Contribution of whole genome sequencing in the molecular diagnosis of mosaic partial deletion of the NF1 gene in neurofibromatosis type 1.

Neurofibromatosis type 1 (NF1) is an autosomal dominant disease with complete penetrance but highly variable expressivity. In most patients, Next Generation Sequencing (NGS) technologies allow the identification of a loss-of-function pathogenic variant in the NF1 gene, a negative regulator of the RAS-MAPK pathway. We describe the 5-year diagnosis wandering of a patient with a clear NF1 clinical diagnosis, but no molecular diagnosis using standard molecular technologies. The patient presented with a typical NF1 phenotype but NF1 targeted NGS, NF1 transcript analysis, MLPA, and array comparative genomic hybridization failed to reveal a genetic aberration. After 5 years of unsuccessful investigations, trio WGS finally identified a de novo mosaic (VAF ~ 14%) 24.6 kb germline deletion encompassing the promoter and first exon of NF1. This case report illustrates the relevance of WGS to detect structural variants including copy number variants that would be missed by alternative approaches. The identification of the causal pathogenic variant allowed a tailored genetic counseling with a targeted non-invasive prenatal diagnosis by detecting the deletion in plasmatic cell-free DNA from the proband's pregnant partner. This report clearly highlights the need to make WGS a clinically accessible test, offering a tremendous opportunity to identify a molecular diagnosis for otherwise unsolved cases.

Cholera Outbreak - Haiti, September 2022-January 2023.

On September 30, 2022, after >3 years with no confirmed cholera cases (1), the Directorate of Epidemiology, Laboratories and Research (DELR) of the Haitian Ministry of Public Health and Population (Ministère de la Santé Publique et de la Population [MSPP]) was notified of two patients with acute, watery diarrhea in the metropolitan area of Port-au-Prince. Within 2 days, Haiti's National Public Health Laboratory confirmed the bacterium Vibrio cholerae O1 in specimens from the two patients with suspected cholera infection, and an outbreak investigation began immediately. As of January 3, 2023, >20,000 suspected cholera cases had been reported throughout the country, and 79% of patients have been hospitalized. The moving 14-day case fatality ratio (CFR) was 3.0%. Cholera, which is transmitted through ingestion of water or food contaminated with fecal matter, can cause acute, severe, watery diarrhea that can rapidly lead to dehydration, shock, and death if not treated promptly (2). Haiti is currently facing ongoing worsening of gang violence, population displacement, social unrest, and insecurity, particularly in the metropolitan area of Port-au-Prince, including Belair, Bas-Delmas, Centre-Ville, Martissant, Cité Soleil, Croix-des Bouquets, and Tabarre, creating an environment that has facilitated the current resurgence of cholera (3). This report describes the initial investigation, ongoing outbreak, and public health response to cholera in Haiti. Cholera outbreak responses require a multipronged, multisectoral approach including surveillance; case management; access to safe water, sanitation, and hygiene (WASH) services; targeted oral cholera vaccine (OCV) campaigns; risk communication; and community engagement. This activity was reviewed by CDC and was conducted consistent with applicable federal law and CDC policy.

Contribution of Whole-Genome Sequencing and Transcript Analysis to Decipher Retinal Diseases Associated with MFSD8 Variants.

Biallelic gene defects in MFSD8 are not only a cause of the late-infantile form of neuronal ceroid lipofuscinosis, but also of rare isolated retinal degeneration. We report clinical and genetic data of seven patients compound heterozygous or homozygous for variants in MFSD8, issued from a French cohort with inherited retinal degeneration, and two additional patients retrieved from a Swiss cohort. Next-generation sequencing of large panels combined with whole-genome sequencing allowed for the identification of twelve variants from which seven were novel. Among them were one deep intronic variant c.998+1669A>G, one large deletion encompassing exon 9 and 10, and a silent change c.750A>G. Transcript analysis performed on patients' lymphoblastoid cell lines revealed the creation of a donor splice site by c.998+1669A>G, resulting in a 140 bp pseudoexon insertion in intron 10. Variant c.750A>G produced exon 8 skipping. In silico and in cellulo studies of these variants allowed us to assign the pathogenic effect, and showed that the combination of at least one severe variant with a moderate one leads to isolated retinal dystrophy, whereas the combination in trans of two severe variants is responsible for early onset severe retinal dystrophy in the context of late-infantile neuronal ceroid lipofuscinosis.

