(2) test and logistic regression. We checked for any effects on the association by tumour stage. RESULTS: Associations that were found confirmed previous research that pointed to the 8p21-23 region. Two MSs: D8S136 (odds ratio (OR), 0.69; P=4.00 × 10(-28)), and D8S520 (OR, 0.80; P=3.37 × 10(-11)), were consistently and strongly related with prostate cancer. Genotype analysis showed an additive effect for D8S136 (P-trend=6.22 × 10(-03)) and D8S520 (P-trend=2.62 × 10(-22)), suggesting an increased risk for people with a short number of repeats on both alleles at those markers. CONCLUSIONS: This study provides strong evidence that the 8p21-23 region is likely to harbour prostate cancer genes.

Rapidly screening variants of uncertain significance in the MAP3K1 gene for phenotypic effects.

DNA sequencing of candidate genes or whole exomes on a diagnostic or investigational basis will yield a plethora of variants of uncertain significance whose potential phenotypic roles cannot be readily demonstrated by prediction programs, SNP databases nor conventional genetic analysis. Many variants may produce phenotypic changes in the encoded proteins by affecting the quantity, post-translational modification or protein interactions. Here, we establish the application of the method of flow cytometry following immunoprecipitation to show that known protein interactions are altered in the B-lymphoblastoid cells of patients with 46,XY gonadal dysgenesis arising from mutations in the MAP3K1 gene. This method can be scaled readily to test multiple interactions for many variants simultaneously from available tissues as well as quantify the effects of variants on protein accumulation and post-translational modification, thus providing an efficient means for screening variants of uncertain significance for phenotypic effects.

The impact of Converso Jews on the genomes of modern Latin Americans.

Modern day Latin America resulted from the encounter of Europeans with the indigenous peoples of the Americas in 1492, followed by waves of migration from Europe and Africa. As a result, the genomic structure of present day Latin Americans was determined both by the genetic structure of the founding populations and the numbers of migrants from these different populations. Here, we analyzed DNA collected from two well-established communities in Colorado (33 unrelated individuals) and Ecuador (20 unrelated individuals) with a measurable prevalence of the BRCA1 c.185delAG and the GHR c.E180 mutations, respectively, using Affymetrix Genome-wide Human SNP 6.0 arrays to identify their ancestry. These mutations are thought to have been brought to these communities by Sephardic Jewish progenitors. Principal component analysis and clustering methods were employed to determine the genome-wide patterns of continental ancestry within both populations using single nucleotide polymorphisms, complemented by determination of Y-chromosomal and mitochondrial DNA haplotypes. When examining the presumed European component of these two communities, we demonstrate enrichment for Sephardic Jewish ancestry not only for these mutations, but also for other segments as well. Although comparison of both groups to a reference Hispanic/Latino population of Mexicans demonstrated proximity and similarity to other modern day communities derived from a European and Native American two-way admixture, identity-by-descent and Y-chromosome mapping demonstrated signatures of Sephardim in both communities. These findings are consistent with historical accounts of Jewish migration from the realms that comprise modern Spain and Portugal during the Age of Discovery. More importantly, they provide a rationale for the occurrence of mutations typically associated with the Jewish Diaspora in Latin American communities.

The genetics of mitral valve prolapse.

Mitral valve prolapse (MVP) is a very common clinical condition that refers to a systolic billowing of one or both mitral valve leaflets into the left atrium. Improvements of echocardiographic techniques and new insights in mitral valve anatomy and physiology have rendered the diagnosis of this condition more accurate and reliable. MVP can be sporadic or familial, demonstrating autosomal dominant and X-linked inheritance. Three different loci on chromosomes 16, 11 and 13 have been found to be linked to MVP, but no specific gene has been described. Another locus on chromosome X was found to cosegregate with a rare form of MVP called 'X-linked myxomatous valvular dystrophy'. MVP is more frequent in patients with connective tissue disorders including Marfan syndrome, Ehlers-Danlos and osteogenesis imperfecta. The purpose of this review is to describe previous studies on the genetics and prevalence of MVP. The report warrants the need for further genetically based studies on this common, albeit not fully understood, clinical entity.

