A likely HOXC4 predisposition variant for Chiari malformations.

OBJECTIVE: Inherited variants predisposing patients to type 1 or 1.5 Chiari malformation (CM) have been hypothesized but have proven difficult to confirm. The authors used a unique high-risk pedigree population resource and approach to identify rare candidate variants that likely predispose individuals to CM and protein structure prediction tools to identify pathogenicity mechanisms. METHODS: By using the Utah Population Database, the authors identified pedigrees with significantly increased numbers of members with CM diagnosis. From a separate DNA biorepository of 451 samples from CM patients and families, 32 CM patients belonging to 1 or more of 24 high-risk Chiari pedigrees were identified. Two high-risk pedigrees had 3 CM-affected relatives, and 22 pedigrees had 2 CM-affected relatives. To identify rare candidate predisposition gene variants, whole-exome sequence data from these 32 CM patients belonging to 24 CM-affected related pairs from high-risk pedigrees were analyzed. The I-TASSER package for protein structure prediction was used to predict the structures of both the wild-type and mutant proteins found here. RESULTS: Sequence analysis of the 24 affected relative pairs identified 38 rare candidate Chiari predisposition gene variants that were shared by at least 1 CM-affected pair from a high-risk pedigree. The authors found a candidate variant in HOXC4 that was shared by 2 CM-affected patients in 2 independent pedigrees. All 4 of these CM cases, 2 in each pedigree, exhibited a specific craniocervical bony phenotype defined by a clivoaxial angle less than 125°. The protein structure prediction results suggested that the mutation considered here may reduce the binding affinity of HOXC4 to DNA. CONCLUSIONS: Analysis of unique and powerful Utah genetic resources allowed identification of 38 strong candidate CM predisposition gene variants. These variants should be pursued in independent populations. One of the candidates, a rare HOXC4 variant, was identified in 2 high-risk CM pedigrees, with this variant possibly predisposing patients to a Chiari phenotype with craniocervical kyphosis.

Significant familial clustering of Peyronie's disease in close and distant relatives.

BACKGROUND: Peyronie's disease (PD) has previously been observed to co-aggregate in a small number of first-degree relative pairs (e.g., father-son). However, the familial aggregation of PD in more distant relatives, as well as the aggregation of Dupuytren's disease (DD) in probands and relatives, has not been thoroughly investigated. OBJECTIVE: This study explored the evidence for familial clustering of PD and DD in close and distant relatives. MATERIALS AND METHODS: The Utah Population Database, which includes genealogy information linked to electronic medical records (available since 1995), was used to identify men and their relatives with PD and DD based on ICD9/10 codes. All cases were required to have high-quality genealogy data. We estimated relative risk (RR) of PD and DD in first- through fifth-degree relatives compared to matched population rates of disease. We also investigated the average relatedness of cases compared to the average relatedness of sets of matched controls. Outcome measures include estimation of relative risk and excessive relatedness as measured by a Genealogical Index of Familiality (GIF) analysis. RESULTS: We analyzed 307 individuals with PD, and their first- through fifth-degree relatives. Approximately 0.12% of the population had PD, 95% of these were diagnosed over the age of 30 years (age range: 10-92 years), and 1.3% of PD probands had a comorbid diagnosis of DD. RR estimates for PD were significant for first- and fifth-degree relatives. RR estimates for DD were significant only for probands. The average relatedness of cases was significantly greater than matched controls, even after removing first- and second-degree relatives. We also found that 74.9% of identified PD probands belonged to pedigrees with a statistical excess of PD. CONCLUSION: Despite the low prevalence of PD in our healthcare records, the results provide evidence that support a genetic contribution to at least a subset of PD cases.

Adiposity and Endometriosis Severity and Typology.

STUDY OBJECTIVE: Prior research has collectively shown that endometriosis is inversely related to women's adiposity. The aim of this study was to assess whether this inverse relationship holds true by disease severity and typology. DESIGN: Cross-sectional study among women with no prior diagnosis of endometriosis. SETTING: Fourteen clinical centers in Salt Lake City, UT, and San Francisco, CA. PATIENTS: A total of 495 women (of which 473 were analyzed), aged 18-44 years, were enrolled in the operative cohort of the Endometriosis, Natural History, Diagnosis, and Outcomes (ENDO) Study. INTERVENTIONS: Gynecologic laparoscopy/laparotomy regardless of clinical indication. MEASUREMENTS AND MAIN RESULTS: Participants underwent anthropometric assessments, body composition measurements, and evaluations of body fat distribution ratios before surgery. Surgeons completed a standardized operative report immediately after surgery to capture revised American Society for Reproductive Medicine staging (I-IV) and typology of disease (superficial endometriosis [SE], ovarian endometrioma [OE], and deep infiltrating endometriosis [DIE]). Linear mixed models, taking into account within-clinical-center correlation, were used to generate least square means (95% confidence intervals) to assess differences in adiposity measures by endometriosis stage (no endometriosis, I-IV) and typology (no endometriosis, SE, DIE, OE, OE + DIE) adjusting for age, race/ethnicity, and parity. Although most confidence intervals were wide and overlapping, 3 general impressions emerged: (1) women with incident endometriosis had the lowest anthropometric/body composition indicators compared with those without incident endometriosis, (2) women with stage I or IV endometriosis had lower indicators compared with women with stage II or III, and (3) women with OE and/or DIE tended to have the lowest indicators, whereas women with SE had the highest indicators. CONCLUSION: Our research highlights that the relationship between women's adiposity and endometriosis severity and typology may be more complicated than prior research indicates.

