Prenatal phenotype analysis and mutation identification of a fetus with meckel gruber syndrome.

Ciliopathies are a class of inherited severe human disorders that occur due to defective formation or function of cilia. The RPGRIP1L (retinitis pigmentosa GTPase regulator-interacting protein1-like) gene encodes for a ciliary protein involved in regulating cilia formation and function. Mutations in RPGRIP1L cause ciliopathies associated with severe embryonic defects, such as Meckel-Gruber Syndrome (MKS). Here we report RPGRIP1L mutation analysis in a family diagnosed with MKS. The clinical manifestations of the fetus included thoraco-lumbar open neural tube defect with associated Chiari type II malformation and hydrocephalus, bilateral club feet, and single right kidney/ureter. Analysis of the parental DNA samples revealed that the father carried a previously reported mutation R1236C/+ whereas the mother had a novel splice site mutation IVS6+1 G > A/+ in RPGRIP1L. The splice site mutation resulted in the exclusion of in-frame exon 6 of RPGRIP1L (RPGRIP1L-∆Ex6) but expressed a stable protein in fibroblasts derived from the parents' skin biopsies. The GFP-RPGRIP1L-∆Ex6 mutant protein exhibited relatively reduced ciliary localization in transiently-transfected cultured RPE-1 cells. Taken together, this study identifies a novel RPGRIP1L variant RPGRIP1L-∆Ex6, which in combination with RPGRIP1L-R1236C is associated with MKS. We also suggest that the deletion of exon 6 of RPGRIP1L leads to reduced ciliary localization of RPGRIP1L, indicating a plausible mechanism of associated disease.

Variant-specific changes in RAC3 function disrupt corticogenesis in neurodevelopmental phenotypes.

Variants in RAC3, encoding a small GTPase RAC3 which is critical for the regulation of actin cytoskeleton and intracellular signal transduction, are associated with a rare neurodevelopmental disorder with structural brain anomalies and facial dysmorphism. We investigated a cohort of 10 unrelated participants presenting with global psychomotor delay, hypotonia, behavioural disturbances, stereotyped movements, dysmorphic features, seizures and musculoskeletal abnormalities. MRI of brain revealed a complex pattern of variable brain malformations, including callosal abnormalities, white matter thinning, grey matter heterotopia, polymicrogyria/dysgyria, brainstem anomalies and cerebellar dysplasia. These patients harboured eight distinct de novo RAC3 variants, including six novel variants (NM_005052.3): c.34G > C p.G12R, c.179G > A p.G60D, c.186_188delGGA p.E62del, c.187G > A p.D63N, c.191A > G p.Y64C and c.348G > C p.K116N. We then examined the pathophysiological significance of these novel and previously reported pathogenic variants p.P29L, p.P34R, p.A59G, p.Q61L and p.E62K. In vitro analyses revealed that all tested RAC3 variants were biochemically and biologically active to variable extent, and exhibited a spectrum of different affinities to downstream effectors including p21-activated kinase 1. We then focused on the four variants p.Q61L, p.E62del, p.D63N and p.Y64C in the Switch II region, which is essential for the biochemical activity of small GTPases and also a variation hot spot common to other Rho family genes, RAC1 and CDC42. Acute expression of the four variants in embryonic mouse brain using in utero electroporation caused defects in cortical neuron morphology and migration ending up with cluster formation during corticogenesis. Notably, defective migration by p.E62del, p.D63N and p.Y64C were rescued by a dominant negative version of p21-activated kinase 1. Our results indicate that RAC3 variants result in morphological and functional defects in cortical neurons during brain development through variant-specific mechanisms, eventually leading to heterogeneous neurodevelopmental phenotypes.

Novel FGF9 variant contributes to multiple synostoses syndrome 3.

Multiple synostoses syndromes (SYNS) are autosomal dominant syndromes characterized by multiple joint fusions commonly involving the carpal-tarsal, interphalangeal, humeroradial, and cervical spine joints. They display genetic heterogeneity with pathogenic variants reported in four separate genes (NOG, GDF5, FGF9, and GDF6) defining four different SYNS forms. FGF9 variants have been reported in SYNS3, a SYNS with multiple synostoses, normal cognition, normal hearing, and craniosynostosis. Here, we report a novel FGF9 c.569G > C p.(Arg190Thr) variant identified by whole-exome sequencing in a patient with multiple bony abnormalities. The patient initially presented with elbow instability and decreased range of motion. Imaging revealed bilateral radial head deformities, carpal-tarsal fusions, brachydactyly, and osteoarthritis of the sacroiliac joints. In silico protein modeling of the identified FGF9 variant predicts decreased stability of ligand-receptor binding supporting the pathogenicity of this finding. This finding expands the repertoire of FGF9 variants and phenotypic information reported for SYNS3 and suggest that genotype phenotype correlations due to localization seem less likely and more so due to the consequence of the pathogenic variant on the receptor. This is useful in the counseling in families as more de novo variants emerge.

Missense variant contribution to USP9X-female syndrome.

USP9X is an X-chromosome gene that escapes X-inactivation. Loss or compromised function of USP9X leads to neurodevelopmental disorders in males and females. While males are impacted primarily by hemizygous partial loss-of-function missense variants, in females de novo heterozygous complete loss-of-function mutations predominate, and give rise to the clinically recognisable USP9X-female syndrome. Here we provide evidence of the contribution of USP9X missense and small in-frame deletion variants in USP9X-female syndrome also. We scrutinise the pathogenicity of eleven such variants, ten of which were novel. Combined application of variant prediction algorithms, protein structure modelling, and assessment under clinically relevant guidelines universally support their pathogenicity. The core phenotype of this cohort overlapped with previous descriptions of USP9X-female syndrome, but exposed heightened variability. Aggregate phenotypic information of 35 currently known females with predicted pathogenic variation in USP9X reaffirms the clinically recognisable USP9X-female syndrome, and highlights major differences when compared to USP9X-male associated neurodevelopmental disorders.

