Rare germline heterozygous missense variants in BRCA1-associated protein 1, BAP1, cause a syndromic neurodevelopmental disorder.

Nuclear deubiquitinase BAP1 (BRCA1-associated protein 1) is a core component of multiprotein complexes that promote transcription by reversing the ubiquitination of histone 2A (H2A). BAP1 is a tumor suppressor whose germline loss-of-function variants predispose to cancer. To our knowledge, there are very rare examples of different germline variants in the same gene causing either a neurodevelopmental disorder (NDD) or a tumor predisposition syndrome. Here, we report a series of 11 de novo germline heterozygous missense BAP1 variants associated with a rare syndromic NDD. Functional analysis showed that most of the variants cannot rescue the consequences of BAP1 inactivation, suggesting a loss-of-function mechanism. In T cells isolated from two affected children, H2A deubiquitination was impaired. In matching peripheral blood mononuclear cells, histone H3 K27 acetylation ChIP-seq indicated that these BAP1 variants induced genome-wide chromatin state alterations, with enrichment for regulatory regions surrounding genes of the ubiquitin-proteasome system (UPS). Altogether, these results define a clinical syndrome caused by rare germline missense BAP1 variants that alter chromatin remodeling through abnormal histone ubiquitination and lead to transcriptional dysregulation of developmental genes.

Trichothiodystrophy causative TFIIEß mutation affects transcription in highly differentiated tissue.

The rare recessive developmental disorder Trichothiodystrophy (TTD) is characterized by brittle hair and nails. Patients also present a variable set of poorly explained additional clinical features, including ichthyosis, impaired intelligence, developmental delay and anemia. About half of TTD patients are photosensitive due to inherited defects in the DNA repair and transcription factor II H (TFIIH). The pathophysiological contributions of unrepaired DNA lesions and impaired transcription have not been dissected yet. Here, we functionally characterize the consequence of a homozygous missense mutation in the general transcription factor II E, subunit 2 (GTF2E2/TFIIEß) of two unrelated non-photosensitive TTD (NPS-TTD) families. We demonstrate that mutant TFIIEß strongly reduces the total amount of the entire TFIIE complex, with a remarkable temperature-sensitive transcription defect, which strikingly correlates with the phenotypic aggravation of key clinical symptoms after episodes of high fever. We performed induced pluripotent stem (iPS) cell reprogramming of patient fibroblasts followed by in vitro erythroid differentiation to translate the intriguing molecular defect to phenotypic expression in relevant tissue, to disclose the molecular basis for some specific TTD features. We observed a clear hematopoietic defect during late-stage differentiation associated with hemoglobin subunit imbalance. These new findings of a DNA repair-independent transcription defect and tissue-specific malfunctioning provide novel mechanistic insight into the etiology of TTD.

Phenotype delineation of ZNF462 related syndrome.

Zinc finger protein 462 (ZNF462) is a relatively newly discovered vertebrate specific protein with known critical roles in embryonic development in animal models. Two case reports and a case series study have described the phenotype of 10 individuals with ZNF462 loss of function variants. Herein, we present 14 new individuals with loss of function variants to the previous studies to delineate the syndrome of loss of function in ZNF462. Collectively, these 24 individuals present with recurring phenotypes that define a multiple congenital anomaly syndrome. Most have some form of developmental delay (79%) and a minority has autism spectrum disorder (33%). Characteristic facial features include ptosis (83%), down slanting palpebral fissures (58%), exaggerated Cupid's bow/wide philtrum (54%), and arched eyebrows (50%). Metopic ridging or craniosynostosis was found in a third of study participants and feeding problems in half. Other phenotype characteristics include dysgenesis of the corpus callosum in 25% of individuals, hypotonia in half, and structural heart defects in 21%. Using facial analysis technology, a computer algorithm applying deep learning was able to accurately differentiate individuals with ZNF462 loss of function variants from individuals with Noonan syndrome and healthy controls. In summary, we describe a multiple congenital anomaly syndrome associated with haploinsufficiency of ZNF462 that has distinct clinical characteristics and facial features.

Mutations in N-acetylglucosamine (O-GlcNAc) transferase in patients with X-linked intellectual disability.

