45,X/46,XY mosaicism: Clinical manifestations and long term follow-up.

45,X/46,XY chromosomal mosaicism presents a range of clinical manifestations, including phenotypes from Turner syndrome through genital abnormalities to apparently unaffected phenotypic males; however, the full clinical spectrum has not yet been fully delineated since prior studies on the clinical phenotype and associated risk of gonadal tumors included small cohorts and limited follow-up. To better describe the clinical manifestations and long-term outcome of patients with 45,X/46,XY mosaicism. We conducted a retrospective chart review of patients with 45,X/46,XY from three health centers (Hospital for Sick Children and Mount Sinai Hospital in Canada, and University of Pittsburgh Medical Center in United States). Of 100 patients with 45,X/46,XY karyotype, 47 were raised as females and 53 as males. Females were significantly shorter than males (p = 0.04) and height Z-score was significantly decreased with age for both genders (p = 0.02). Growth hormone (GH) treatment did not result in a significant height increase compared to the untreated group (p = 0.5). All females required puberty induction in contrast to majority of males. Five females were diagnosed with gonadal tumors, while no males were affected. Around 58% of patients exhibited at least one Turner syndrome stigmata. This study expands the clinical spectrum, long-term outcomes, and associated tumor risk in a large cohort of patients with 45,X/46,XY mosaicism. Additionally, it highlights our experience with GH therapy and prophylactic gonadectomy.

A POT1 mutation implicates defective telomere end fill-in and telomere truncations in Coats plus.

Coats plus (CP) can be caused by mutations in the CTC1 component of CST, which promotes polymerase α (polα)/primase-dependent fill-in throughout the genome and at telomeres. The cellular pathology relating to CP has not been established. We identified a homozygous POT1 S322L substitution (POT1(CP)) in two siblings with CP. POT1(CP)induced a proliferative arrest that could be bypassed by telomerase. POT1(CP)was expressed at normal levels, bound TPP1 and telomeres, and blocked ATR signaling. POT1(CP)was defective in regulating telomerase, leading to telomere elongation rather than the telomere shortening observed in other telomeropathies. POT1(CP)was also defective in the maintenance of the telomeric C strand, causing extended 3' overhangs and stochastic telomere truncations that could be healed by telomerase. Consistent with shortening of the telomeric C strand, metaphase chromosomes showed loss of telomeres synthesized by leading strand DNA synthesis. We propose that CP is caused by a defect in POT1/CST-dependent telomere fill-in. We further propose that deficiency in the fill-in step generates truncated telomeres that halt proliferation in cells lacking telomerase, whereas, in tissues expressing telomerase (e.g., bone marrow), the truncations are healed. The proposed etiology can explain why CP presents with features distinct from those associated with telomerase defects (e.g., dyskeratosis congenita).

CYP26B1-related disorder: expanding the ends of the spectrum through clinical and molecular evidence.

CYP26B1 metabolizes retinoic acid in the developing embryo to regulate its levels. A limited number of individuals with pathogenic variants in CYP26B1 have been documented with a varied phenotypic spectrum, spanning from a severe manifestation involving skull anomalies, craniosynostosis, encephalocele, radio-humeral fusion, oligodactyly, and a narrow thorax, to a milder presentation characterized by craniosynostosis, restricted radio-humeral joint mobility, hearing loss, and intellectual disability. Here, we report two families with CYP26B1-related phenotypes and describe the data obtained from functional studies of the variants. Exome and Sanger sequencing were used for variant identification in family 1 and family 2, respectively. Family 1 reflects a mild phenotype, which includes craniofacial dysmorphism with brachycephaly (without craniosynostosis), arachnodactyly, reduced radioulnar joint movement, conductive hearing loss, learning disability-and compound heterozygous CYP26B1 variants: (p.[(Pro118Leu)];[(Arg234Gln)]) were found. In family 2, a stillborn fetus presented a lethal phenotype with spina bifida occulta, hydrocephalus, poor skeletal mineralization, synostosis, limb defects, and a synonymous homozygous variant in CYP26B1: c.1083C > A. A minigene assay revealed that the synonymous variant created a new splice site, removing part of exon 5 (p.Val361_Asp382del). Enzymatic activity was assessed using a luciferase assay, demonstrating a notable reduction in exogenous retinoic acid metabolism for the variant p.Val361_Asp382del. (~ 3.5 × decrease compared to wild-type); comparatively, the variants p.(Pro118Leu) and p.(Arg234Gln) demonstrated a partial loss of metabolism (1.7× and 2.3× reduction, respectively). A proximity-dependent biotin identification assay reaffirmed previously reported ER-resident protein interactions. Additional work into these interactions is critical to determine if CYP26B1 is involved with other biological events on the ER. Immunofluorescence assay suggests that mutant CYP26B1 is still localized in the endoplasmic reticulum. These results indicate that novel pathogenic variants in CYP26B1 result in varying levels of enzymatic activity that impact retinoic acid metabolism and relate to the distinct phenotypes observed.