De Novo Variants in the ATPase Module of MORC2 Cause a Neurodevelopmental Disorder with Growth Retardation and Variable Craniofacial Dysmorphism.

MORC2 encodes an ATPase that plays a role in chromatin remodeling, DNA repair, and transcriptional regulation. Heterozygous variants in MORC2 have been reported in individuals with autosomal-dominant Charcot-Marie-Tooth disease type 2Z and spinal muscular atrophy, and the onset of symptoms ranges from infancy to the second decade of life. Here, we present a cohort of 20 individuals referred for exome sequencing who harbor pathogenic variants in the ATPase module of MORC2. Individuals presented with a similar phenotype consisting of developmental delay, intellectual disability, growth retardation, microcephaly, and variable craniofacial dysmorphism. Weakness, hyporeflexia, and electrophysiologic abnormalities suggestive of neuropathy were frequently observed but were not the predominant feature. Five of 18 individuals for whom brain imaging was available had lesions reminiscent of those observed in Leigh syndrome, and five of six individuals who had dilated eye exams had retinal pigmentary abnormalities. Functional assays revealed that these MORC2 variants result in hyperactivation of epigenetic silencing by the HUSH complex, supporting their pathogenicity. The described set of morphological, growth, developmental, and neurological findings and medical concerns expands the spectrum of genetic disorders resulting from pathogenic variants in MORC2.

Noninvasive Prenatal Detection of Trisomy 21 by Targeted Semiconductor Sequencing: A Technical Feasibility Study.

OBJECTIVE: To develop an alternate noninvasive prenatal testing method for the assessment of trisomy 21 (T21) using a targeted semiconductor sequencing approach. METHODS: A customized AmpliSeq panel was designed with 1,067 primer pairs targeting specific regions on chromosomes 21, 18, 13, and others. A total of 235 samples, including 30 affected with T21, were sequenced with an Ion Torrent Proton sequencer, and a method was developed for assessing the probability of fetal aneuploidy via derivation of a risk score. RESULTS: Application of the derived risk score yields a bimodal distribution, with the affected samples clustering near 1.0 and the unaffected near 0. For a risk score cutoff of 0.345, above which all would be considered at "high risk," all 30 T21-positive pregnancies were correctly predicted to be affected, and 199 of the 205 non-T21 samples were correctly predicted. The average hands-on time spent on library preparation and sequencing was 19 h in total, and the average number of reads of sequence obtained was 3.75 million per sample. CONCLUSION: With the described targeted sequencing approach on the semiconductor platform using a custom-designed library and a probabilistic statistical approach, we have demonstrated the feasibility of an alternate method of assessment for fetal T21.

Mutations in the Chromatin Regulator Gene BRPF1 Cause Syndromic Intellectual Disability and Deficient Histone Acetylation.

Identification of over 500 epigenetic regulators in humans raises an interesting question regarding how chromatin dysregulation contributes to different diseases. Bromodomain and PHD finger-containing protein 1 (BRPF1) is a multivalent chromatin regulator possessing three histone-binding domains, one non-specific DNA-binding module, and several motifs for interacting with and activating three lysine acetyltransferases. Genetic analyses of fish brpf1 and mouse Brpf1 have uncovered an important role in skeletal, hematopoietic, and brain development, but it remains unclear how BRPF1 is linked to human development and disease. Here, we describe an intellectual disability disorder in ten individuals with inherited or de novo monoallelic BRPF1 mutations. Symptoms include infantile hypotonia, global developmental delay, intellectual disability, expressive language impairment, and facial dysmorphisms. Central nervous system and spinal abnormalities are also seen in some individuals. These clinical features overlap with but are not identical to those reported for persons with KAT6A or KAT6B mutations, suggesting that BRPF1 targets these two acetyltransferases and additional partners in humans. Functional assays showed that the resulting BRPF1 variants are pathogenic and impair acetylation of histone H3 at lysine 23, an abundant but poorly characterized epigenetic mark. We also found a similar deficiency in different lines of Brpf1-knockout mice. These data indicate that aberrations in the chromatin regulator gene BRPF1 cause histone H3 acetylation deficiency and a previously unrecognized intellectual disability syndrome.

Melanocortin-4 Receptor Deficiency Phenotype with an Interstitial 18q Deletion: A Case Report of Severe Childhood Obesity and Tall Stature.

Childhood obesity is a growing health concern, associated with significant physical and psychological morbidity. Childhood obesity is known to have a strong genetic component, with mutations in the melanocortin-4 receptor (MC4R) gene being the most common monogenetic cause of obesity. Over 166 different MC4R mutations have been identified in persons with hyperphagia, severe childhood obesity, and increased linear growth. However, it is unclear whether the MC4-R deficiency phenotype is due to haploinsufficiency or dominant-negative effects by the mutant receptor. We report the case of a four-and-a-half-year-old boy with an interstitial deletion involving the long arm of chromosome 18 (46,XY,del(18)(q21.32q22.1)) encompassing the MC4R gene. This patient presented with tall stature and hyperphagia within his first 18 months of life leading to significant obesity. This case supports haploinsufficiency of MC4-R as it describes a MC4-R deficiency phenotype in a patient heterozygous for a full MC4R gene deletion. The intact functional allele with MC4-R haploinsufficiency has the potential to favor a therapeutic response to gastric surgery. Currently, small molecule MC4-R agonists are under development for pharmacologic therapy.