N-Acetylglucosamine (O-GlcNAc) transferase (OGT) regulates protein O-GlcNAcylation, an essential and dynamic post-translational modification. The O-GlcNAc modification is present on numerous nuclear and cytosolic proteins and has been implicated in essential cellular functions such as signaling and gene expression. Accordingly, altered levels of protein O-GlcNAcylation have been associated with developmental defects and neurodegeneration. However, mutations in the OGT gene have not yet been functionally confirmed in humans. Here, we report on two hemizygous mutations in OGT in individuals with X-linked intellectual disability (XLID) and dysmorphic features: one missense mutation (p.Arg284Pro) and one mutation leading to a splicing defect (c.463-6T>G). Both mutations reside in the tetratricopeptide repeats of OGT that are essential for substrate recognition. We observed slightly reduced levels of OGT protein and reduced levels of its opposing enzyme O-GlcNAcase in both patient-derived fibroblasts, but global O-GlcNAc levels appeared to be unaffected. Our data suggest that mutant cells attempt to maintain global O-GlcNAcylation by down-regulating O-GlcNAcase expression. We also found that the c.463-6T>G mutation leads to aberrant mRNA splicing, but no stable truncated protein was detected in the corresponding patient-derived fibroblasts. Recombinant OGT bearing the p.Arg284Pro mutation was prone to unfolding and exhibited reduced glycosylation activity against a complex array of glycosylation substrates and proteolytic processing of the transcription factor host cell factor 1, which is also encoded by an XLID-associated gene. We conclude that defects in O-GlcNAc homeostasis and host cell factor 1 proteolysis may play roles in mediation of XLID in individuals with OGT mutations.

Prioritization and burden analysis of rare variants in 208 candidate genes suggest they do not play a major role in CAKUT.

The leading cause of end-stage renal disease in children is attributed to congenital anomalies of the kidney and urinary tract (CAKUT). Familial clustering and mouse models support the presence of monogenic causes. Genetic testing is insufficient as it mainly focuses on HNF1B and PAX2 mutations that are thought to explain CAKUT in 5­15% of patients. To identify novel, potentially pathogenic variants in additional genes, we designed a panel of genes identified from studies on familial forms of isolated or syndromic CAKUT and genes suggested by in vitro and in vivo CAKUT models. The coding exons of 208 genes were analyzed in 453 patients with CAKUT using next-generation sequencing. Rare truncating, splice-site variants, and non-synonymous variants, predicted to be deleterious and conserved, were prioritized as the most promising variants to have an effect on CAKUT. Previously reported disease-causing mutations were detected, but only five were fully penetrant causal mutations that improved diagnosis. We prioritized 148 candidate variants in 151 patients, found in 82 genes, for follow-up studies. Using a burden test, no significant excess of rare variants in any of the genes in our cohort compared with controls was found. Thus, in a study representing the largest set of genes analyzed in CAKUT patients to date, the contribution of previously implicated genes to CAKUT risk was significantly smaller than expected, and the disease may be more complex than previously assumed.

Phenotypic variability in patients with ADA2 deficiency due to identical homozygous R169Q mutations.

OBJECTIVE: To determine the genotype-phenotype association in patients with adenosine deaminase-2 (ADA2) deficiency due to identical homozygous R169Q mutations inCECR1 METHODS: We present a case series of nine ADA2-deficient patients with an identical homozygous R169Q mutation. Clinical and diagnostic data were collected and available MRI studies were reviewed. We performed genealogy and haplotype analyses and measured serum ADA2 activity. ADA2 activity values were correlated to clinical symptoms. RESULTS: Age of presentation differed widely between the nine presented patients (range: 0 months to 8 years). The main clinical manifestations were (hepato)splenomegaly (8/9), skin involvement (8/9) and neurological involvement (8/9, of whom 6 encountered stroke). Considerable variation was seen in type, frequency and intensity of other symptoms, which included aplastic anaemia, acute myeloid leukaemia and cutaneous ulcers. Common laboratory abnormalities included cytopenias and hypogammaglobulinaemia. ADA2 enzyme activity in patients was significantly decreased compared with healthy controls. ADA2 activity levels tended to be lower in patients with stroke compared with patients without stroke. Genealogical studies did not identify a common ancestor; however, based on allele frequency, a North-West European founder effect can be noted. Three patients underwent haematopoietic cell transplantation, after which ADA2 activity was restored and clinical symptoms resolved. CONCLUSION: This case series revealed large phenotypic variability in patients with ADA2 deficiency though they were homozygous for the same R169Q mutation inCECR1 Disease modifiers, including epigenetic and environmental factors, thus seem important in determining the phenotype. Furthermore, haematopoietic cell transplantation appears promising for those patients with a severe clinical phenotype.

