Genetic Variants in SRD5A2 in a Spectrum of DSD Patients from Australian Clinics Highlight Importance of Genetic Testing alongside Typical First-Line Investigations.

INTRODUCTION: Steroid 5-alpha reductase deficiency (5α-R2D) is a rare condition caused by genetic variants that reduce the activity of the enzyme that converts testosterone into dihydrotestosterone. The clinical spectrum of 5α-R2D is known to overlap with other 46,XY differences of sex development (DSD) such as androgen insensitivity or gonadal dysgenesis. However, the clinical trajectories of the aetiologies can differ, with 5α-R2D presenting its own challenges. METHODS: In this study, we have collated clinical information for five individuals with variants in SRD5A2 identified using research genetic testing in an Australian paediatric setting. RESULTS: We describe how a genetic finding resolved or confirmed a diagnosis for these individuals and how it guided clinical management and family counselling. CONCLUSION: This work highlights the importance of early genetic testing in children born with 46,XY DSD where it complements traditional first-line testing.

Babies born with ambiguous genitalia: Developing an educational resource for Australian midwives.

PROBLEM AND BACKGROUND: The birth of a baby with ambiguous genitalia is rare and usually unexpected. Parents often receive inconsistent language from health-professionals after the birth. Initial interaction with the birth team has long-term consequences for families with babies born with ambiguous genitalia. AIM: Understand the current practices on the day of birth and explore knowledge gaps for midwives regarding babies born with ambiguous genitalia. Develop educational content that can enable midwives to respond appropriately when the sex of a baby is unclear. METHODS: This study included two phases, utilising qualitative descriptive research design with semi-structured interviews to understand the experiences of midwives caring for babies with ambiguous genitalia and their families. The findings informed the development a midwifery educational resource using these qualitative findings. FINDINGS: Our analysis of 14 interviews with Australian midwives identified that they had no formal education to support families with a baby with ambiguous genitalia. Emotional support, advocacy and medical information translation were areas midwives perceived as essential skills to support these families. DISCUSSION: Midwives provide a unique role in parental birth experiences. Themes that arose emphasised their psychosocial support role but lacked formal education and guidance on this topic. Midwives had learnt from the media about babies born with ambiguous genitalia and wanted evidence-based education to support parents. Midwife education focusing on both psychosocial and clinical care for parents and their baby with ambiguous genitalia is crucial. CONCLUSION: Midwives can play a pivotal role in supporting parents with a baby with ambiguous genitalia. Themes from this qualitative study informed the development of a midwifery education digital resource.

Premature Ovarian Insufficiency in CLPB Deficiency: Transcriptomic, Proteomic and Phenotypic Insights.

CONTEXT: Premature ovarian insufficiency (POI) is a common form of female infertility that usually presents as an isolated condition but can be part of various genetic syndromes. Early diagnosis and treatment of POI can minimize comorbidity and improve health outcomes. OBJECTIVE: We aimed to determine the genetic cause of syndromic POI, intellectual disability, neutropenia, and cataracts. METHODS: We performed whole-exome sequencing (WES) followed by functional validation via RT-PCR, RNAseq, and quantitative proteomics, as well as clinical update of previously reported patients with variants in the caseinolytic peptidase B (CLPB) gene. RESULTS: We identified causative variants in CLPB, encoding a mitochondrial disaggregase. Variants in this gene are known to cause an autosomal recessive syndrome involving 3-methylglutaconic aciduria, neurological dysfunction, cataracts, and neutropenia that is often fatal in childhood; however, there is likely a reporting bias toward severe cases. Using RNAseq and quantitative proteomics we validated causation and gained insight into genotype:phenotype correlation. Clinical follow-up of patients with CLPB deficiency who survived to adulthood identified POI and infertility as a common postpubertal ailment. CONCLUSION: A novel splicing variant is associated with CLPB deficiency in an individual who survived to adulthood. POI is a common feature of postpubertal female individuals with CLPB deficiency. Patients with CLPB deficiency should be referred to pediatric gynecologists/endocrinologists for prompt POI diagnosis and hormone replacement therapy to minimize associated comorbidities.

Identifying the Resource Needs of Young People with Differences of Sex Development.