Novel TTLL5 Variants Associated with Cone-Rod Dystrophy and Early-Onset Severe Retinal Dystrophy.

Variants of the TTLL5 gene, which encodes tubulin tyrosine ligase-like family member five, are a rare cause of cone dystrophy (COD) or cone-rod dystrophy (CORD). To date, only a few TTLL5 patients have been clinically and genetically described. In this study, we report five patients harbouring biallelic variants of TTLL5. Four adult patients presented either COD or CORD with onset in the late teenage years. The youngest patient had a phenotype of early onset severe retinal dystrophy (EOSRD). Genetic analysis was performed by targeted next generation sequencing of gene panels and assessment of copy number variants (CNV). We identified eight variants, of which six were novel, including two large multiexon deletions in patients with COD or CORD, while the EOSRD patient harboured the novel homozygous p.(Trp640*) variant and three distinct USH2A variants, which might explain the observed rod involvement. Our study highlights the role of TTLL5 in COD/CORD and the importance of large deletions. These findings suggest that COD or CORD patients lacking variants in known genes may harbour CNVs to be discovered in TTLL5, previously undetected by classical sequencing methods. In addition, variable phenotypes in TTLL5-associated patients might be due to the presence of additional gene defects.

Mutations in KARS cause a severe neurological and neurosensory disease with optic neuropathy.

Mutations in genes encoding aminoacyl-tRNA synthetases have been reported in several neurological disorders. KARS is a dual localized lysyl-tRNA synthetase and its cytosolic isoform belongs to the multiple aminoacyl-tRNA synthetase complex (MSC). Biallelic mutations in the KARS gene were described in a wide phenotypic spectrum ranging from nonsyndromic deafness to complex impairments. Here, we report on a patient with severe neurological and neurosensory disease investigated by whole-exome sequencing and found to carry biallelic mutations c.683C>T (p.Pro228Leu) and c.871T>G (p.Phe291Val), the second one being novel, in the KARS gene. The patient presented with an atypical clinical presentation with an optic neuropathy not previously reported. At the cellular level, we show that cytoplasmic KARS was expressed at a lower level in patient cells and displayed decreased interaction with MSC. In vitro, these two KARS variants have a decreased aminoacylation activity compared with wild-type KARS, the p.Pro228Leu being the most affected. Our data suggest that dysfunction of cytoplasmic KARS resulted in a decreased level of translation of the nuclear-encoded lysine-rich proteins belonging to the respiratory chain complex, thus impairing mitochondria functions.

Where are the missing gene defects in inherited retinal disorders? Intronic and synonymous variants contribute at least to 4% of CACNA1F-mediated inherited retinal disorders.

Inherited retinal disorders (IRD) represent clinically and genetically heterogeneous diseases. To date, pathogenic variants have been identified in ~260 genes. Albeit that many genes are implicated in IRD, for 30-50% of the cases, the gene defect is unknown. These cases may be explained by novel gene defects, by overlooked structural variants, by variants in intronic, promoter or more distant regulatory regions, and represent synonymous variants of known genes contributing to the dysfunction of the respective proteins. Patients with one subgroup of IRD, namely incomplete congenital stationary night blindness (icCSNB), show a very specific phenotype. The major cause of this condition is the presence of a hemizygous pathogenic variant in CACNA1F. A comprehensive study applying direct Sanger sequencing of the gene-coding regions, exome and genome sequencing applied to a large cohort of patients with a clinical diagnosis of icCSNB revealed indeed that seven of the 189 CACNA1F-related cases have intronic and synonymous disease-causing variants leading to missplicing as validated by minigene approaches. These findings highlight that gene-locus sequencing may be a very efficient method in detecting disease-causing variants in clinically well-characterized patients with a diagnosis of IRD, like icCSNB.

Trends in Prevalence of Advanced HIV Disease at Antiretroviral Therapy Enrollment - 10 Countries, 2004-2015.

