De novo variants in the non-coding spliceosomal snRNA gene RNU4-2 are a frequent cause of syndromic neurodevelopmental disorders.

Around 60% of individuals with neurodevelopmental disorders (NDD) remain undiagnosed after comprehensive genetic testing, primarily of protein-coding genes1. Increasingly, large genome-sequenced cohorts are improving our ability to discover new diagnoses in the non-coding genome. Here, we identify the non-coding RNA RNU4-2 as a novel syndromic NDD gene. RNU4-2 encodes the U4 small nuclear RNA (snRNA), which is a critical component of the U4/U6.U5 tri-snRNP complex of the major spliceosome2. We identify an 18 bp region of RNU4-2 mapping to two structural elements in the U4/U6 snRNA duplex (the T-loop and Stem III) that is severely depleted of variation in the general population, but in which we identify heterozygous variants in 119 individuals with NDD. The vast majority of individuals (77.3%) have the same highly recurrent single base-pair insertion (n.64_65insT). We estimate that variants in this region explain 0.41% of individuals with NDD. We demonstrate that RNU4-2 is highly expressed in the developing human brain, in contrast to its contiguous counterpart RNU4-1 and other U4 homologs, supporting RNU4-2's role as the primary U4 transcript in the brain. Overall, this work underscores the importance of non-coding genes in rare disorders. It will provide a diagnosis to thousands of individuals with NDD worldwide and pave the way for the development of effective treatments for these individuals.

Reprograming skin fibroblasts into Sertoli cells: a patient-specific tool to understand effects of genetic variants on gonadal development.

BACKGROUND: Disorders/differences of sex development (DSD) are congenital conditions in which the development of chromosomal, gonadal, or anatomical sex is atypical. With overlapping phenotypes and multiple genes involved, poor diagnostic yields are achieved for many of these conditions. The current DSD diagnostic regimen can be augmented by investigating transcriptome/proteome in vivo, but it is hampered by the unavailability of affected gonadal tissue at the relevant developmental stage. We try to mitigate this limitation by reprogramming readily available skin tissue-derived dermal fibroblasts into Sertoli cells (SC), which could then be deployed for different diagnostic strategies. SCs form the target cell type of choice because they act like an organizing center of embryonic gonadal development and many DSD arise when these developmental processes go awry. METHODS: We employed a computational predictive algorithm for cell conversions called Mogrify to predict the transcription factors (TFs) required for direct reprogramming of human dermal fibroblasts into SCs. We established trans-differentiation culture conditions where stable transgenic expression of these TFs was achieved in 46, XY adult dermal fibroblasts using lentiviral vectors. The resulting Sertoli like cells (SLCs) were validated for SC phenotype using several approaches. RESULTS: SLCs exhibited Sertoli-like morphological and cellular properties as revealed by morphometry and xCelligence cell behavior assays. They also showed Sertoli-specific expression of molecular markers such as SOX9, PTGDS, BMP4, or DMRT1 as revealed by IF imaging, RNAseq and qPCR. The SLC transcriptome shared about two thirds of its differentially expressed genes with a human adult SC transcriptome and expressed markers typical of embryonic SCs. Notably, SLCs lacked expression of most markers of other gonadal cell types such as Leydig, germ, peritubular myoid or granulosa cells. CONCLUSIONS: The trans-differentiation method was applied to a variety of commercially available 46, XY fibroblasts derived from patients with DSD and to a 46, XX cell line. The DSD SLCs displayed altered levels of trans-differentiation in comparison to normal 46, XY-derived SLCs, thus showcasing the robustness of this new trans-differentiation model. Future applications could include using the SLCs to improve definitive diagnosis of DSD in patients with variants of unknown significance.