Familiality analysis of provoked vestibulodynia treated by vestibulectomy supports genetic predisposition.

BACKGROUND: Provoked vestibulodynia is a poorly understood disease that affects 8-15% of women in their lifetime. There is significant inflammation and nerve growth in vestibular biopsies from affected women treated by vestibulectomy compared with matched female population controls without vestibulodynia. The triggers leading to this neurogenic inflammation are unknown, but they are likely multifactorial. OBJECTIVE: Our objective was to determine whether vestibulodynia is more common in close and distantly related female relatives of women diagnosed with the disease and those specifically treated by vestibulectomy. Excess familial clustering would support a potential genetic predisposition for vestibulodynia and warrant further studies to isolate risk alleles. STUDY DESIGN: Using population-based genealogy linked to University of Utah Hospital CPT coded data, we estimated the relative risk of vestibulectomy in female relatives of affected women. We also compared the average pairwise relatedness of cases to the expected relatedness of the population and identified high-disease-burden pedigrees. RESULTS: A total of 183 potential vestibulectomy probands were identified using CPT codes. The relative risk of vestibulectomy was elevated in first-degree (20 [6.6-47], P < .00001), second-degree (4.5 [0.5-16], P = .07), and third-degree female relatives (3.4 [1.2-8.8], P = .03). Seventy of these 183 CPT-based probands had available clinical history to confirm a diagnosis of moderate to severe vestibulodynia. Notably, this smaller group of confirmed probands (n = 70) revealed a similar familiality in first-degree (54 [17.5-126], P < .00001), second-degree (19.7 [2.4-71], P = .005), and third-degree relatives (12 [3.3-31], P = .0004), despite less statistical power for analysis. Overall, the average pairwise relatedness of affected women was significantly higher than expected (P < .001) and a number of high-disease-burden Utah families were identified. CONCLUSION: Our data suggest that vestibulodynia treated by vestibulectomy has a genetic predisposition. Future studies will identify candidate genes by linkage analysis in affected families and sequencing of distantly related probands.

Phenotyping clinical disorders: lessons learned from pelvic organ prolapse.

Genetic epidemiology, the study of genetic contributions to risk for disease, is an innovative area in medicine. Although research in this arena has advanced in other disciplines, few genetic epidemiological studies have been conducted in obstetrics and gynecology. It is crucial that we study the genetic susceptibility for issues in women's health because this information will shape the new frontier of personalized medicine. To date, preterm birth may be one of the best examples of genetic susceptibility in obstetrics and gynecology, but many areas are being evaluated including endometriosis, fibroids, polycystic ovarian syndrome, and pelvic floor disorders. An essential component to genetic epidemiological studies is to characterize, or phenotype, the disorder to identify genetic effects. Given the growing importance of genomics and genetic epidemiology, we discuss the importance of accurate phenotyping of clinical disorders and highlight critical considerations and opportunities in phenotyping, using pelvic organ prolapse as a clinical example.

Mutations in TFIIIA that increase stability of the TFIIIA-5 S rRNA gene complex: unusual effects on the kinetics of complex assembly and dissociation.

We have identified four mutations in Xenopus TFIIIA that increase the stability of TFIIIA-5 S rRNA gene complexes. In each case, the mutation has a relatively modest effect on equilibrium binding affinity. In three cases, these equilibrium binding effects can be ascribed primarily to decreases in the rate constant for protein-DNA complex dissociation. In the fourth case, however, a substitution of phenylalanine for the wild-type leucine at position 148 in TFIIIA results in much larger compensating changes in the kinetics of complex assembly and dissociation. The data support a model in which a relatively unstable population of complexes with multi-component dissociation kinetics forms rapidly; complexes then undergo a slow conformational change that results in very stable, kinetically homogeneous TFIIIA-DNA complexes. The L148F mutant protein acts as a particularly potent transcriptional activator when it is fused to the VP16 activation domain and expressed in yeast cells. Substitution of L148 to tyrosine or tryptophan produces an equally strong transcriptional activator. Substitution to histidine results in genetic and biochemical effects that are more modest than, but similar to, those observed with the L148F mutation. We propose that an amino acid with a planar side chain at position 148 can intercalate between adjacent base pairs in the intermediate element of the 5 S rRNA gene. Intercalation occurs slowly but results in a very stable DNA-protein complex. These results suggest that transcriptional activation by a cis-acting sequence element is largely dependent on the kinetic, rather than the thermodynamic, stability of the complex formed with an activator protein. Thus, transcriptional activation is dependent in large part on the lifetime of the activator-DNA complex rather than on binding site occupancy at steady state. Introduction of intercalating amino acids into zinc finger proteins may be a useful tool for producing artificial transcription factors with particularly high in vivo activity.

