Successful therapeutic intervention in new mouse models of frizzled 2-associated congenital malformations.

Frizzled 2 (FZD2) is a transmembrane Wnt receptor. We previously identified a pathogenic human FZD2 variant in individuals with FZD2-associated autosomal dominant Robinow syndrome. The variant encoded a protein with a premature stop and loss of 17 amino acids, including a region of the consensus dishevelled-binding sequence. To model this variant, we used zygote microinjection and i-GONAD-based CRISPR/Cas9-mediated genome editing to generate a mouse allelic series. Embryos mosaic for humanized Fzd2W553* knock-in exhibited cleft palate and shortened limbs, consistent with patient phenotypes. We also generated two germline mouse alleles with small deletions: Fzd2D3 and Fzd2D4. Homozygotes for each allele exhibit a highly penetrant cleft palate phenotype, shortened limbs compared with wild type and perinatal lethality. Fzd2D4 craniofacial tissues indicated decreased canonical Wnt signaling. In utero treatment with IIIC3a (a DKK inhibitor) normalized the limb lengths in Fzd2D4 homozygotes. The in vivo replication represents an approach for further investigating the mechanism of FZD2 phenotypes and demonstrates the utility of CRISPR knock-in mice as a tool for investigating the pathogenicity of human genetic variants. We also present evidence for a potential therapeutic intervention.

Using human sequencing to guide craniofacial research.

A recent convergence of technological innovations has re-energized the ability to apply genetics to research in human craniofacial development. Next-generation exome and whole genome sequencing have significantly dropped in price, making it relatively trivial to sequence and analyze patients and families with congenital craniofacial anomalies. A concurrent revolution in genome editing with the use of the CRISPR-Cas9 system enables the rapid generation of animal models, including mouse, which can precisely recapitulate human variants. Here, we summarize the choices currently available to the research community. We illustrate this approach with the study of a family with a novel craniofacial syndrome with dominant inheritance pattern. The genomic analysis suggested a causal variant in AMOTL1 which we modeled in mice. We also made a novel deletion allele of Amotl1. Our results indicate that Amotl1 is not required in the mouse for survival to weaning. Mice carrying the variant identified in the human sequencing studies, however, do not survive to weaning in normal ratios. The cause of death is not understood for these mice complicating our conclusions about the pathogenicity in the index patient. Thus, we highlight some of the powerful opportunities and confounding factors confronting current craniofacial genetic research.

Routine HIV counseling and testing during antenatal care in Ukraine: a qualitative study of the experiences and perspectives of pregnant women and antenatal care providers.

Ukraine has one of the highest levels of HIV prevalence in Europe. Provider-initiated HIV testing and counseling (PITC) is routine during antenatal care (ANC) in Ukraine, with nearly all pregnant women receiving HIV testing. National policies state that testing is voluntary, with consent and confidentiality protections, but little is known about actual testing implementation and adherence to these policies. A qualitative study was conducted to explore women's and providers' experiences of HIV testing during ANC, with a focus on consent, counseling, and confidentiality. In-depth interviews were conducted at two urban women's clinics and one semi-urban women's clinic in Donetsk, one of the most HIV-affected regions in Ukraine. Interviews targeted HIV-positive and HIV-negative women recently tested during ANC, as well as health providers involved in administering HIV tests during ANC. Interview analysis revealed gaps between policies and practices, as well as differences in perceptions of aspects of PITC between women and providers. Notable findings included: wide variation in informed consent procedures; providers reporting feeling unequipped to provide adequate counseling; and post-testing practices that may jeopardize patient confidentiality, particularly in the semi-urban site. These findings suggest the need for additional training and support, especially outside major cities, to ensure that HIV testing practices during ANC adhere to Ukrainian policy and global principles regarding PITC.

Multi-city assessment of lifetime pregnancy involvement among street youth, Ukraine.

Although street youth are at increased risk of lifetime pregnancy involvement (LPI), or ever becoming or getting someone pregnant, no reports to date describe the epidemiology of LPI among systematically sampled street youth from multiple cities outside of North America. The purpose of our assessment was to describe the prevalence of and risk factors associated with LPI among street youth from three Ukrainian cities. We used modified time-location sampling to conduct a cross-sectional assessment in Odesa, Kyiv, and Donetsk that included citywide mapping of 91 public venue locations frequented by street youth, random selection of 74 sites, and interviewing all eligible and consenting street youth aged 15-24 years found at sampled sites (n = 929). Characteristics of youth and prevalence of LPI overall and by demographic, social, sexual, and substance use risk factors, were estimated separately for males and females. Adjusted odds ratios (AORs) were calculated with multivariable logistic regression and effect modification by gender was examined. Most (96.6%) eligible youth consented to participate. LPI was reported for 41.7% of females (93/223) and 23.5% of males (166/706). For females, LPI was significantly elevated and highest (>70%) among those initiating sexual activity at ≤12 years and for those reporting lifetime anal sex and exchanging sex for goods. For males, LPI was significantly elevated and highest (>40%) among those who reported lifetime anal sex and history of a sexually transmitted infection. Overall, risk factors associated with LPI were similar for females and males. Among the total sample (females and males combined), significant independent risk factors with AORs ≥2.5 included female gender, being aged 20-24 years, having five to six total adverse childhood experiences, initiating sex at age ≤12 or 13-14 years, lifetime anal sex, most recent sex act unprotected, and lifetime exchange of sex for goods. Among street youth with LPI (n = 259), the most recent LPI event was reported to be unintended by 63.3% and to have ended in abortion by 43.2%. In conclusion, our assessment documented high rates of LPI among Ukrainian street youth who, given the potential for negative outcomes and the challenges of raising a child on the streets, are in need of community-based pregnancy prevention programs and services. Promising preventive strategies are discussed, which are likely applicable to other urban populations of street-based youth as well.

HIV seroprevalence among orphaned and homeless youth: no place like home.

OBJECTIVES: We evaluated the combined influences of orphaned status and homelessness on HIV seroprevalence and risk among street-involved Ukrainian youth in 2008. DESIGN: Systematic, multicity, community-based, cross-sectional assessment. METHODS: Time-location sampling was used to identify eligible youth aged 15-24 after city-wide mapping of 91 sites where street-involved youth gathered in Odessa, Kiev, and Donetsk. Universal sampling identified 961 youth in 74 randomly selected sites; 97% consented. Youth reporting one or both parents dead were classified as orphaned; those without a stable residence or sleeping outside their residence at least two nights per week were classified as homeless. Trained staff provided HIV counseling and rapid testing via mobile vans. Adjusted odds ratios (AORs) were calculated using logistic regression, accounting for intracluster homogeneity. RESULTS: We found 32% (300 of 929) were both orphaned and homeless; 48% either (but not both) homeless [37% (343 of 929)] or orphaned [11% (104 of 929)]; and [20% (182 of 929)] neither orphaned nor homeless. HIV seroprevalences were 7% for neither orphaned/homeless; 16 and 17%, respectively, for either orphaned/homeless; 28% for both orphaned/homeless (P for trend <0.0001). AORs for HIV infection were 1 for neither; 2.3 and 2.4 for either homeless [95% confidence interval (CI) 1.7-2.9] or orphaned (CI 1.8-3.3); 3.3 for both orphaned/homeless (CI 2.3-4.4). Ever-use of injection drugs increased from 15 to 32 to 48% for those who neither, either, or both orphaned and homeless, respectively (P for trend <0.0001). CONCLUSIONS: One of four youths who were both homeless and orphaned was HIV-infected; these youths were significantly more likely to be HIV infected and to report injection drug use than those with adequate housing and living parents.