Adolescents with differences of sex development (DSD) often have complex medical, surgical, and psychological care needs and require age-appropriate resources. This cross-sectional study describes the past and current experiences of adolescents and young adults with DSD and their need for information and support. Participants aged 14−30 years with DSD diagnoses were identified, either from departmental records at the Royal Children's Hospital (RCH), Melbourne, Australia, or from the private practice of a gynecologist linked to RCH. Anonymized data were collected from a specifically designed online survey. Of the 314 successfully traced patients, 91 (28.9%) completed the survey. Amongst respondents, older age was strongly correlated with higher levels of distress at the time of disclosure (b = 0.67, p < 0.001). People who reported greater understanding of their condition (b = −0.45, p = 0.010) and higher levels of support (b = −0.40, p = 0.003) identified lower levels of current distress. Respondents preferred to receive information from a specialist doctor, GP, or websites and reported information needs being highest during adolescence. Only one in four respondents recalled ever being offered psychological support. A number of perceived barriers to accessing support were identified. Our findings indicate that young people's information and support needs may be best met by improving online resources, as well as increasing introductions to knowledgeable and appropriate primary care physicians, psychological services, and peer support groups. Further work to promote and increase engagement with psychological and peer support for those with DSD will be important.

Role of cross-campus multidisciplinary team meetings in decision-making for children and adolescents with differences of sex development/intersex.

AIM: Management of children with differences/disorders of sex development (DSD) is complex with limited evidence to guide clinical decisions. Regular multidisciplinary team meetings were set up in Sydney and Melbourne paediatric hospitals to enable systematic peer review of complex decision-making. We aim to describe the workload and role of these meetings. METHODS: The multidisciplinary team forum includes invited representatives from endocrinology, urology, gynaecology, genetics, psychology, social work, clinical ethics, laboratory and hospital executive and meetings occur 1-3 times monthly. Descriptive data were collected from de-identified meeting referrals and minutes between August 2012 to August 2018 (Sydney) and January 2014 to August 2018 (Melbourne). RESULTS: A total of 192 referrals (142 new and 50 follow-ups) aged 1 week to 17 years were discussed across the two sites. 46, XY DSD (n = 81) was the most common sub-classification. Consideration of surgical options and optimal management of gonads with malignant potential were amongst the common reasons for referral to the multidisciplinary team meetings. Surgical interventions were considered but not recommended after review for 38 of 154 (24.7%) procedures. Gonad retention to allow potential functional benefit was recommended in 15/46 (32.6%) referrals. Evidence of premalignant or malignant changes was found in 20/57 (35%) gonads removed, with dysgenetic features and atrophy/streak features in 6 (10.5%) and 27 (47.4%) gonads respectively. CONCLUSION: Formal DSD multidisciplinary team meetings provide a framework and opportunity for multi and interdisciplinary discussions amongst representatives from several specialities to help make complex decision-making.

Genomic sequencing highlights the diverse molecular causes of Perrault syndrome: a peroxisomal disorder (PEX6), metabolic disorders (CLPP, GGPS1), and mtDNA maintenance/translation disorders (LARS2, TFAM).

Perrault syndrome is a rare heterogeneous condition characterised by sensorineural hearing loss and premature ovarian insufficiency. Additional neuromuscular pathology is observed in some patients. There are six genes in which variants are known to cause Perrault syndrome; however, these explain only a minority of cases. We investigated the genetic cause of Perrault syndrome in seven affected individuals from five different families, successfully identifying the cause in four patients. This included previously reported and novel causative variants in known Perrault syndrome genes, CLPP and LARS2, involved in mitochondrial proteolysis and mitochondrial translation, respectively. For the first time, we show that pathogenic variants in PEX6 can present clinically as Perrault syndrome. PEX6 encodes a peroxisomal biogenesis factor, and we demonstrate evidence of peroxisomal dysfunction in patient serum. This study consolidates the clinical overlap between Perrault syndrome and peroxisomal disorders, and highlights the need to consider ovarian function in individuals with atypical/mild peroxisomal disorders. The remaining patients had variants in candidate genes such as TFAM, involved in mtDNA transcription, replication, and packaging, and GGPS1 involved in mevalonate/coenzyme Q10 biosynthesis and whose enzymatic product is required for mouse folliculogenesis. This genomic study highlights the diverse molecular landscape of this poorly understood syndrome.

TP63-truncating variants cause isolated premature ovarian insufficiency.