Individuals with disorders/differences of sex development (DSD) frequently do not get a specific genetic diagnostic. A limitation in the field is that the relevant cell types that would be needed to study the molecular events occurring at the time of onset of many DSD are found in the embryonic gonad. This, of course, is not accessible for research or diagnostic purposes. We set out to develop a method for directly transforming more accessible cells, from adult skin, into the cells known to organize the male gonad in the embryo, Sertoli cells. A combination of unique transcription factors was stably integrated into skin fibroblasts, and culture under appropriate conditions allowed differentiation into Sertoli-like cells (SLC), but not other gonadal cell types. The SLCs recapitulated known patterns of gene expression, shape, and behavior of Sertoli cells. The method was also tested on commercially available fibroblasts from a variety of DSD genetic backgrounds. The resulting cells exhibited condition-specific behavior (gene expression, adhesion to substrate, division rate…). This method provides a new tool to study molecular events occurring at the time of onset of DSD in the embryonic gonad, and the impact of patient-specific mutations on those. It could allow identification of new developmental pathways (and, thus, new candidate genes for DSD), as well as a provide models to validate the impact of variants of unknown significance, or to test approaches to correct the genetic anomaly in patient cells.

Using a chat-based informed consent tool in large-scale genomic research.

OBJECTIVE: We implemented a chatbot consent tool to shift the time burden from study staff in support of a national genomics research study. MATERIALS AND METHODS: We created an Institutional Review Board-approved script for automated chat-based consent. We compared data from prospective participants who used the tool or had traditional consent conversations with study staff. RESULTS: Chat-based consent, completed on a user's schedule, was shorter than the traditional conversation. This did not lead to a significant change in affirmative consents. Within affirmative consents and declines, more prospective participants completed the chat-based process. A quiz to assess chat-based consent user understanding had a high pass rate with no reported negative experiences. CONCLUSION: Our report shows that a structured script can convey important information while realizing the benefits of automation and burden shifting. Analysis suggests that it may be advantageous to use chatbots to scale this rate-limiting step in large research projects.

Objective documentation of hypospadias anatomy with three-dimensional scanning.

INTRODUCTION: The absence of a standardized classification of hypospadias hinders understanding of the anatomic differences among patients and the evaluation of outcomes following surgical repair. In working towards a standardized, objective method of recording patients' hypospadias anatomy, we describe our initial experience using a non-invasive three-dimensional scanner. MATERIAL AND METHODS: An Artec3D Space Spider scanner was used to obtain 3D scans in 29 patients undergoing hypospadias repair. Measurements of the urethral plate width, urethral plate length, glans width, penile shaft length, and penile shaft width were made by 2 pediatric urology attendings and 1 pediatric urology fellow. Measurements were compared and inter-rater reliability was calculated. RESULTS: A total of 435 measurements were made on 29 successfully generated 3D scans, ranging from distal to proximal hypospadias. The inter-rater reliability of measurements from the generated 3D models shown good inter-rater reliability of urethral plate width (ICC0.87 [95%CI:0.76,0.93]), penile shaft length (ICC0.87 [95%CI:0.70,0.94]) and glans width (ICC0.83 [95%CI:0.68,0.92]), excellent inter-rater reliability of urethral plate length (ICC0.96) and moderate inter-rater reliability of penile shaft width (ICC0.69 [95%CI:0.44,0.84]). DISCUSSION: There was a high degree of reliability of measurements made across multiple users. Calculation of the ratio of the urethral plate length/total penile shaft length objectively defined the initial position of the urethral meatus. When compared to the 3-dimensional volume of the glans, a more proximally positioned urethral meatus was associated with a lower glans volume. CONCLUSION: 3D scanning offers a rapid, reproducible, and non-invasive method of documenting hypospadias anatomy. The ability to evaluate three dimensional features (i.e. glans volume) offers an exciting opportunities for robust investigation of hypospadias outcomes and further understanding of the relationship between a patient's genotype and phenotype.

Benchmarking long-read genome sequence alignment tools for human genomics applications.