A C-terminal fragment of an intron-encoded maturase is sufficient for promoting group I intron splicing.

Group I introns often encode proteins that catalyze site-specific DNA hydrolysis. Some of these proteins have acquired the ability to promote splicing of their cognate intron, but whether these two activities reside in different regions of the protein remains obscure. A crystal structure of I-AniI, a dual function intron-encoded protein, has shown that the protein has two pseudo-symmetric domains of equal size. Each domain contacts its DNA substrate on either side of two cleavage sites. As a first step to identify the RNA binding surface, the N- and C-terminal domains of I-AniI were separately expressed and tested for promoting the splicing of the mitochondrial (mt) COB pre-RNA. The N-terminal protein showed no splicing activation or RNA binding, suggesting that this domain plays a minimal role in activity or is improperly folded. Remarkably, the 16-kDa C-terminal half facilitates intron splicing with a rate similar to that of the full-length protein. Both the C-terminal fragment and full-length proteins bind tightly to the COB intron. RNase footprinting shows that the C-terminal and full-length proteins bind to the same regions and induce the same conformational changes in the COB intron. Together, these results show that the C-terminal fragment of I-AniI is necessary and sufficient for maturase activity and suggests that I-AniI acquired splicing function by utilizing a relatively small protein surface that likely represents a novel RNA binding motif. This fragment of I-AniI represents the smallest group I intron splicing cofactor described to date.

Is there a dynamic DNA-protein interface in the transcription factor IIIA-5 S rRNA gene complex?

Others have proposed that several amino acid side chains exhibit considerable conformational mobility at the DNA-protein interface in the transcription factor IIIA.5 S rRNA gene complex and that the rapid movements of these side chains permit them to make fluctuating contacts with adjacent bp in the DNA target site. This "dynamic interface" model makes biochemical predictions concerning the consequences of truncating specific amino acid side chains and the effects of these truncations on sequence selectivity in DNA binding. The model also makes predictions concerning the effects of DNA sequence context on the apparent energetic contributions to binding made by individual bp. We have tested these predictions, and our results are inconsistent with any significant energetic role being played by the contact of multiple bp by conformationally mobile amino acid side chains. They do, however, show that some individual amino acids affect the recognition of multiple bp through mechanisms other than direct interaction.

Structural and biochemical analyses of DNA and RNA binding by a bifunctional homing endonuclease and group I intron splicing factor.

We determined the crystal structure of a bifunctional group I intron splicing factor and homing endonuclease, termed the I-AniI maturase, in complex with its DNA target at 2.6 A resolution. The structure demonstrates the remarkable structural conservation of the beta-sheet DNA-binding motif between highly divergent enzyme subfamilies. DNA recognition by I-AniI was further studied using nucleoside deletion and DMS modification interference analyses. Correlation of these results with the crystal structure provides information on the relative importance of individual nucleotide contacts for DNA recognition. Alignment and modeling of two homologous maturases reveals conserved basic surface residues, distant from the DNA-binding surface, that might be involved in RNA binding. A point mutation that introduces a single negative charge in this region uncouples the maturase and endonuclease functions of the protein, inhibiting RNA binding and splicing while maintaining DNA binding and cleavage.

Functionally distinct nucleic acid binding sites for a group I intron encoded RNA maturase/DNA homing endonuclease.

A large number of group I introns encode a family of homologous proteins that either promote intron splicing (maturases) or are site-specific DNA endonucleases that function in intron mobility (a process called "homing"). Genetic studies have shown that some of these proteins have both activities, yet how a single protein carries out both functions remains obscure. The similarity between respective DNA-binding sites and the RNA structure near the 5' and 3' splice sites has fueled speculation that such proteins may use analogous interactions to perform both functions. The Aspergillus nidulans mitochondrial COB group I intron encodes a bi-functional protein, I-AniI, that has both RNA maturase and site-specific DNA endonuclease activities in vitro. Here, we show that I-AniI shows distinctive features of the endonuclease family to which it belongs, including highly specific, tight binding and sequential DNA strand cleavage. Competition experiments demonstrate that I-AniI binds the COB intron RNA even in saturating concentrations of its DNA target site substrate, suggesting that the protein has a separate binding site for RNA. In addition, we provide evidence that two different DNA-binding site mutants of I-AniI have little effect on the protein's RNA maturation activity. Since RNA splicing is likely a secondary adaptation of the protein, these observations support a model in which homing endonucleases may have developed maturase function by utilizing a hitherto "non-functional" protein surface.