Biallelic variants in CSMD1 are implicated in a neurodevelopmental disorder with intellectual disability and variable cortical malformations.

CSMD1 (Cub and Sushi Multiple Domains 1) is a well-recognized regulator of the complement cascade, an important component of the innate immune response. CSMD1 is highly expressed in the central nervous system (CNS) where emergent functions of the complement pathway modulate neural development and synaptic activity. While a genetic risk factor for neuropsychiatric disorders, the role of CSMD1 in neurodevelopmental disorders is unclear. Through international variant sharing, we identified inherited biallelic CSMD1 variants in eight individuals from six families of diverse ancestry who present with global developmental delay, intellectual disability, microcephaly, and polymicrogyria. We modeled CSMD1 loss-of-function (LOF) pathogenesis in early-stage forebrain organoids differentiated from CSMD1 knockout human embryonic stem cells (hESCs). We show that CSMD1 is necessary for neuroepithelial cytoarchitecture and synchronous differentiation. In summary, we identified a critical role for CSMD1 in brain development and biallelic CSMD1 variants as the molecular basis of a previously undefined neurodevelopmental disorder.

Is joint hypermobility associated with vesico-ureteral reflux? An assessment of 50 patients.

OBJECTIVE: To assess whether there is an increased prevalence of joint hypermobility in patients with vesico-ureteric reflux (VUR). MATERIALS AND METHODS: We studied 50 patients with primary VUR and matched controls drawn from a reference population. Joint mobility was assessed using the Bulbena hypermobility score. RESULTS: We identified significantly more patients with VUR with generalized joint hypermobility than controls (24% vs 6.7%, P= 0.007). CONCLUSION: Our findings confirm our clinical observation of an increased rate of joint hypermobility in patients with VUR. We speculate that an altered composition of the connective tissue may contribute to the severity of the (pre-existing) VUR phenotype.

Familial Male-limited Precocious Puberty (FMPP) and Testicular Germ Cell Tumors.

OBJECTIVE: The purpose of this study is to report development of a malignant testicular germ cell tumor (GCT) in 2 young adult males with familial male-limited precocious puberty (FMPP) because of LHCGR pathogenic variants in 2 families. Secondarily, to study the possible relation between FMPP and testicular tumors and to investigate whether FMPP might predispose to development of malignant testicular tumors in adulthood a literature review is conducted. METHODS: Data on 6 cases in 2 families are obtained from the available medical records. In addition, a database search is performed in Cochrane, PubMed, and Embase for studies that report on a possible link between FMPP and testicular tumors. RESULTS: The characteristics of 6 males with FMPP based on activating LH receptor (LHCGR) germline pathogenic variants are described, as are details of the testicular GCTs. Furthermore, a literature review identified 4 more patients with signs of FMPP and a (precursor of) testicular GCT in adolescence or adulthood (age 15-35 years). Additionally, 12 patients with signs of precocious puberty and, simultaneously, occurrence of a Leydig cell adenoma or Leydig cell hyperplasia are reported. CONCLUSION: There is a strong suggestion that FMPP might increase the risk of development of testicular GCTs in early adulthood compared with the risk in the general population. Therefore, prolonged patient monitoring from mid-pubertal age onward including instruction for self-examination and periodic testicular ultrasound investigation in patients with a germline LHCGR pathogenic variant might contribute to early detection and thus early treatment of testicular GCT.

Attitudes of Klinefelter men and their relatives towards TESE-ICSI.

PURPOSE: At the start of the implementation of TESE-ICSI for Klinefelter men in the Netherlands, we aimed to evaluate their wish to father children and their attitudes towards this artificial reproduction technique. METHODS: Questionnaires were distributed to members of the Dutch Klinefelter Association (n = 365) and to Klinefelter cases known at our Department (n = 58). Questions addressed several aspects: socio-demographic characteristics, ascertainment of diagnosis, children and child wish, and TESE-ICSI. Data were characterized using descriptive statistics. RESULTS: A total of 260 questionnaires (corresponding to 194 cases, 46%) were returned. A possible wish to father children was reported by 90% of Klinefelter men. 70% of Klinefelter men and 74% of their partners would (probably) opt for TESE-ICSI. CONCLUSION: The majority of Dutch Klinefelter men and their partners desire to have children and have a positive attitude towards TESE-ICSI. Concerns include the risk of congenital malformations/developmental delay of the child and the limited success rate of TESE-ICSI.