Barriers in applying to genetic counseling Master's degree programs: Perceptions of prospective applicants when compared with Canadian admissions committee members.

The goal of this study was to identify potential barriers in applying to a genetic counseling (GC) Master's degree program to inform strategies for increasing diversity and inclusiveness in the GC student recruitment process. Participants included prospective GC program applicants and admissions committee members from the four Canadian accredited programs. The study was conducted using a quantitative survey-based approach. Twenty-five prospective applicants who previously applied to a GC Master's degree program, 26 who had not applied, and 48 admissions committee members completed the survey. The small number of positions in GC programs was perceived by all groups as highly likely to impact an applicant's ability to gain acceptance to a program as was the limited number of GC training programs. Prospective applicants perceived additional barriers as significantly more likely to impact an individual's ability to apply to/attend a program when compared with admissions committee members including: cost of the application process, the applicant being a visible minority and the applicant having a physical disability. These findings highlight a number of perceived barriers related to applying to a GC Master's degree program. To our knowledge, this is the first study surveying prospective applicants and admissions committee members on barriers faced during the application process. The data from this study can also be used to inform the application process for other health professions.

The Clinician-reported Genetic testing Utility InDEx (C-GUIDE): Preliminary evidence of validity and reliability.

PURPOSE: Demonstrating the clinical utility of genetic testing is fundamental to clinical adoption and reimbursement, but standardized definitions and measurement strategies for this construct do not exist. The Clinician-reported Genetic testing Utility InDEx (C-GUIDE) offers a novel measure to fill this gap. This study assessed its validity and inter-rater reliability. METHODS: Genetics professionals completed C-GUIDE after disclosure of test results to patients. Construct validity was assessed using regression analysis to measure associations between C-GUIDE and global item scores as well as potentially explanatory variables. Inter-rater reliability was assessed by administering a vignette-based survey to genetics professionals and calculating Krippendorff's α. RESULTS: On average, a 1-point increase in the global item score was associated with an increase of 3.0 in the C-GUIDE score (P < .001). Compared with diagnostic results, partially/potentially diagnostic and nondiagnostic results were associated with a reduction in C-GUIDE score of 9.5 (P < .001) and 10.2 (P < .001), respectively. Across 19 vignettes, Krippendorff's α was 0.68 (95% CI: 0.63-0.72). CONCLUSION: C-GUIDE showed acceptable validity and inter-rater reliability. Although further evaluation is required, C-GUIDE version 1.2 can be useful as a standardized approach to assess the clinical utility of genetic testing.

De novo pathogenic variant in TUBB2A presenting with arthrogryposis multiplex congenita, brain abnormalities, and severe developmental delay.

Disorders of brain formation can occur from pathogenic variants in various alpha and beta tubulin genes. Heterozygous pathogenic variants in the beta tubulin isotype A gene, TUBB2A, have been recently implicated in brain malformations, seizures, and developmental delay. Limited information is known regarding the phenotypic spectrum associated with pathogenic variants in this gene given the rarity of the condition. We report the sixth individual with a de novo heterozygous TUBB2A pathogenic variant, who presented with a severe neurological phenotype along with unique features of arthrogryposis multiplex congenita, optic nerve hypoplasia, dysmorphic facial features, and vocal cord paralysis, thereby expanding the gene-related phenotype.