Monitoring prevalence of advanced human immunodeficiency virus (HIV) disease (i.e., CD4+ T-cell count <200 cells/µL) among persons starting antiretroviral therapy (ART) is important to understand ART program outcomes, inform HIV prevention strategy, and forecast need for adjunctive therapies.*,†,§ To assess trends in prevalence of advanced disease at ART initiation in 10 high-burden countries during 2004-2015, records of 694,138 ART enrollees aged ≥15 years from 797 ART facilities were analyzed. Availability of national electronic medical record systems allowed up-to-date evaluation of trends in Haiti (2004-2015), Mozambique (2004-2014), and Namibia (2004-2012), where prevalence of advanced disease at ART initiation declined from 75% to 34% (p<0.001), 73% to 37% (p<0.001), and 80% to 41% (p<0.001), respectively. Significant declines in prevalence of advanced disease during 2004-2011 were observed in Nigeria, Swaziland, Uganda, Vietnam, and Zimbabwe. The encouraging declines in prevalence of advanced disease at ART enrollment are likely due to scale-up of testing and treatment services and ART-eligibility guidelines encouraging earlier ART initiation. However, in 2015, approximately a third of new ART patients still initiated ART with advanced HIV disease. To reduce prevalence of advanced disease at ART initiation, adoption of World Health Organization (WHO)-recommended "treat-all" guidelines and strategies to facilitate earlier HIV testing and treatment are needed to reduce HIV-related mortality and HIV incidence.

"This Method, I Think, Can Shed New Light": Haitian-American Women's Reflections on Risk, Culture, and Family Planning Decisions From a Short-Term Trial of a Cervical Barrier (Femcap™).

Improving the reproductive health of immigrant populations requires understanding the specific context of risk and need. As part of a field trial of the FemCap™, a woman-initiated cervical barrier contraceptive, we conducted postintervention focus group discussions (FGDs) with 20 women (five FGDs) of Haitian background, the majority of whom were born in Haiti and spoke Haitian Créole at home, at a community health center in south Florida. Participants discussed the role of religion and inequitable gender norms in Haitian traditions about family planning decisions and provided important insights into the gender-power nuances of their partnership dynamics vis à vis the use of female barrier methods. Encouraged by more equitable gender norms in the United States, participants were eager to serve as health education agents, with strong altruistic sentiments toward other Haitian girls and women who they felt could be encouraged to negotiate for greater reproductive decision-making power.

Exome sequencing of Bardet-Biedl syndrome patient identifies a null mutation in the BBSome subunit BBIP1 (BBS18).

BACKGROUND: Bardet-Biedl syndrome (BBS) is a recessive and genetically heterogeneous ciliopathy characterised by retinitis pigmentosa, obesity, kidney dysfunction, postaxial polydactyly, behavioural dysfunction and hypogonadism. 7 of the 17 BBS gene products identified to date assemble together with the protein BBIP1/BBIP10 into the BBSome, a protein complex that ferries signalling receptors to and from cilia. METHODS AND RESULTS: Exome sequencing performed on a sporadic BBS case revealed for the first time a homozygous stop mutation (NM_001195306: c.173T>G, p.Leu58*) in the BBIP1 gene. This mutation is pathogenic since no BBIP1 protein could be detected in fibroblasts from the patient, and BBIP1[Leu58*] is unable to associate with the BBSome subunit BBS4. CONCLUSIONS: These findings identify BBIP1 as the 18th BBS gene (BBS18) and suggest that BBSome assembly may represent a unifying pathomechanism for BBS.

'Men don't need to know everything': a field trial of a discreet, female-initiated, contraceptive barrier method (FemCap™) among Haitian-American women.

Worldwide, women report the need for safe, non-hormonal, woman-initiated methods of family planning. Cervical barriers provide such technology but are under-researched and under-promoted. In the USA, there are few studies of cervical barriers among women at high unmet need for contraception. A feasibility study of the FemCap™ was conducted among US women of Haitian origin. Participants were heterosexual and seeking to avoid pregnancy. At first visit, participants completed baseline assessments, underwent group counselling and were fitted with FemCap™. Women were asked to insert or use the cap at home. The second visit (2-3 weeks) included an interviewer-administered questionnaire and a focus-group discussion. Participants (n  =  20) were Haitian-born (70%), married (55%) and parous (85%). Their mean age was 32.6 years. Seventy percent reported recent unprotected sex. All women inserted the device at home and 9 women used it during intercourse, including 5 without prior partner negotiation. Of 20 women, 11 liked FemCap™ very much or somewhat; 7 considered it 'OK'; 2 disliked it. Best-liked attributes were comfort, discreet wear and reusability. Difficulties with removal abated over time. Qualitative data revealed a high value placed on lack of systemic side effects. Use of FemCap™ was feasible and acceptable, supporting expansion of research, particularly among relevant populations with unmet need.