Deficiency of TET3 leads to a genome-wide DNA hypermethylation episignature in human whole blood.

TET3 encodes an essential dioxygenase involved in epigenetic regulation through DNA demethylation. TET3 deficiency, or Beck-Fahrner syndrome (BEFAHRS; MIM: 618798), is a recently described neurodevelopmental disorder of the DNA demethylation machinery with a nonspecific phenotype resembling other chromatin-modifying disorders, but inconsistent variant types and inheritance patterns pose diagnostic challenges. Given TET3's direct role in regulating 5-methylcytosine and recent identification of syndrome-specific DNA methylation profiles, we analyzed genome-wide DNA methylation in whole blood of TET3-deficient individuals and identified an episignature that distinguishes affected and unaffected individuals and those with mono-allelic and bi-allelic pathogenic variants. Validation and testing of the episignature correctly categorized known TET3 variants and determined pathogenicity of variants of uncertain significance. Clinical utility was demonstrated when the episignature alone identified an affected individual from over 1000 undiagnosed cases and was confirmed upon distinguishing TET3-deficient individuals from those with 46 other disorders. The TET3-deficient signature - and the signature resulting from activating mutations in DNMT1 which normally opposes TET3 - are characterized by hypermethylation, which for BEFAHRS involves CpG sites that may be biologically relevant. This work expands the role of epi-phenotyping in molecular diagnosis and reveals genome-wide DNA methylation profiling as a quantitative, functional readout for characterization of this new biochemical category of disease.

The clinical spectrum of missense mutations of the first aspartic acid of cbEGF-like domains in fibrillin-1 including a recessive family.

Marfan syndrome (MFS) is a dominant disorder with a recognizable phenotype. In most patients with the classical phenotype mutations are found in the fibrillin-1 gene (FBN1) on chromosome 15q21. It is thought that most mutations act in a dominant negative way or through haploinsufficiency. In 9 index cases referred for MFS we detected heterozygous missense mutations in FBN1 predicted to substitute the first aspartic acid of different calcium-binding Epidermal Growth Factor-like (cbEGF) fibrillin-1 domains. A similar mutation was found in homozygous state in 3 cases in a large consanguineous family. Heterozygous carriers of this mutation had no major skeletal, cardiovascular or ophthalmological features of MFS. In the literature 14 other heterozygous missense mutations are described leading to the substitution of the first aspartic acid of a cbEGF domain and resulting in a Marfan phenotype. Our data show that the phenotypic effect of aspartic acid substitutions in the first position of a cbEGF domain can range from asymptomatic to a severe neonatal phenotype. The recessive nature with reduced expression of FBN1 in one of the families suggests a threshold model combined with a mild functional defect of this specific mutation.

A de novo variant in the human HIST1H4J gene causes a syndrome analogous to the HIST1H4C-associated neurodevelopmental disorder.

We report here a de novo missense variant in HIST1H4J resulting in a complex syndrome combining growth delay, microcephaly and intellectual disability. Trio whole exome sequencing (WES) revealed that the proband was heterozygous for a de novo c.274 A > G p.(K91E) variant in HIST1H4J, a gene not yet associated with human disease. The patient presented with profound intellectual disability, microcephaly, and dysmorphic facial features. Functional consequences of the identified de novo missense variant were evaluated in zebrafish embryos, where they affected general development, especially resulting in defective head organs and reduced body axis length. Our results show that the monoallelic p.K91E substitution on HIST1H4J underlies a human syndrome that is genetically and phenotypically akin to the HIST1H4C-associated neurodevelopmental disorder resulting from p.K91A and p.K91Q substitions in HIST1H4C. The highly overlapping patient phenotypes highlight functional similarities between HIST1H4J and HIST1H4C perturbations, establishing the singular importance of K91 across histone H4 genes for vertebrate development.

Maternal risk associated with the VACTERL association: A case-control study.