Background: The utility of long-read genome sequencing platforms has been shown in many fields including whole genome assembly, metagenomics, and amplicon sequencing. Less clear is the applicability of long reads to reference-guided human genomics, which is the foundation of genomic medicine. Here, we benchmark available platform-agnostic alignment tools on datasets from nanopore and single-molecule real-time platforms to understand their suitability in producing a genome representation. Results: For this study, we leveraged publicly-available data from sample NA12878 generated on Oxford Nanopore and sample NA24385 on Pacific Biosciences platforms. We employed state of the art sequence alignment tools including GraphMap2, long-read aligner (LRA), Minimap2, CoNvex Gap-cost alignMents for Long Reads (NGMLR), and Winnowmap2. Minimap2 and Winnowmap2 were computationally lightweight enough for use at scale, while GraphMap2 was not. NGMLR took a long time and required many resources, but produced alignments each time. LRA was fast, but only worked on Pacific Biosciences data. Each tool widely disagreed on which reads to leave unaligned, affecting the end genome coverage and the number of discoverable breakpoints. No alignment tool independently resolved all large structural variants (1,001-100,000 base pairs) present in the Database of Genome Variants (DGV) for sample NA12878 or the truthset for NA24385. Conclusions: These results suggest a combined approach is needed for LRS alignments for human genomics. Specifically, leveraging alignments from three tools will be more effective in generating a complete picture of genomic variability. It should be best practice to use an analysis pipeline that generates alignments with both Minimap2 and Winnowmap2 as they are lightweight and yield different views of the genome. Depending on the question at hand, the data available, and the time constraints, NGMLR and LRA are good options for a third tool. If computational resources and time are not a factor for a given case or experiment, NGMLR will provide another view, and another chance to resolve a case. LRA, while fast, did not work on the nanopore data for our cluster, but PacBio results were promising in that those computations completed faster than Minimap2. Due to its significant burden on computational resources and slow run time, Graphmap2 is not an ideal tool for exploration of a whole human genome generated on a long-read sequencing platform.

Genome-wide neonatal epigenetic changes associated with maternal exposure to the COVID-19 pandemic.

BACKGROUND: During gestation, stressors to the fetus, including viral exposure or maternal psychological distress, can fundamentally alter the neonatal epigenome, and may be associated with long-term impaired developmental outcomes. The impact of in utero exposure to the COVID-19 pandemic on the newborn epigenome has yet to be described. METHODS: This study aimed to determine whether there are unique epigenetic signatures in newborns who experienced otherwise healthy pregnancies that occurred during the COVID-19 pandemic (Project RESCUE). The pre-pandemic control and pandemic cohorts (Project RESCUE) included in this study are part of a prospective observational and longitudinal cohort study that evaluates the impact of elevated prenatal maternal stress during the COVID-19 pandemic on early childhood neurodevelopment. Using buccal swabs collected at birth, differential DNA methylation analysis was performed using the Infinium MethylationEPIC arrays and linear regression analysis. Pathway analysis and gene ontology enrichment were performed on resultant gene lists. RESULTS: Widespread differential methylation was found between neonates exposed in utero to the pandemic and pre-pandemic neonates. In contrast, there were no apparent epigenetic differences associated with maternal COVID-19 infection during pregnancy. Differential methylation was observed among genomic sites that underpin important neurological pathways that have been previously reported in the literature to be differentially methylated because of prenatal stress, such as NR3C1. CONCLUSIONS: The present study reveals potential associations between exposure to the COVID-19 pandemic during pregnancy and subsequent changes in the newborn epigenome. While this finding warrants further investigation, it is a point that should be considered in any study assessing newborn DNA methylation studies obtained during this period, even in otherwise healthy pregnancies.

Beyond the exome: What's next in diagnostic testing for Mendelian conditions.