A cellular study of human testis development.

This study catalogs the cellular events underlying the formation of a human testis. These events were identified by immunocytochemistry using antibodies that served as markers for specific cell types, then contrasted with the events occurring in the developing mouse testis. The presence of germ cells in the embryonic gonadal ridge and of coelomic epithelial cells that give rise to Sertoli cells was observed at 7 weeks. This was followed by the appearance of Sertoli cells in testicular tubules and of Leydig cells at 9 weeks and by the appearance of vascular endothelial cells and peritubular myoid cells at 12 weeks. Overall the temporal sequence of events in humans was similar, albeit longer, than what occurs in mice. Notably, Leydig cell differentiation occurs earlier in the sequence of events and germ cell maturation occurs during fetal life. The candidate testis-determining genes, FGF9, GATA4, FOG2, EMX2, and CBX2 were expressed at 7 weeks suggesting a role in early gonadal development, such as that observed in mice. In addition, expression of FGF9 in germ cells following testis determination suggests a role in germ cell maturation.

Human embryo and early fetus research.

Studies of human embryos and fetuses have highlighted developmental differences between humans and model organisms. In addition to describing the normal biology of our own species, a justification in itself, studies of early human development have aided identification of candidate disease genes mapped by positional cloning strategies, understanding pathophysiology, where human disorders are not faithfully reproduced by models in other species, and, more recently, potential therapies based on human embryonic stem and embryonic germ cells. In this article, we review these applications. We also discuss when and how to study human embryo and early fetuses and some of the regulations of this research.

Will the new cytogenetics replace the old cytogenetics?

With the advent of array-based comparative genomic hybridization technology, the analog cytogenetic analysis that has been used for the past 100 years could be replaced by the quantitative, microarray-based molecular analysis. Major advantages of the new array-based cytogenetic technologies are the high resolution and the high throughput. This technology is the first to offer an autonomous whole-chromosome analysis in one hybridization reaction for the detection of submicroscopic gains/losses. However, as with any new technology, it needs to be validated with regard to its performance in various applications (e.g. clinical genetic testing and cancer applications), comparative cost, and the data interpretation.

Genetic variants in SOD2, MPO, and NQO1, and risk of ovarian cancer.

OBJECTIVE: One way in which parity and use of oral contraceptives may protect against ovarian cancer is by preventing inflammation and oxidative stress associated with ovulation. Since the genes superoxide dismutase (SOD2), myeloperoxidase (MPO), and NAD(P)H:quinone oxidoreductase 1 (NQO1) are involved in inflammation and oxidative stress, we investigated whether variants of these genes are associated with risk of ovarian cancer. METHODS: In a hospital-based case-control study, we compared 125 cases and 193 controls with respect to prevalence of (1) the T-->C (val-->ala) substitution at the -9 position in the signal sequence of SOD2; (2) the G-->A substitution at the -463 position in the promoter region of MPO; and (3) the C-->T (pro-->ser) change in exon 6 of NQO1. Genotyping was done using PCR and gel electrophoresis for MPO and NQO1 and using MALDI-TOF mass spectrometry for SOD2. RESULTS: For SOD2, women with the TC (val/ala) or CC (ala/ala) genotypes were at increased risk [odds ratio (OR) 2.1, 95% confidence interval (CI) 1.1-4.0]. Results for MPO and NQO1 were in the hypothesized directions but were not statistically significant. For MPO, there was a small inverse association among women with GA or AA genotypes (OR = 0.72, 95% CI 0.43-1.2). For NQO1, the TT (ser/ser) genotype was associated with somewhat increased risk (OR = 2.3, 95% CI 0.69-7.6). CONCLUSIONS: While these results need to be confirmed in other studies, they point to a possible role for genes involved in oxidative stress in the development of ovarian cancer.

The frequency of GJB2 and GJB6 mutations in the New York State newborn population: feasibility of genetic screening for hearing defects.