Experience with genetic counseling: the adolescent perspective.

Adolescence is a complex period of development that involves creating a sense of identity, autonomy, relationships and values. This stage of adjustment can be complicated by having a genetic condition. Genetic counseling can play an important role in providing information and support to this patient population; however, resources and guidelines are currently limited. In order to appropriately establish genetic counseling approaches and resource development, we investigated the experiences and perspectives of adolescents with a genetic condition with respect to their genetic counseling interactions. Using a qualitative exploratory approach, eleven semi-structured interviews were conducted with adolescents diagnosed with a genetic condition who received genetic counseling between the ages of 12 and 18 years at The Hospital for Sick Children. Transcripts were analyzed thematically using qualitative content analysis, from which three major interrelated themes emerged: 1) understanding the genetic counselor's role; 2) increasing perceived personal control; and 3) adolescent-specific factors influencing adaptation to one's condition. Additionally, a list of suggested tools and strategies for genetic counseling practice were elucidated. Our findings can contribute to the development of an adolescent-focused framework to enhance emerging genetic counseling approaches for this patient population, and can also facilitate the transition process from pediatric to adult care within patient and family-centered contexts.

Missense mutations in ß-1,3-N-acetylglucosaminyltransferase 1 (B3GNT1) cause Walker-Warburg syndrome.

Several known or putative glycosyltransferases are required for the synthesis of laminin-binding glycans on alpha-dystroglycan (αDG), including POMT1, POMT2, POMGnT1, LARGE, Fukutin, FKRP, ISPD and GTDC2. Mutations in these glycosyltransferase genes result in defective αDG glycosylation and reduced ligand binding by αDG causing a clinically heterogeneous group of congenital muscular dystrophies, commonly referred to as dystroglycanopathies. The most severe clinical form, Walker-Warburg syndrome (WWS), is characterized by congenital muscular dystrophy and severe neurological and ophthalmological defects. Here, we report two homozygous missense mutations in the ß-1,3-N-acetylglucosaminyltransferase 1 (B3GNT1) gene in a family affected with WWS. Functional studies confirmed the pathogenicity of the mutations. First, expression of wild-type but not mutant B3GNT1 in human prostate cancer (PC3) cells led to increased levels of αDG glycosylation. Second, morpholino knockdown of the zebrafish b3gnt1 orthologue caused characteristic muscular defects and reduced αDG glycosylation. These functional studies identify an important role of B3GNT1 in the synthesis of the uncharacterized laminin-binding glycan of αDG and implicate B3GNT1 as a novel causative gene for WWS.

Mutations in EZH2 cause Weaver syndrome.

We used trio-based whole-exome sequencing to analyze two families affected by Weaver syndrome, including one of the original families reported in 1974. Filtering of rare variants in the affected probands against the parental variants identified two different de novo mutations in the enhancer of zeste homolog 2 (EZH2). Sanger sequencing of EZH2 in a third classically-affected proband identified a third de novo mutation in this gene. These data show that mutations in EZH2 cause Weaver syndrome.

Characterization of human disease phenotypes associated with mutations in TREX1, RNASEH2A, RNASEH2B, RNASEH2C, SAMHD1, ADAR, and IFIH1.