Despite advances in clinical genetic testing, including the introduction of exome sequencing (ES), more than 50% of individuals with a suspected Mendelian condition lack a precise molecular diagnosis. Clinical evaluation is increasingly undertaken by specialists outside of clinical genetics, often occurring in a tiered fashion and typically ending after ES. The current diagnostic rate reflects multiple factors, including technical limitations, incomplete understanding of variant pathogenicity, missing genotype-phenotype associations, complex gene-environment interactions, and reporting differences between clinical labs. Maintaining a clear understanding of the rapidly evolving landscape of diagnostic tests beyond ES, and their limitations, presents a challenge for non-genetics professionals. Newer tests, such as short-read genome or RNA sequencing, can be challenging to order, and emerging technologies, such as optical genome mapping and long-read DNA sequencing, are not available clinically. Furthermore, there is no clear guidance on the next best steps after inconclusive evaluation. Here, we review why a clinical genetic evaluation may be negative, discuss questions to be asked in this setting, and provide a framework for further investigation, including the advantages and disadvantages of new approaches that are nascent in the clinical sphere. We present a guide for the next best steps after inconclusive molecular testing based upon phenotype and prior evaluation, including when to consider referral to research consortia focused on elucidating the underlying cause of rare unsolved genetic disorders.

Increased diagnostic yield from negative whole genome-slice panels using automated reanalysis.

We evaluated the diagnostic yield using genome-slice panel reanalysis in the clinical setting using an automated phenotype/gene ranking system. We analyzed whole genome sequencing (WGS) data produced from clinically ordered panels built as bioinformatic slices for 16 clinically diverse, undiagnosed cases referred to the Pediatric Mendelian Genomics Research Center, an NHGRI-funded GREGoR Consortium site. Genome-wide reanalysis was performed using Moon™, a machine-learning-based tool for variant prioritization. In five out of 16 cases, we discovered a potentially clinically significant variant. In four of these cases, the variant was found in a gene not included in the original panel due to phenotypic expansion of a disorder or incomplete initial phenotyping of the patient. In the fifth case, the gene containing the variant was included in the original panel, but being a complex structural rearrangement with intronic breakpoints outside the clinically analyzed regions, it was not initially identified. Automated genome-wide reanalysis of clinical WGS data generated during targeted panels testing yielded a 25% increase in diagnostic findings and a possibly clinically relevant finding in one additional case, underscoring the added value of analyses versus those routinely performed in the clinical setting.

"Development and Implementation of Novel Chatbot-based Genomic Research Consent".

Objective: To conduct a retrospective analysis comparing traditional human-based consenting to an automated chat-based consenting process. Materials and Methods: We developed a new chat-based consent using our IRB-approved consent forms. We leveraged a previously developed platform (GiaⓇ, or "Genetic Information Assistant") to deliver the chat content to candidate participants. The content included information about the study, educational information, and a quiz to assess understanding. We analyzed 144 families referred to our study during a 6-month time period. A total of 37 families completed consent using the traditional process, while 35 families completed consent using Gia. Results: Engagement rates were similar between both consenting methods. The median length of the consent conversation was shorter for Gia users compared to traditional (44 vs. 76 minutes). Additionally, the total time from referral to consent completion was faster with Gia (5 vs. 16 days). Within Gia, understanding was assessed with a 10-question quiz that most participants (96%) passed. Feedback about the chat consent indicated that 86% of participants had a positive experience. Discussion: Using Gia resulted in time savings for both the participant and study staff. The chatbot enables studies to reach more potential candidates. We identified five key features related to human-centered design for developing a consent chat. Conclusion: This analysis suggests that it is feasible to use an automated chatbot to scale obtaining informed consent for a genomics research study. We further identify a number of advantages when using a chatbot.

Beyond the exome: what's next in diagnostic testing for Mendelian conditions.