Premature ovarian insufficiency involves amenorrhea and elevated follicle-stimulating hormone before age 40, and its genetic basis is poorly understood. Here, we study 13 premature ovarian insufficiency (POI) patients using whole-exome sequencing. We identify PREPL and TP63 causative variants, and variants in other potentially novel POI genes. PREPL deficiency is a known cause of syndromic POI, matching the patients' phenotype. A role for TP63 in ovarian biology has previously been proposed but variants have been described in multiorgan syndromes, and not isolated POI. One patient with isolated POI harbored a de novo nonsense TP63 variant in the terminal exon and an unrelated patient had a different nonsense variant in the same exon. These variants interfere with the repression domain while leaving the activation domain intact. We expand the phenotypic spectrum of TP63-related disorders, provide a new genotype:phenotype correlation for TP63 and identify a new genetic cause of isolated POI.

Identification of Candidate Genes for Mayer-Rokitansky-Küster-Hauser Syndrome Using Genomic Approaches.

Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome is a disorder of sex development which affects 1 in 4,500 females and is characterized by agenesis of müllerian structures, including the uterus, cervix, and upper vagina. It can occur in isolation (type 1) or in conjunction with various anomalies (type 2), with a subset of these comprising müllerian, renal, and cervicothoracic abnormalities (MURCS) association. The genetic causes of MRKH have been investigated previously yielding limited results, with massive parallel sequencing becoming increasingly utilized. We sought to identify genetic contributions to MRKH using a combination of microarray and whole exome sequencing (WES) on a cohort of 8 unrelated women with MRKH and MURCS. WES data were analysed using a candidate gene approach to identify potential contributing variants. Microarray analysis identified a 0.6-Mb deletion in the previously implicated 16p11.2 region in a patient with MRKH type 2. WES revealed 16 rare nonsynonymous variants in MRKH candidate genes across the cohort. These included variants in several genes, such as LRP10 and DOCK4, associated with disorders with müllerian anomalies. Further functional studies of these variants will help to delineate their biological significance and expand the genotypic spectrum of MRKH.

Identification of variants in pleiotropic genes causing "isolated" premature ovarian insufficiency: implications for medical practice.

Next-generation sequencing (NGS) is increasingly being used in a clinical setting for the molecular diagnosis of patients with heterogeneous disorders, such as premature ovarian insufficiency (POI). We performed NGS of ~1000 candidate genes in four unrelated patients with POI. We discovered the genetic cause of "isolated" POI in two cases, both of which had causative variants in surprising genes. In the first case, a homozygous nonsense variant in NBN was causative. Recessive function-altering NBN variants typically cause Nijmegen breakage syndrome characterized by microcephaly, cancer predisposition, and immunodeficiency, none of which are evident in the patient. At a cellular level, we found evidence of chromosomal instability. In the second case, compound heterozygous variants in EIF2B2 were causative. Recessive EIF2B2 function-altering variants usually cause leukoencephalopathy with episodic decline. Subsequent MRI revealed subclinical neurological abnormalities. These cases demonstrate that variants in NBN and EIF2B2, which usually cause severe syndromes, can cause apparently isolated POI, and that (1) NGS can precede clinical diagnosis and guide patient management, (2) NGS can redefine the phenotypic spectrum of syndromes, and (3) NGS may make unanticipated diagnoses that must be sensitively communicated to patients. Although there is rigorous debate about the handling of secondary/incidental findings using NGS, there is little discussion of the management of causative pleiotropic gene variants that have broader implications than that for which genetic studies were sought.

Functional characterization of novel NR5A1 variants reveals multiple complex roles in disorders of sex development.

Variants in the NR5A1 gene encoding SF1 have been described in a diverse spectrum of disorders of sex development (DSD). Recently, we reported the use of a targeted gene panel for DSD where we identified 15 individuals with a variant in NR5A1, nine of which are novel. Here, we examine the functional effect of these changes in relation to the patient phenotype. All novel variants tested had reduced trans-activational activity, while several had altered protein level, localization, or conformation. In addition, we found evidence of new roles for SF1 protein domains including a region within the ligand binding domain that appears to contribute to SF1 regulation of Müllerian development. There was little correlation between the severity of the phenotype and the nature of the NR5A1 variant. We report two familial cases of NR5A1 deficiency with evidence of variable expressivity; we also report on individuals with oligogenic inheritance. Finally, we found that the nature of the NR5A1 variant does not inform patient outcomes (including pubertal androgenization and malignancy risk). This study adds nine novel pathogenic NR5A1 variants to the pool of diagnostic variants. It highlights a greater need for understanding the complexity of SF1 function and the additional factors that contribute.