Homozygosity mapping and candidate prioritization identify mutations, missed by whole-exome sequencing, in SMOC2, causing major dental developmental defects.

Inherited dental malformations constitute a clinically and genetically heterogeneous group of disorders. Here, we report on a severe developmental dental defect that results in a dentin dysplasia phenotype with major microdontia, oligodontia, and shape abnormalities in a highly consanguineous family. Homozygosity mapping revealed a unique zone on 6q27-ter. The two affected children were found to carry a homozygous mutation in SMOC2. Knockdown of smoc2 in zebrafish showed pharyngeal teeth that had abnormalities reminiscent of the human phenotype. Moreover, smoc2 depletion in zebrafish affected the expression of three major odontogenesis genes: dlx2, bmp2, and pitx2.

Migration patterns among Floridians with AIDS, 1993-2007: implications for HIV prevention and care.

OBJECTIVES: To characterize migration patterns among people diagnosed as having and who died of acquired immunodeficiency syndrome (AIDS) from 1993 to 2007 because migrating to a new community can disrupt human immunodeficiency virus/AIDS care delivery and patients' adherence to care and affect migrants' social services and healthcare needs. METHODS: Florida AIDS surveillance data were used to describe patterns of migration among people diagnosed as having and who died of AIDS from 1993 to 2007. Individual and community characteristics were compared between residence at the time of AIDS diagnosis and residence at the time of death by type of migration. RESULTS: Of 31,816 people in the cohort, 2510 (7.9%) migrated to another county in Florida and 1306 (4.1%) migrated to another state. Interstate migrants were more likely to be men, 20 to 39 years old, non-Hispanic white, and born in the United States, to have had a transmission mode of injection drug use (IDU) or men who have sex with men with IDU (MSM&IDU), and to have been diagnosed before 1999. Intercounty migrants were more likely to be non-Hispanic white, younger than 60 years, have had a transmission mode of MSM, IDU, or MSM&IDU, have higher CD4 counts/percentages, and to have lived in areas with low levels of poverty or low physician density. There was a small net movement from urban to rural areas within the state. CONCLUSIONS: A sizable percentage of people, particularly younger people and people with a transmission mode of IDU and IDU&MSM, migrated at least once between the time of their AIDS diagnosis and death. This has important implications for care and treatment, as well as efforts to prevent the disease. Further research is needed to explore barriers and facilitators to access to care upon migration and to assess the need for programs to help people transfer their human immunodeficiency virus/AIDS care, ensuring continuity of care and adherence.

Factors explaining racial/ethnic disparities in rates of physician recommendation for colorectal cancer screening.

OBJECTIVES: Physician recommendation plays a crucial role in receiving endoscopic screening for colorectal cancer (CRC). This study explored factors associated with racial/ethnic differences in rates of screening recommendation. METHODS: Data on 5900 adults eligible for endoscopic screening were obtained from the National Health Interview Survey. Odds ratios of receiving an endoscopy recommendation were calculated for selected variables. Planned, sequenced logistic regressions were conducted to examine the extent to which socioeconomic and health care variables account for racial/ethnic disparities in recommendation rates. RESULTS: Differential rates were observed for CRC screening and screening recommendations among racial/ethnic groups. Compared with Whites, Hispanics were 34% less likely (P < .01) and Blacks were 26% less likely (P < .05) to receive this recommendation. The main predictors that emerged in sequenced analysis were education for Hispanics and Blacks and income for Blacks. After accounting for the effects of usual source of care, insurance coverage, and education, the disparity reduced and became statistically insignificant. CONCLUSIONS: Socioeconomic status and access to health care may explain major racial/ethnic disparities in CRC screening recommendation rates.

Adoribatella, Ferolocella, Joelia and Ophidiotrichus (Acari, Oribatida, Oribatellidae) of North America.