Aicardi-Goutières syndrome is an inflammatory disease occurring due to mutations in any of TREX1, RNASEH2A, RNASEH2B, RNASEH2C, SAMHD1, ADAR or IFIH1. We report on 374 patients from 299 families with mutations in these seven genes. Most patients conformed to one of two fairly stereotyped clinical profiles; either exhibiting an in utero disease-onset (74 patients; 22.8% of all patients where data were available), or a post-natal presentation, usually within the first year of life (223 patients; 68.6%), characterized by a sub-acute encephalopathy and a loss of previously acquired skills. Other clinically distinct phenotypes were also observed; particularly, bilateral striatal necrosis (13 patients; 3.6%) and non-syndromic spastic paraparesis (12 patients; 3.4%). We recorded 69 deaths (19.3% of patients with follow-up data). Of 285 patients for whom data were available, 210 (73.7%) were profoundly disabled, with no useful motor, speech and intellectual function. Chilblains, glaucoma, hypothyroidism, cardiomyopathy, intracerebral vasculitis, peripheral neuropathy, bowel inflammation and systemic lupus erythematosus were seen frequently enough to be confirmed as real associations with the Aicardi-Goutieres syndrome phenotype. We observed a robust relationship between mutations in all seven genes with increased type I interferon activity in cerebrospinal fluid and serum, and the increased expression of interferon-stimulated gene transcripts in peripheral blood. We recorded a positive correlation between the level of cerebrospinal fluid interferon activity assayed within one year of disease presentation and the degree of subsequent disability. Interferon-stimulated gene transcripts remained high in most patients, indicating an ongoing disease process. On the basis of substantial morbidity and mortality, our data highlight the urgent need to define coherent treatment strategies for the phenotypes associated with mutations in the Aicardi-Goutières syndrome-related genes. Our findings also make it clear that a window of therapeutic opportunity exists relevant to the majority of affected patients and indicate that the assessment of type I interferon activity might serve as a useful biomarker in future clinical trials.

Mutations in genes encoding ribonuclease H2 subunits cause Aicardi-Goutières syndrome and mimic congenital viral brain infection.

Aicardi-Goutières syndrome (AGS) is an autosomal recessive neurological disorder, the clinical and immunological features of which parallel those of congenital viral infection. Here we define the composition of the human ribonuclease H2 enzyme complex and show that AGS can result from mutations in the genes encoding any one of its three subunits. Our findings demonstrate a role for ribonuclease H in human neurological disease and suggest an unanticipated relationship between ribonuclease H2 and the antiviral immune response that warrants further investigation.

Deletions in 16q24.2 are associated with autism spectrum disorder, intellectual disability and congenital renal malformation.

BACKGROUND: The contribution of copy-number variation (CNV) to disease has been highlighted with the widespread adoption of array-based comparative genomic hybridisation (aCGH) and microarray technology. Contiguous gene deletions involving ANKRD11 in 16q24.3 are associated with autism spectrum disorder (ASD) and intellectual disability (ID), while 16q24.1 deletions affecting FOXF1 are associated with congenital renal malformations, alveolar capillary dysplasia, and various other abnormalities. The disease associations of deletions in the intervening region, 16q24.2, have only been defined to a limited extent. AIM: To determine whether deletions affecting 16q24.2 are correlated with congenital anomalies. METHODS: 35 individuals, each having a deletion in 16q24.2, were characterised clinically and by aCGH and/or SNP-genotyping microarray. RESULTS: Several of the 35 16q24.2 deletions identified here closely abut or overlap the coding regions of FOXF1 and ANKRD11, two genes that have been previously associated with the disease. 25 patients were reported to have ASD/ID, and three were found to have bilateral hydronephrosis. 14 of the deletions associated with ASD/ID overlap the coding regions of FBXO31 and MAP1LC3B. These same genes and two others, C16orf95 and ZCCHC14, are also included in the area of minimal overlap of the three deletions associated with hydronephrosis. CONCLUSIONS: Our data highlight 16q24.2 as a region of interest for ASD, ID and congenital renal malformations. These conditions are associated, albeit without complete penetrance, with deletions affecting C16orf95, ZCCHC14, MAP1LC3B and FBXO31. The function of each gene in development and disease warrants further investigation.

Parental Preferences for Expanded Newborn Screening: What Are the Limits?

The use of next-generation sequencing technologies such as genomic sequencing in newborn screening (NBS) could enable the detection of a broader range of conditions. We explored parental preferences and attitudes towards screening for conditions for which varying types of treatment exist with a cross-sectional survey completed by 100 parents of newborns who received NBS in Ontario, Canada. The survey included four vignettes illustrative of hypothetical screening targets, followed by questions assessing parental attitudes. Chi-square tests were used to compare frequency distributions of preferences. Results show that most parents supported NBS for conditions for which only supportive interventions are available, but to a significantly lesser degree than those with disease-specific treatments (99% vs. 82-87%, p ≤ 0.01). For conditions without an effective treatment, the type of supportive care and age of onset of the condition did not significantly alter parent perceptions of risks and benefits. Parents are interested in expanded NBS for conditions with only supportive interventions in childhood, despite lower levels of perceived benefit for the child and greater anticipated anxiety from screen-positive results. These preferences suggest that the expansion of NBS may require ongoing deliberation of perceived benefits and risks and enhanced approaches to education, consent, and support.