Despite advances in clinical genetic testing, including the introduction of exome sequencing (ES), more than 50% of individuals with a suspected Mendelian condition lack a precise molecular diagnosis. Clinical evaluation is increasingly undertaken by specialists outside of clinical genetics, often occurring in a tiered fashion and typically ending after ES. The current diagnostic rate reflects multiple factors, including technical limitations, incomplete understanding of variant pathogenicity, missing genotype-phenotype associations, complex gene-environment interactions, and reporting differences between clinical labs. Maintaining a clear understanding of the rapidly evolving landscape of diagnostic tests beyond ES, and their limitations, presents a challenge for non-genetics professionals. Newer tests, such as short-read genome or RNA sequencing, can be challenging to order and emerging technologies, such as optical genome mapping and long-read DNA or RNA sequencing, are not available clinically. Furthermore, there is no clear guidance on the next best steps after inconclusive evaluation. Here, we review why a clinical genetic evaluation may be negative, discuss questions to be asked in this setting, and provide a framework for further investigation, including the advantages and disadvantages of new approaches that are nascent in the clinical sphere. We present a guide for the next best steps after inconclusive molecular testing based upon phenotype and prior evaluation, including when to consider referral to a consortium such as GREGoR, which is focused on elucidating the underlying cause of rare unsolved genetic disorders.

Precocious Puberty in a Boy With Bilateral Leydig Cell Tumors due to a Somatic Gain-of-Function LHCGR Variant.

Context: Autosomal dominant and rarely de novo gain-of-function variants in the LHCGR gene are associated with precocious male puberty, while somatic LHCGR variants have been found in isolated Leydig cell adenomas and Leydig cell hyperplasia. Bilateral diffuse Leydig cell tumor formation in peripheral precocious male puberty has not been reported. Case Description: We present a boy with gonadotropin-independent precocious puberty and rapid virilization beginning in infancy resistant to standard therapy. Treatment with abiraterone in addition to letrozole and bicalutamide proved effective. Bilateral diffuse Leydig cell tumors were identified at age 5 years. Results: Whole-genome sequencing of tumor and blood samples was performed. The patient was confirmed to have bilateral, diffuse Leydig cell tumors harboring the somatic, gain-of-function p.Asp578His variant in the LHCGR gene. Digital droplet polymerase chain reaction of the LHCGR variant performed in tumor and blood samples detected low levels of this same variant in the blood. Conclusion: We report a young boy with severe gonadotropin-independent precocious puberty beginning in infancy who developed bilateral diffuse Leydig cell tumors at age 5 years due to a somatic gain-of-function p.Asp578His variant in LHCGR. The gain-of-function nature of the LHCGR variant and the developmental timing of the somatic mutation likely play a role in the risk of tumor formation. Abiraterone (a CYP17A1 inhibitor), in combination with an antiandrogen, aromatase inhibitor, and glucocorticoid, appears to be an effective therapy for severe peripheral precocious puberty in boys.

Towards improved genetic diagnosis of human differences of sex development.

Despite being collectively among the most frequent congenital developmental conditions worldwide, differences of sex development (DSD) lack recognition and research funding. As a result, what constitutes optimal management remains uncertain. Identification of the individual conditions under the DSD umbrella is challenging and molecular genetic diagnosis is frequently not achieved, which has psychosocial and health-related repercussions for patients and their families. New genomic approaches have the potential to resolve this impasse through better detection of protein-coding variants and ascertainment of under-recognized aetiology, such as mosaic, structural, non-coding or epigenetic variants. Ultimately, it is hoped that better outcomes data, improved understanding of the molecular causes and greater public awareness will bring an end to the stigma often associated with DSD.

Paucity and Disparity of Publicly Available Sex-Disaggregated Data for the COVID-19 Epidemic Hamper Evidence-Based Decision-Making.