BACKGROUND: The VACTERL association (VACTERL) includes at least three of these congenital anomalies: vertebral, anal, cardiac, trachea-esophageal, renal, and limb anomalies. Assisted reproductive techniques (ART), pregestational diabetes mellitus, and chronic lower obstructive pulmonary disorders (CLOPD) have been associated with VACTERL. We aimed to replicate these findings and were interested in additional maternal risk factors. METHODS: A case-control study using self-administered questionnaires was performed including 142 VACTERL cases and 2,135 population-based healthy controls. Multivariable logistic regression analyses were performed to estimate confounder adjusted odds ratios (aOR) and 95% confidence intervals (95%CI). RESULTS: Parents who used invasive ART had an increased risk of VACTERL in offspring (aOR 4.4 [95%CI 2.1-8.8]), whereas the increased risk for mothers with CLOPD could not be replicated. None of the case mothers had pregestational diabetes mellitus. Primiparity (1.5 [1.1-2.1]) and maternal pregestational overweight and obesity (1.8 [1.2-2.8] and 1.8 [1.0-3.4]) were associated with VACTERL. Consistent folic acid supplement use during the advised periconceptional period may reduce the risk of VACTERL (0.5 [0.3-1.0]). Maternal smoking resulted in an almost twofold increased risk of VACTERL. CONCLUSION: We identified invasive ART, primiparity, pregestational overweight and obesity, lack of folic acid supplement use, and smoking as risk factors for VACTERL.

Psychiatric morbidity and X-chromosomal origin in a Klinefelter sample.

The presence of an additional X-chromosome in Klinefelter patients provides an opportunity to study the influence of this chromosome on psychiatric disorders. Previous studies have reported an excess of Klinefelter patients in psychiatric patient groups. We report an increased prevalence of psychiatric disorders including psychotic disorders in a sample of Klinefelter patients but could not find evidence of an effect of the parental origin of the extra X-chromosome on the psychiatric phenotype. Nevertheless, these findings provide further support for the role of the X-chromosome in the susceptibility to psychiatric disorders in general and psychotic disorders in particular.

Germline mutations affecting the histone H4 core cause a developmental syndrome by altering DNA damage response and cell cycle control.

Covalent modifications of histones have an established role as chromatin effectors, as they control processes such as DNA replication and transcription, and repair or regulate nucleosomal structure. Loss of modifications on histone N tails, whether due to mutations in genes belonging to histone-modifying complexes or mutations directly affecting the histone tails, causes developmental disorders or has a role in tumorigenesis. More recently, modifications affecting the globular histone core have been uncovered as being crucial for DNA repair, pluripotency and oncogenesis. Here we report monoallelic missense mutations affecting lysine 91 in the histone H4 core (H4K91) in three individuals with a syndrome of growth delay, microcephaly and intellectual disability. Expression of the histone H4 mutants in zebrafish embryos recapitulates the developmental anomalies seen in the patients. We show that the histone H4 alterations cause genomic instability, resulting in increased apoptosis and cell cycle progression anomalies during early development. Mechanistically, our findings indicate an important role for the ubiquitination of H4K91 in genomic stability during embryonic development.

Tetraploid/Diploid Mosaicism in Cultured Genital Skin Fibroblasts: Is It Causally Related to Penoscrotal Hypospadias?

Tetraploid/diploid mosaicism is a rare chromosomal abnormality that is infrequently reported in patients with severe developmental delay, growth retardation, and short life span. Here, we present a 6-year-old patient with severe penoscrotal hypospadias and a coloboma of the left eye but with normal growth, normal psychomotor development, and without dysmorphisms. We considered a local, mosaic sex chromosomal aneuploidy as a possible cause of his genital anomaly and performed karyotyping in cultured fibroblasts from the genital skin, obtained during surgical correction. Tetraploid/diploid (92,XXYY/46,XY) mosaicism was found in 43/57 and 6/26 metaphases in 2 separate cultures, respectively. Buccal smear cells, blood lymphocytes, and cells from urine sediment all showed diploidy. We investigated whether this chromosomal abnormality could be found in other patients with severe hypospadias and karyotyped genital fibroblasts of 6 additional patients but found only low frequencies (<11%) of tetraploid cells, not statistically different from those found in control males with no hypospadias. This is the first time tetraploid mosaicism is found in such a high percentage in a patient without psychomotor retardation, dysmorphisms or growth delay. Although the relationship between this observed mosaicism in cultured cells and the underlying pathogenetic mechanism in penoscrotal hypospadias remains to be determined, our data clearly illustrate the power of cytogenetic techniques in detecting mosaicism compared to next-generation sequencing techniques, in which DNA pooled from multiple cells is used.