In the US, approximately one in every 1000 children has hearing loss sufficiently severe to interfere with the acquisition of normal speech [Ann NY Acad Sci 630 (1991) 16]. The causes of non-syndromic hearing loss (NSHL) are known to be heterogeneous, with genetic factors accounting for 50-75%[Am J Med Genet 46 (1993) 486]. Often individuals with NSHL thought to be caused by mutations in GJB2 have only one detectable mutant allele [Am J Hum Genet 62 (1998) 792, Hum Mol Genet 6 (12) (1997) 2173]. Another gene that has been identified as a possible cause of NSHL is GJB6 that codes for the gap junction protein, connexin 30. A consecutive series of anonymous newborn dried blood specimens (n = 2089) was tested for two GJB2 mutations: (i) 35delG, a pan-ethnic mutation; and (ii) 167delT, a mutation more frequently found in individuals of Ashkenazi Jewish and Mediterranean descents. Mutation detection was validated using allele-specific oligonucleotide hybridization in single wells. Once the positive samples had been identified, the samples were pooled and retested. All positives in the individual experiment were correctly identified in the pooled experiment. The same random set of anonymous newborn dried blood specimens plus some additional samples were tested (n = 2112) for the 342-kb deletion in the GJB6 gene.

Mapping a gene for 46,XY gonadal dysgenesis by linkage analysis.

46,XY gonadal dysgenesis was transmitted as an autosomal-dominant trait in a large family with multiple affected members. Expressivity of the trait was highly variable, ranging from pure to partial gonadal dysgenesis associated with normal female genitalia or sexual ambiguity, to mild hypospadias in otherwise normal males. The phenotypic features of this trait appeared to be confined to the genitourinary system. Multipoint parametric analysis using markers D5S664, D5S633, and D5D2102 yielded an LOD score of 4.47, assuming sex-limited, autosomal-dominant inheritance with a penetrance of 0.6. Because mutation in testis-determining genes leads to gonadal dysgenesis in 46,XY individuals, we postulate that the gene mapped by this study normally plays a role in gonadal differentiation.

Genetic counseling for prostate cancer risk.

Major risk factors for developing prostate cancer, including positive family history and African-American ethnicity, can be quantified for genetic counseling. Factors increasing familial risk for prostate cancer are closer degree of kinship, number of affected relatives, and early age of onset (< 50 years) among the affected relatives. Genetic testing may be useful for modification of risk, but currently should be performed only within the context of a well-designed research study that will determine penetrance and genotype-phenotype correlation of specific mutations. Even in the absence of genetic testing, African-American men and men with a strong family history of prostate cancer may opt to initiate screening by prostate specific antigen (PSA) and digital rectal exam (DRE) screening at age 40.

Identifying genes for male sex determination in humans.

The convergence of genetic and molecular technologies has led to the identification of a number of genes for male sex determination. The observation of chromosomal translocations, deletions, and duplications in sex reversed individuals was instrumental for the positional cloning of SRY, SOX9, WT1, and DAX1. Cloning by protein-DNA interaction was required for the identification of SF1. The observation of an extended phenotype for the alpha thalassemia-mental retardation syndrome assigned a role for XH2 in the testicular determining process. Over the next several years, new sex determining genes will be identified by linkage analysis in large families with multiple sex reversed members, comparative genomic hybridization of sex reversed individuals, and database searches for genes that encode interacting proteins or paralogs of other species. Given the apparent differences in the sex determining mechanisms of even closely related species, the roles of all of these genes will require confirmation by demonstrating expression in human gonadal ridge at the critical time, and that mutations result in sex reversal.

A genetic profile of contemporary Jewish populations.

The Jews are an ancient people with a history spanning several millennia. Genetic studies over the past 50 years have shed light on Jewish origins, the relatedness of Jewish communities and the genetic basis of Mendelian disorders among Jewish peoples. In turn, these observations have been used to develop genetic testing programmes and, more recently, to attempt to discover new genes for susceptibility to common diseases.

Invited review: sex-based differences in gene expression.