COVID-19 has joined the long list of sexually dimorphic human disorders. Higher lethality in men, evident in the first reports from China, was confirmed in the subsequent Italian outbreak. Newspapers and scientific journals commented on this finding and the preexisting conditions, biological processes, and behavioral differences that may underlie it. However, little appeared to be released about sex differences in severity of disease, comorbidities, rate of recovery, length of hospital stay, or number of tests performed. Systematic analysis of official websites for 20 countries and 6 US states revealed a wide disparity in sex-disaggregated data made available to the public and scholars. Only a handful reported cases by sex. None of the other characteristics, including deaths, were stratified by sex at the time. Beyond suboptimal sex disaggregation, we found a paucity of usable raw data sets and a generalized lack of standardization of captured data, making comparisons difficult. A second round of data capture in April found more complete, but even more disparate, information. Our analysis revealed a wide range of sex ratios among confirmed cases. In countries where a male bias was initially reported, the proportion of women dramatically increased in 3 weeks. Analysis also revealed a complex pattern of sex ratio variation with age. Accurate, peer-reviewed, analysis of harmonized, sex-disaggregated data for characteristics of epidemics, such as availability of testing, suspected source of infection, or comorbidities, will be critical to understand where the observed disparities come from and to generate evidence-based recommendations for decision-making by governments.

nanotatoR: a tool for enhanced annotation of genomic structural variants.

BACKGROUND: Whole genome sequencing is effective at identification of small variants, but because it is based on short reads, assessment of structural variants (SVs) is limited. The advent of Optical Genome Mapping (OGM), which utilizes long fluorescently labeled DNA molecules for de novo genome assembly and SV calling, has allowed for increased sensitivity and specificity in SV detection. However, compared to small variant annotation tools, OGM-based SV annotation software has seen little development, and currently available SV annotation tools do not provide sufficient information for determination of variant pathogenicity. RESULTS: We developed an R-based package, nanotatoR, which provides comprehensive annotation as a tool for SV classification. nanotatoR uses both external (DGV; DECIPHER; Bionano Genomics BNDB) and internal (user-defined) databases to estimate SV frequency. Human genome reference GRCh37/38-based BED files are used to annotate SVs with overlapping, upstream, and downstream genes. Overlap percentages and distances for nearest genes are calculated and can be used for filtration. A primary gene list is extracted from public databases based on the patient's phenotype and used to filter genes overlapping SVs, providing the analyst with an easy way to prioritize variants. If available, expression of overlapping or nearby genes of interest is extracted (e.g. from an RNA-Seq dataset, allowing the user to assess the effects of SVs on the transcriptome). Most quality-control filtration parameters are customizable by the user. The output is given in an Excel file format, subdivided into multiple sheets based on SV type and inheritance pattern (INDELs, inversions, translocations, de novo, etc.). nanotatoR passed all quality and run time criteria of Bioconductor, where it was accepted in the April 2019 release. We evaluated nanotatoR's annotation capabilities using publicly available reference datasets: the singleton sample NA12878, mapped with two types of enzyme labeling, and the NA24143 trio. nanotatoR was also able to accurately filter the known pathogenic variants in a cohort of patients with Duchenne Muscular Dystrophy for which we had previously demonstrated the diagnostic ability of OGM. CONCLUSIONS: The extensive annotation enables users to rapidly identify potential pathogenic SVs, a critical step toward use of OGM in the clinical setting.

Newborn Screening Protocols and Positive Predictive Value for Congenital Adrenal Hyperplasia Vary across the United States.

Newborn screening for congenital adrenal hyperplasia (CAH) caused by 21-hydroxylase deficiency is mandated throughout the US. Filter paper blood specimens are assayed for 17-hydroxyprogesterone (17OHP). Prematurity, low birth weight, or critical illness cause falsely elevated results. The purpose of this report is to highlight differences in protocols among US state laboratories. We circulated a survey to state laboratory directors requesting qualitative and quantitative information about individual screening programs. Qualitative and quantitative information provided by 17 state programs were available for analysis. Disease prevalence ranged from 1:9941 to 1:28,661 live births. Four state laboratories mandated a second screen regardless of the initial screening results; most others did so for infants in intensive care units. All but one program utilized birthweight cut-points, but cutoffs varied widely: 17OHP values of 25 to 75 ng/mL for birthweights >2250-2500 g. The positive predictive values for normal birthweight infants varied from 0.7% to 50%, with the highest predictive values based in two of the states with a mandatory second screen. Data were unavailable for negative predictive values. These data imply differences in sensitivity and specificity in CAH screening in the US. Standardization of newborn screening protocols could improve the positive predictive value.