Challenges experienced by genetic counselors while they provided counseling about mosaic embryos.

Objective: To survey genetic counselors (GCs) who have counseled about mosaic embryos regarding the challenges they faced in counseling this patient population and assess their need for more resources to support their practice. Design: Self-administered online survey. Setting: Academic university. Study Population: Seventy-eight GCs primarily from the United States and Canada. Interventions: Genetic counselors completed a quantitative survey with an embedded qualitative component. Quantitative data were analyzed by descriptive statistics. An inductive thematic analysis was performed on open-text responses. Main Outcome Measures: Genetic counselors were asked what clinical activities relating to mosaic embryos they performed. They were then asked to rate how challenging each activity was to perform using a 5-point scale; a rating of 4 or 5 was defined as highly challenging. Open-text questions enabled GCs to describe factors that they felt contributed to these challenges. Results: The challenges reported by GCs included the uncertainty of outcomes in offspring after mosaic embryo transfer, limited guidelines available to assist clinicians with counseling about mosaic embryos, and ranking mosaic embryos by suitability for transfer. The contributing factors suggested by participants included limited outcome data, limited GC involvement in pretest counseling for preimplantation genetic testing for aneuploidy (PGT-A), and perceived inconsistency in counseling practices across clinics. Genetic counselors differed in their genetic testing recommendations for pregnancies conceived after mosaic embryo transfer. Amniocentesis and postnatal assessment were recommended by 85% and 49% of GCs, respectively, and 15% recommended chorionic villus sampling and noninvasive prenatal testing. Almost all (92%) reported a need for more resources, such as standardized guidelines, more outcome data, and continuing education on PGT-A and mosaicism. Conclusions: This study describes challenges experienced by GCs while they counseled about mosaic embryos. Our findings demonstrate a need for more outcome data on mosaic embryo pregnancies and for evidence-based clinical guidelines. The differing recommendations for prenatal genetic testing among GCs in the study warrant further research into contributing factors. We strongly recommend that pretest counseling, including a discussion regarding mosaicism, is provided to all couples considering PGT-A to reduce counseling challenges and to promote patients' informed decision-making.

XX male sex reversal with genital abnormalities associated with a de novo SOX3 gene duplication.

Differentiation of the bipotential gonad into testis is initiated by the Y chromosome-linked gene SRY (Sex-determining Region Y) through upregulation of its autosomal direct target gene SOX9 (Sry-related HMG box-containing gene 9). Sequence and chromosome homology studies have shown that SRY most probably evolved from SOX3, which in humans is located at Xq27.1. Mutations causing SOX3 loss-of-function do not affect the sex determination in mice or humans. However, transgenic mouse studies have shown that ectopic expression of Sox3 in the bipotential gonad results in upregulation of Sox9, resulting in testicular induction and XX male sex reversal. However, the mechanism by which these rearrangements cause sex reversal and the frequency with which they are associated with disorders of sex development remains unclear. Rearrangements of the SOX3 locus were identified recently in three cases of human XX male sex reversal. We report on a case of XX male sex reversal associated with a novel de novo duplication of the SOX3 gene. These data provide additional evidence that SOX3 gain-of-function in the XX bipotential gonad causes XX male sex reversal and further support the hypothesis that SOX3 is the evolutionary antecedent of SRY.

Middle and inner ear malformations in mutation-proven branchio-oculo-facial (BOF) syndrome: case series and review of the literature.

Hearing impairment is common in individuals with branchio-oculo-facial (BOF) syndrome. The majority of described individuals have conductive hearing impairment due to malformed ossicles and/or external canal stenosis or atresia, although a sensorineural component to the hearing impairment in BOF syndrome is increasingly being reported. Sophisticated computed tomography (CT) of the temporal bone has revealed middle and inner ear malformations in three previous reports. We present middle and inner ear abnormalities in three additional individuals with mutation-proven BOF syndrome. We suggest that temporal bone CT imaging be included in the medical workup of a child with BOF syndrome, in order to guide management.