Certain diseases are more prevalent among women than men. The reasons for this increased prevalence are unknown, but there could be a genetic basis. Increased expression of X-linked genes in females, protective effects of Y-linked genes in males, or sex-limited gene expression that is developmentally or hormonally regulated could all account for these differences. Analysis of individuals with and without genetic sex reversal provides a means for distinguishing between genetic and hormonal causes. This can be complemented by genetic linkage and gene expression profiling to aid in the identification of candidate genes.

Sex determination: lessons from families and embryos.

Genetic studies in familial cases of sex reversal and in human embryos have contributed to the understanding of human sex determination and its disorders. For some heritable disorders of sex reversal, the gonadal phenotype was frequently overlooked until sex reversal was discovered fortuitously by chromosome analysis, often resulting in preventable complications. Within families, the phenotypes are variable and, in some instances, these can be explained by known genetic mechanisms. When a novel molecular marker is shared by family members affected with sex reversal, the level of confidence is higher that this marker may play a role in the development of the phenotype. The identification of pedigrees with sufficient power to generate significant linkage of disorder (lod) scores from genomewide screens can now lead to the identification of novel sex-determining genes. Studies of the gonads of 46,XY human embryos have shown that SOX9 expression follows a pattern similar to that of SRY and, in both instances, stands in contrast to the expression observed in the mouse. Differences between human and mouse embryonic gonads have also been observed for the temporal expression of DAX1, suggesting that the mechanisms of action of SRY, SOX9, and DAX1 may vary between these and other species.

Jewish and Middle Eastern non-Jewish populations share a common pool of Y-chromosome biallelic haplotypes.

Haplotypes constructed from Y-chromosome markers were used to trace the paternal origins of the Jewish Diaspora. A set of 18 biallelic polymorphisms was genotyped in 1,371 males from 29 populations, including 7 Jewish (Ashkenazi, Roman, North African, Kurdish, Near Eastern, Yemenite, and Ethiopian) and 16 non-Jewish groups from similar geographic locations. The Jewish populations were characterized by a diverse set of 13 haplotypes that were also present in non-Jewish populations from Africa, Asia, and Europe. A series of analyses was performed to address whether modern Jewish Y-chromosome diversity derives mainly from a common Middle Eastern source population or from admixture with neighboring non-Jewish populations during and after the Diaspora. Despite their long-term residence in different countries and isolation from one another, most Jewish populations were not significantly different from one another at the genetic level. Admixture estimates suggested low levels of European Y-chromosome gene flow into Ashkenazi and Roman Jewish communities. A multidimensional scaling plot placed six of the seven Jewish populations in a relatively tight cluster that was interspersed with Middle Eastern non-Jewish populations, including Palestinians and Syrians. Pairwise differentiation tests further indicated that these Jewish and Middle Eastern non-Jewish populations were not statistically different. The results support the hypothesis that the paternal gene pools of Jewish communities from Europe, North Africa, and the Middle East descended from a common Middle Eastern ancestral population, and suggest that most Jewish communities have remained relatively isolated from neighboring non-Jewish communities during and after the Diaspora.

SRY, SOX9, and DAX1 expression patterns during human sex determination and gonadal development.

SRY, SOX9, and DAX1 are key genes in human sex determination, by virtue of their associated male-to-female sex reversal phenotypes when mutated (SRY, SOX9) or over-expressed (DAX1). During human sex determination, SRY is expressed in 46,XY gonads coincident with sex cord formation, but also persists as nuclear protein within Sertoli cells at 18 weeks gestation. High-level SOX9 expression in the sex cords of the testis parallels that seen during mouse development, however in humans, SOX9 transcripts also are detected in the developing ovary. Low-level DAX1 expression predates peak SRY expression by at least 10 days, and persists in Sertoli cells throughout the entire sex determination period. In Dosage Sensitive Sex reversal, the anti-testis properties of DAX1 over-expression could act prior to the peak effects of SRY and continue during the period of SOX9 expression. These findings highlight expression differences for the SRY, SOX9, and DAX1 genes during sex determination in humans and mice. These results provide a direct framework for future investigation into the mechanisms underlying normal and abnormal human sex determination.