Loss-of-Function Variants in PPP1R12A: From Isolated Sex Reversal to Holoprosencephaly Spectrum and Urogenital Malformations.

In two independent ongoing next-generation sequencing projects for individuals with holoprosencephaly and individuals with disorders of sex development, and through international research collaboration, we identified twelve individuals with de novo loss-of-function (LoF) variants in protein phosphatase 1, regulatory subunit 12a (PPP1R12A), an important developmental gene involved in cell migration, adhesion, and morphogenesis. This gene has not been previously reported in association with human disease, and it has intolerance to LoF as illustrated by a very low observed-to-expected ratio of LoF variants in gnomAD. Of the twelve individuals, midline brain malformations were found in five, urogenital anomalies in nine, and a combination of both phenotypes in two. Other congenital anomalies identified included omphalocele, jejunal, and ileal atresia with aberrant mesenteric blood supply, and syndactyly. Six individuals had stop gain variants, five had a deletion or duplication resulting in a frameshift, and one had a canonical splice acceptor site loss. Murine and human in situ hybridization and immunostaining revealed PPP1R12A expression in the prosencephalic neural folds and protein localization in the lower urinary tract at critical periods for forebrain division and urogenital development. Based on these clinical and molecular findings, we propose the association of PPP1R12A pathogenic variants with a congenital malformations syndrome affecting the embryogenesis of the brain and genitourinary systems and including disorders of sex development.

Translating genomics to the clinical diagnosis of disorders/differences of sex development.

The medical and psychosocial challenges faced by patients living with Disorders/Differences of Sex Development (DSD) and their families can be alleviated by a rapid and accurate diagnostic process. Clinical diagnosis of DSD is limited by a lack of standardization of anatomical and endocrine phenotyping and genetic testing, as well as poor genotype/phenotype correlation. Historically, DSD genes have been identified through positional cloning of disease-associated variants segregating in families and validation of candidates in animal and in vitro modeling of variant pathogenicity. Owing to the complexity of conditions grouped under DSD, genome-wide scanning methods are better suited for identifying disease causing gene variant(s) and providing a clinical diagnosis. Here, we review a number of established genomic tools (karyotyping, chromosomal microarrays and exome sequencing) used in clinic for DSD diagnosis, as well as emerging genomic technologies such as whole-genome (short-read) sequencing, long-read sequencing, and optical mapping used for novel DSD gene discovery. These, together with gene expression and epigenetic studies can potentiate the clinical diagnosis of DSD diagnostic rates and enhance the outcomes for patients and families.

New technologies to uncover the molecular basis of disorders of sex development.

The elegant developmental biology experiments conducted in the 1940s by French physiologist Alfred Jost demonstrated that the sexual phenotype of a mammalian embryo depended whether the embryonic gonad develops into a testis or not. In humans, anomalies in the processes that regulate development of chromosomal, gonadal or anatomic sex result in a spectrum of conditions termed Disorders/Differences of Sex Development (DSD). Each of these conditions is rare, and understanding of their genetic etiology is still incomplete. Historically, DSD diagnoses have been difficult to establish due to the lack of standardization of anatomical and endocrine phenotyping procedures as well as genetic testing. Yet, a definitive diagnosis is critical for optimal management of the medical and psychosocial challenges associated with DSD conditions. The advent in the clinical realm of next-generation sequencing methods, with constantly decreasing price and turnaround time, has revolutionized the diagnostic process. Here we review the successes and limitations of the genetic methods currently available for DSD diagnosis, including Sanger sequencing, karyotyping, exome sequencing and chromosomal microarrays. While exome sequencing provides higher diagnostic rates, many patients still remain undiagnosed. Newer approaches, such as whole-genome sequencing and whole-genome mapping, along with gene expression studies, have the potential to identify novel DSD-causing genes and significantly increase total diagnostic yield, hopefully shortening the patient's journey to an accurate diagnosis and enhancing health-related quality-of-life outcomes for patients and families.