Interstitial deletion of 11q-implicating the KIRREL3 gene in the neurocognitive delay associated with Jacobsen syndrome.

Jacobsen syndrome (JS) is a rare contiguous gene disorder characterized by a deletion within the distal part of the long arm of chromosome 11 ranging in size from 7 to 20 Mb. The clinical findings include characteristic dysmorphic features, growth and psychomotor delays and developmental anomalies involving the brain, eyes, heart, kidneys, immune, hematologic, endocrine, and gastrointestinal systems. The majority of cases are due to a terminal deletion of 11q; however interstitial deletions have also been reported. We report on a child with clinical manifestations consistent with JS who had a 2.899 Mb interstitial deletion at 11q24.2-q24.3 which is the smallest interstitial deletion reported so far to our knowledge. This deletion includes the KIRREL3 gene, and given our patient's history of neurocognitive delay and autism spectrum disorder, it raises the possibility that this gene is a candidate for the social and expressive language delay observed in our patient.

Long QT, syndactyly, joint contractures, stroke and novel CACNA1C mutation: expanding the spectrum of Timothy syndrome.

Timothy syndrome (TS) is an autosomal dominant condition with the constellation of features including prolonged QT interval, hand and foot abnormalities, and mental retardation or autism. Splawski et al. [2004] previously described two phenotypes associated with TS distinguished by two unique and different mutations within the CACNA1C gene. We report on a newborn who presented with prolonged QT interval and associated polymorphic ventricular tachycardia, dysmorphic facial features, syndactyly of the hands and feet, and joint contractures, suggestive of TS. He developed a stroke, subsequent intractable seizures, and was found to have cortical blindness and later profound developmental delay. Initial targeted mutation analysis did not identify either of the previously described TS associated mutations; however, full gene sequencing detected a novel CACNA1C gene mutation (p.Ala1473Gly). The clinical and genetic findings in our case expand both the clinical and molecular knowledge of TS.

Genetic counselling for infertile men of known and unknown etiology.

Of the couples trying to conceive (had frequent, unprotected sexual intercourse for a year or longer) 15% will experience infertility with the annual incidence of infertility estimated at 1.2 couples per 1,000 total in the general population. Male factors contribute to over 50% of the cases with 7% of the male population experiencing infertility. Not being able to conceive a child is emotionally traumatic and frustrating and can affect the person's self esteem and the couple's relationship. Major progress has been achieved in identifying the etiology of male infertility and especially the genetic causes. However, in about 40% of the male infertility cases, the etiology remains unknown and both the diagnosis and/or treatment are a challenge. Genetic testing to determine the underlying genetic cause of infertility is not 100% and genes involved are still being discovered. Consequently, negative genetic test results do not rule out a genetic cause. Thus, genetic counselling should include information regarding the genetic etiology, if known, and the treatment options available. Furthermore, when the infertile couple/male is seeking assisted reproductive technology (ART) using intracytoplasmic sperm injection (ICSI), genetic counselling should include information regarding the risk of transmitting the genetic disorder, causing the male infertility, to the offspring. Therefore, the provision of genetic counselling is an integral component in the investigation and treatment of male infertility. This article will discuss the genetic counselling approach in cases with male infertility.

Attitudes regarding predictive testing for retinitis pigmentosa.

PURPOSE: To assess attitudes towards predictive testing for autosomal dominant retinitis pigmentosa (ADRP). METHODS: A prospective questionnaire study of 46 affected adults and their adult family members identified from pedigrees clearly consistent with ADRP or who had had DNA-testing confirmation of ADRP before the study commenced. RESULTS: High proportions of unaffected siblings (73%) and patients (67%) agreed to prenatal testing. Patients agreed to prenatal testing. Patients agreed significantly more often than unaffected siblings that treatment should be available prior to initiating predictive testing. Psychoemotional distress was reported in 57% of the affected adults and their family members in recollecting their own predictive testing as children. CONCLUSIONS: ADRP families indicate a favorable attitude towards testing presymptomatic children with counseling to lessen the psychological and social impact of results.