In North America, species diversity in the oribatid family Oribatellidae is primarily in the genus Oribatella Banks, but the genera Adoribatella Woolley, Ferolocella Grabowski, Joelia Oudemans and Ophidiotrichus Grandjean are also represented. I provide detailed diagnoses for these genera and the previously described species, Adoribatella punctata Woolley, known from Colorado and Oregon, USA, and Alberta, Canada, Ferolocella tessalata Berlese known from Missouri, Virginia, West Virginia, Georgia, Wisconsin, Ohio, Alabama, Arkansas and Texas, USA, and Ophidiotrichus exastus Woolley, known from North Carolina, Alabama, Missouri, Tennessee and Georgia, USA. I describe Joelia appalachia sp. nov., based on adult specimens, from West Virginia. A key is given to distinguish adults of these genera from those of Oribatella. Character states shared by adults of Oribatellidae are discussed, the synonymy of Gendzella Kuliev with Ferolocella is rejected, and arguments are presented for movement of Adoribatella from Oribatellidae to the Ceratozetoidea.

Phylogenetic relationships of Tectoribates: nymphal characters of new North American species place the genus in Tegoribatidae (Acari, Oribatida).

Species in the oribatid mite genus Tectoribates are primarily Palaearctic and Neotropical, with scattered, unidentified records from North America. Herein, we describe 3 new Tectoribates species from dry forest and prairie habitats in North America: T. alcecampestris sp. nov., from Alberta, T. borealis sp. nov., from southern Alberta and Ontario, both on the basis of adults and nymphs, and T. campestris sp. nov., from dry grassland habitats in Ontario and Kansas, on the basis of adults. We provide a revised and expanded diagnosis for adults of Tectoribates. We assess relationships of Tectoribates, using characters of adults and newly discovered apheredermous, plicate immatures. We include observations on Pseudotectoribates which is closely related to Tectoribates. The closest relatives of these genera are hypothesised to be among the Tegoribatidae (Achipterioidea) rather than among the Achipteriidae (Achipterioidea), Oribatellidae (Oribatelloidea), or Ceratozetoidea, as suggested in previous classifications. Finally, we give a key to adults of the world fauna of Tectoribates.

Description of Oromurcia magadanensis sp. nov. (Acari, Oribatida, Ceratozetidae) from Russia, with remarks on biogeography of the genus Oromurcia Thor, 1930.

Species of the genus Oromurcia Thor, 1930 (Oribatida, Ceratozetidae) mainly have an arctic or alpine distribution in the Western Palaearctic region and Greenland. We describe a new species of Oromurcia from Northeast Asia (Magadan Region, Russia) based on adult and juvenile instars. Numerous populations of Oromurcia magadanensis sp. nov. were found under arctic-alpine plants in a narrow stream gully with large snow accumulation on the Ola Plateau (1023 m a.s.l.). Adults of Oromurcia magadanensis sp. nov. differ from those of Oromurcia bicuspidata and O. sudetica by their smaller size, the presence of clavate, distally broadly rounded bothridial seta, tutorium with several teeth distally, and the absence of striations on the lamella. Juvenile instars of the new species differ from those of O. bicuspidata and O. sudetica by the presence of medium-sized gastronotic setae in the larva, and long lateral and posterior gastronotic setae in nymphs. We compare adult and juveniles of Oromurcia magadanensis sp. nov. with those of other members of the ceratozetid subfamily Trichoribatinae Shaldybina, 1966 from arctic or alpine regions, for which ontogeny is known, and provide revised diagnoses for adult and juvenile members of Trichoribatinae. All Oromurcia species are associated with cold wet places (periglacial habitats, bogs and fens, alpine meadows, snowbeds, and, less frequently, montane coniferous forests) in Eastern and Western Palaearctic, but they are absent from High Arctic and Siberian regions that are ultra-cold in winter. This spatial disjunction argues for a former Trans-Palaearctic range that was possibly subdivided by reсurrent cryo-arid Pleistocene episodes.

Hogsbackia africaensis/ gen. nov., sp. nov. (Acari, Oribatida, Ceratozetoidea, Punctoribatidae), from Afromontane forest of South Africa.

A new oribatid mite genus, Hogsbackia gen. nov. (Oribatida, Punctoribatidae), with type species Hogsbackia africaensis sp. nov., is proposed and described from the indigenous Afromontane forest of South Africa. The new genus is closely related to Scotiazetes, from which it is most easily distinguished by body size ratio, number of notogastral and genital setae, type of humeral porose area Ah, position of dorsophragmata, and leg setation. The redescription of Scotiazetes bidens Wallwork, 1966 is presented, based on material from South Georgia (Antarctic region). The generic diagnosis of Scotiazetes is revised, and the genus is transferred from Ceratozetidae to Punctoribatidae. Relationships between the genera Hogsbackia gen. nov., Scotiazetes and other punctoribatid genera are discussed.