Identification of novel candidate genes for 46,XY disorders of sex development (DSD) using a C57BL/6J-Y POS mouse model.

BACKGROUND: Disorders of sex development (DSD) have an estimated frequency of 0.5% of live births encompassing a variety of urogenital anomalies ranging from mild hypospadias to a discrepancy between sex chromosomes and external genitalia. In order to identify the underlying genetic etiology, we had performed exome sequencing in a subset of DSD cases with 46,XY karyotype and were able to identify the causative genetic variant in 35% of cases. While the genetic etiology was not ascertained in more than half of the cases, a large number of variants of unknown clinical significance (VUS) were identified in those exomes. METHODS: To investigate the relevance of these VUS in regards to the patient's phenotype, we utilized a mouse model in which the presence of a Y chromosome from the poschiavinus strain (Y POS ) on a C57BL/6J (B6) background results in XY undervirilization and sex reversal, a phenotype characteristic to a large subset of human 46,XY DSD cases. We assessed gene expression differences between B6-Y B6 and undervirilized B6-Y POS gonads at E11.5 and identified 515 differentially expressed genes (308 underexpressed and 207 overexpressed in B6-Y POS males). RESULTS: We identified 15 novel candidate genes potentially involved in 46,XY DSD pathogenesis by filtering the list of human VUS-carrying genes provided by exome sequencing with the list of differentially expressed genes from B6-Y POS mouse model. Additionally, we identified that 7 of the 15 candidate genes were significantly underexpressed in the XY gonads of mice with suppressed Sox9 expression in Sertoli cells suggesting that some of the candidate genes may be downstream of a well-known sex determining gene, Sox9. CONCLUSION: The use of a DSD-specific animal model improves variant interpretation by correlating human sequence variants with transcriptome variation.

Psychosocial Screening in Disorders/Differences of Sex Development: Psychometric Evaluation of the Psychosocial Assessment Tool.

BACKGROUND/AIMS: Utilization of a psychosocial screener to identify families affected by a disorder/difference of sex development (DSD) and at risk for adjustment challenges may facilitate efficient use of team resources to optimize care. The Psychosocial Assessment Tool (PAT) has been used in other pediatric conditions. The current study explored the reliability and validity of the PAT (modified for use within the DSD population; PAT-DSD). METHODS: Participants were 197 families enrolled in the DSD-Translational Research Network (DSD-TRN) who completed a PAT-DSD during a DSD clinic visit. Psychosocial data were extracted from the DSD-TRN clinical registry. Internal reliability of the PAT-DSD was tested using the Kuder-Richardson-20 coefficient. Validity was examined by exploring the correlation of the PAT-DSD with other measures of caregiver distress and child emotional-behavioral functioning. RESULTS: One-third of families demonstrated psychosocial risk (27.9% "Targeted" and 6.1% "Clinical" level of risk). Internal reliability of the PAT-DSD Total score was high (α = 0.86); 4 of 8 subscales met acceptable internal reliability. A priori predicted relationships between the PAT-DSD and other psychosocial measures were supported. The PAT-DSD Total score related to measures of caregiver distress (r = 0.40, p < 0.001) and to both caregiver-reported and patient self-reported behavioral problems (r = 0.61, p < 0.00; r = 0.37, p < 0.05). CONCLUSIONS: This study provides evidence for the reliability and validity of the PAT-DSD. Given variability in the internal reliability across subscales, this measure is best used to screen for overall family risk, rather than to assess specific psychosocial concerns.