Expanding the natural history of CASK-related disorders to the prenatal period

Aim To assess whether microcephaly with pontine and cerebellar hypoplasia (MICPCH) could manifest in the prenatal period in patients with calcium/calmodulin-dependent serine protein kinase (CASK) gene disorders. Method In this international multicentre retrospective study, we contacted a CASK parents' social media group and colleagues with expertise in cerebellar malformations and asked them to supply clinical and imaging information. Centiles and standard deviations (SD) were calculated according to age by nomograms. Results The study consisted of 49 patients (44 females and 5 males). Information regarding prenatal head circumference was available in 19 patients; 11 out of 19 had a fetal head circumference below -2SD (range -4.1SD to -2.02SD, mean gestational age at diagnosis 20 weeks). Progressive prenatal deceleration of head circumference growth rate was observed in 15 out of 19. At birth, 20 out of 42 had a head circumference below -2SD. A total of 6 out of 15 fetuses had a TCD z-score below -2 (range -5.88 to -2.02). Interpretation This study expands the natural history of CASK-related disorders to the prenatal period, showing evidence of progressive deceleration of head circumference growth rate, head circumference below -2SD, or small TCD. Most cases will not be diagnosed according to current recommendations for fetal central nervous system routine assessment. Consecutive measurements and genetic studies are advised in the presence of progressive deceleration of head circumference growth rates or small TCD

Recurrent, founder and hypomorphic variants contribute to the genetic landscape of Joubert syndrome

Background Joubert syndrome (JS) is a neurodevelopmental ciliopathy characterised by a distinctive mid-hindbrain malformation, the 'molar tooth sign'. Over 40 JS-associated genes are known, accounting for two-thirds of cases.Methods While most variants are novel or extremely rare, we report on 11 recurring variants in seven genes, including three known 'founder variants' in the Ashkenazi Jewish, Hutterite and Finnish populations. We evaluated variant frequencies in similar to 550 European patients with JS and compared them with controls (>15 000 Italian plus gnomAD), and with an independent cohort of similar to 600 JS probands from the USA.Results All variants were markedly enriched in the European JS cohort compared with controls. When comparing allele frequencies in the two JS cohorts, the Ashkenazim founder variant (TMEM216 c.218G>T) was significantly enriched in American compared with European patients with JS, while MKS1 c.1476T>G was about 10 times more frequent among European JS. Frequencies of other variants were comparable in the two cohorts. Genotyping of several markers identified four novel European founder haplotypes. Two recurrent variants (MKS1 c.1476T>G and KIAA0586 c.428delG), have been detected in homozygosity in unaffected individuals, suggesting they could act as hypomorphic variants. However, while fibroblasts from a MKS1 c.1476T>G healthy homozygote showed impaired ability to form primary cilia and mildly reduced ciliary length, ciliary parameters were normal in cells from a KIAA0586 c.428delG healthy homozygote.Conclusion This study contributes to understand the complex genetic landscape of JS, explain its variable prevalence in distinct geographical areas and characterise two recurrent hypomorphic variants

Exploring the non-coding regions of the genome: the contribution of cryptic splicing variants to the onset of Joubert syndrome

Joubert syndrome (JS) is a mendelian disease that falls into the category of cerebellar and brainstem congenital defects. It represents a complex syndrome with predominantly autosomal recessive inheritance caused by causative biallelic variants affecting about 40 known genes, involved in the biogenesis and correct functioning of the primary cilium. JS is characterized by a congenital malformation of the brain stem and agenesis or hypoplasia of the cerebellar vermis. The distinctive neuroradiological hallmark of JS is the molar tooth sign (MTS), detectable on encephalic MRI. The main clinical signs are hypotonia, respiratory abnormalities, ataxia, and delayed motor and intellectual development. Although the progresses accomplished in the field of neuroimaging and Next-Generation Sequencing (NGS) molecular diagnostics are continuous, to date, about 30-40% of patients clinically and neuroradiologically diagnosed with JS remain without a genetic diagnosis, thus living the so called “diagnostic odyssey” and leading to increased socioeconomic burden. A subset of these missing diagnoses is likely due to cryptic intronic or structural variants, that, with current criteria, are classified as uncertain clinical significance. This thesis aims to explore the hypothesis that a subset of mutations is cryptic and escape conventional diagnosis. To address this, 32 JS patients from Professor Valente’s cohort (Department of Molecular Medicine, University of Pavia) who were heterozygous for a deleterious or possibly deleterious variant in a JS major gene, whose reported phenotypes were consistent with the patients’, and who did not carry other obvious variants in other genes were selected. Whole Exome Sequencing (WES) was performed on all patients whose genomic DNA was available and the intronic regions included in the sequencing were not filtered out during the bioinformatic analysis. After this process, 7 different cryptic splicing variants were identified and their in silico predicted effects were validated through direct RNA studies, if available, or minigene assays if patient lymphoblastoid cell lines or blood RNA samples were not available. Therefore, it was possible to identify and validate in vitro the second cryptic causative splicing variant in 9 patients, thus ending their diagnostic odyssey. Eventually, the selected probands, who remained without a genetic diagnosis after this screening, represent a very promising cohort for further investigations aimed to unravel other classes of cryptic variants, using innovative technologies capable of sequencing the deepest regions of human genome

SUFU haploinsufficiency causes a recognisable neurodevelopmental phenotype at the mild end of the Joubert syndrome spectrum

Background Joubert syndrome (JS) is a recessively inherited ciliopathy characterised by congenital ocular motor apraxia (COMA), developmental delay (DD), intellectual disability, ataxia, multiorgan involvement, and a unique cerebellar and brainstem malformation. Over 40 JS-associated genes are known with a diagnostic yield of 60%-75%. In 2018, we reported homozygous hypomorphic missense variants of the SUFU gene in two families with mild JS. Recently, heterozygous truncating SUFU variants were identified in families with dominantly inherited COMA, occasionally associated with mild DD and subtle cerebellar anomalies. Methods We reanalysed next generation sequencing (NGS) data in two cohorts comprising 1097 probands referred for genetic testing of JS genes. Results Heterozygous truncating and splice-site SUFU variants were detected in 22 patients from 17 families (1.5%) with strong male prevalence (86%), and in 8 asymptomatic parents. Patients presented with COMA, hypotonia, ataxia and mild DD, and only a third manifested intellectual disability of variable severity. Brain MRI showed consistent findings characterised by vermis hypoplasia, superior cerebellar dysplasia and subtle-to-mild abnormalities of the superior cerebellar peduncles. The same pattern was observed in two out of three tested asymptomatic parents. Conclusion Heterozygous truncating or splice-site SUFU variants cause a novel neurodevelopmental syndrome encompassing COMA and mild JS, which likely represent overlapping entities. Variants can arise de novo or be inherited from a healthy parent, representing the first cause of JS with dominant inheritance and reduced penetrance. Awareness of this condition will increase the diagnostic yield of JS genetic testing, and allow appropriate counselling about prognosis, medical monitoring and recurrence risk