Spectrum of SPATA7 mutations in Leber congenital amaurosis and delineation of the associated phenotype.

Leber congenital amaurosis (LCA) is the earliest and most severe retinal degeneration. It may present as a congenital stationary cone-rod dystrophy (LCA type I) or a progressive yet severe rod-cone dystrophy (LCA type II). Twelve LCA genes have been identified, three of which account for Type I and nine for LCA type II. All proteins encoded by these genes but two are preferentially expressed in the retina and are responsible for non-syndromic LCA only. By contrast LCA5 and CEP290 are widely expressed and mutations in this latter result in a variety of phenotypes from non-syndromic retinal degeneration to pleiotropic disorders including senior-Loken (SNLS) and Joubert syndromes (JBTS). Recently, mutations in the widely expressed gene SPATA7 were reported to cause LCA or juvenile retinitis pigmentosa. The purpose of this study was i) to determine the level of expression of two major alternative SPATA7 transcripts in a large range of tissues and ii) to assess the involvement of this novel gene in a large cohort of unrelated patients affected with LCA (n = 134). Here, we report high SPATA7expression levels in retina, brain and testis with differential expression of the two transcripts. SPATA7 mutations were identified in few families segregating non-syndromic LCA (n = 4/134). Six different mutations were identified, four of which are novel; All affected both SPATA7 transcripts. The clinical evaluation of patients suggested that SPATA7 mutations account for the rod-cone dystrophy type of the disease.

Oribatid mites in soil toxicity testing-the use of Oppia nitens (C.L. Koch) as a new test species.

Few soil invertebrate species are available for the toxic assessment of soils from boreal or other northern ecozones, yet these soils cover the majority of Canada's landmass as well as significant portions of Eurasia. Oppia nitens (C.L. Koch) is an herbivorous and fungivorous oribatid mite found in soil throughout Holarctic regions, including Canada. Soil tests using O. nitens were performed using 15 different forest soil types and horizons to investigate test variability in adult survival and reproduction. Adult survival (86.1 +/- 1.1%) was consistent across soil types, with a coefficient of variation (CV) of 15%. However, reproduction varied significantly, ranging from 2.9 (+/-1.1) to 86.2 (+/-11.7) individuals, with a corresponding CV of 118 and 30%, respectively. Of the soil factors assessed (NH(3), NO(3), pH, phosphorus [P], organic matter content (OM), carbon:nitrogen (C:N), sand, silt, clay, and sodium adsorption ratio), soil organic matter (OM) explained 68% of the variation observed for reproduction. Increasing the OM using Sphagnum sp. peat moss resulted in optimal reproduction at 7% OM (8% peat content) with the lowest variability (CV of 20%). When assessing the toxicity of a reference chemical, boric acid, the effect of peat amendment reduced lethality to adults with no observable difference on reproduction. The use an age-synchronized culture reduced the test variability for reproduction relative to the use of unsynchronized cultures. Oppia nitens is a good candidate species for a standardized test design, with adult survival easily assessed in a relatively simple design. A long-term reproduction test with O. nitens will require the use of a synchronized population and, on occasion, OM amendment when testing soils with low organic matter content.

Sculpteremaeus olszanowskii gen. nov., sp. nov. (Acari, Oribatida, Cymbaeremaeidae) from chaparral in California, USA, with a reassessment of Cymbaeremaeidae.

A new oribatid mite genus, Sculpteremaeus gen. nov. (Oribatida, Cymbaeremaeidae), with Sculpteremaeus olszanowskii sp. nov. as type species, is proposed and described based on adults. It is from sandy soil-litter in chaparral of California, USA, an environment from which no oribatid mites have been recorded previously. Based on an analysis of adults of all genera of Cymbaeremaeidae, the new genus is closely related to Ametroproctus, from which it is most easily distinguished by the morphology of lamellae, size of lamellar cusps, and shape of the tutorium. We expand the Cymbaeremaeidae family diagnosis, and question the inclusion of Scapheremaeus based on extensive molecular evidence showing its closer relationship with Ameronothroidea and Licneremaeoidea.