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Haploinsufficiency of TRIP12 causes a neurodevelopmental disorder characterized by intellectual disability associated with epilepsy, autism spectrum disorder and dysmorphic features, also named Clark-Baraitser syndrome. Only a limited number of cases have been reported to date. We aimed to further delineate the TRIP12-associated phenotype and objectify characteristic facial traits through GestaltMatcher image analysis based on deep-learning algorithms in order to establish a TRIP12 gestalt. 38 individuals between 3 and 66 years (F = 20, M = 18) - 1 previously published and 37 novel individuals - were recruited through an ERN ITHACA call for collaboration. 35 TRIP12 variants were identified, including frameshift (n = 15) and nonsense (n = 6) variants, as well as missense (n = 5) and splice (n = 3) variants, intragenic deletions (n = 4) and two multigene deletions disrupting TRIP12. Though variable in severity, global developmental delay was noted in all individuals, with language deficit most pronounced. About half showed autistic features and susceptibility to obesity seemed inherent to this disorder. A more severe expression was noted in individuals with a missense variant. Facial analysis showed a clear gestalt including deep-set eyes with narrow palpebral fissures and fullness of the upper eyelids, downturned corners of the mouth and large, often low-set ears with prominent earlobes. We report the largest cohort to date of individuals with TRIP12 variants, further delineating the associated phenotype and introducing a facial gestalt. These findings will improve future counseling and patient guidance.

BackgroundThe paired-domain transcription factor paired box gene 6 (PAX6) causes a wide spectrum of ocular developmental anomalies, including congenital aniridia, Peters anomaly and microphthalmia. Here, we aimed to functionally assess the involvement of seven potentially non-canonical splicing variants on missplicing of exon 6, which represents the main hotspot region for loss-of-function PAX6 variants.MethodsBy locus-specific analysis of PAX6 using Sanger and/or targeted next-generation sequencing, we screened a Spanish cohort of 106 patients with PAX6-related diseases. Functional splicing assays were performed by in vitro minigene approaches or directly in RNA from patient-derived lymphocytes cell line, when available.ResultsFive out seven variants, including three synonymous changes, one small exonic deletion and one non-canonical splice variant, showed anomalous splicing patterns yielding partial exon skipping and/or elongation.ConclusionWe describe new spliceogenic mechanisms for PAX6 variants mediated by creating or strengthening five different cryptic donor sites at exon 6. Our work revealed that the activation of cryptic PAX6 splicing sites seems to be a recurrent and underestimated cause of aniridia. Our findings pointed out the importance of functional assessment of apparently silent PAX6 variants to uncover hidden genetic alterations and to improve variant interpretation for genetic counselling in aniridia.

SUMMARYBackgroundKBG syndrome is a highly variable neurodevelopmental disorder and clinical diagnostic criteria have changed as new patients have been published. Both loss-of-function sequence variants and large deletions (CNVs) involving ANKRD11 have been involved in KBG, but no genotype-phenotype correlation has been reported to date. This study presents the clinical and molecular characteristics of 67 new patients with KBG syndrome and the results of the first genotype-phenotype correlation leveraging data on 273 patients previously published.Methods67 patients with KBG syndrome were recruited through a Spanish collaborative effort and were assessed using a custom phenotypic questionnaire. The frequency of all features was calculated. Manifestations present in >50% of the patients and a “severity score” were used to perform a genotype-phenotype correlation in the 340 KBG patients.ResultsNeurodevelopmental delay (95%), comorbidites (82.8%), macrodontia (80.9%), triangular face (71%), characteristic ears (76%), nose (75.9%) and eyebrows (67.3%) were the most prevalent features in the 67 patients. The genotype-phenotype correlation yielded significant associations with the triangular face (71.1% in patients with sequence variants vs 45.2% in CNVs, p=0.015), short stature (62.5% variants in exon 9 vs. 27.8% outside; p=0.009) and macrodontia (with larger deletions, p=0.028), ID/ADHD/ASD (70.4% in c.1903_1907del vs. 89.4%; p=0.012) and a higher phenotypic score in patients with sequence variants compared with CNVs (p=0.005).ConclusionsWe present a detailed phenotypic description of KBG syndrome in the largest series of patients reported to date, provide evidence of a genotype-phenotype correlation between some KBG features and specific ANKRD11 aberrations, and propose updated clinical diagnostic criteria based on our findings.

Purpose We aimed to unravel the molecular basis of sporadic retinitis pigmentosa (sRP) in the largest cohort reported to date. Design Case series. Participants A cohort of 877 unrelated Spanish sporadic cases with a clinical diagnosis of retinitis pigmentosa (RP) and negative family history. Methods The cohort was studied by classic genotyping or targeted next-generation sequencing (NGS). Multiplex ligation-dependent probe amplification (MLPA) and array-based comparative genomic hybridization were performed to confirm copy number variations detected by NGS. Quantitative fluorescent polymerase chain reaction was assessed in sRP cases carrying de novo variants to confirm paternity. Main Outcome Measures The study of the sRP cohort showed a high proportion of causal autosomal dominant (AD) and X-linked (XL) variants, most of them being de novo. Results Causative variants were identified in 38% of the patients studied, segregating recessively in 84.5% of the solved cases. Biallelic variants detected in only 6 different autosomal recessive genes explained 50% of the cases characterized. Causal AD and XL variants were found in 7.6% and 7.9% of cases, respectively. Remarkably, 20 de novo variants were confirmed after trio analysis, explaining 6% of the cases. In addition, 17% of the solved sRP cases were reclassified to a different retinopathy phenotype. Conclusions This study highlights the clinical utility of NGS testing for sRP cases, expands the mutational spectrum, and provides accurate prevalence of mutated genes. Our findings evidence the underestimated role of de novo variants in the etiology of RP, emphasizing the importance of segregation analysis as well as comprehensive screening of genes carrying XL and AD variants in sporadic cases. Such in-depth study is essential for accurate family counseling and future enrollment in gene therapy–based treatments.

Purpose To define genotype–phenotype correlations in the largest cohort study worldwide of patients with biallelic ABCA4 variants, including 434 patients with Stargardt disease (STGD1) and 72 with cone-rod dystrophy (CRD). Design Cohort study. Methods We characterized 506 patients with ABCA4 variants using conventional genetic tools and next-generation sequencing technologies. Medical history and ophthalmologic data were obtained from 372 patients. Genotype–phenotype correlation studies were carried out for the following variables: variant type, age at symptom onset (AO), and clinical phenotype. Results A total of 228 different pathogenic variants were identified in 506 ABCA4 patients, 50 of which were novel. Genotype–phenotype correlations showed that most of the patients with biallelic truncating variants presented with CRD and that these cases had a significantly earlier AO than patients with STGD1. Three missense variants are associated with CRD for the first time (c.1804C>T; p.[Arg602Trp], c.3056C>T; p.[Thr1019Met], and c.6320G>C; p.[Arg2107Pro]). Analysis of the most prevalent ABCA4 variant in Spain, c.3386G>T; p.(Arg1129Leu), revealed that is correlated to STGD1, later AO, and foveal sparing. Conclusions Our study, conducted in the largest ABCA4-associated disease cohort reported to date, updates the genotype–phenotype model established for ABCA4 variants and broadens the mutational spectrum of the gene. According to our observations, patients with ABCA4 presenting with 2 truncating variants may first present features of STGD1 but eventually develop rod dysfunction, and specific missense variants may be associated with a different phenotype, underscoring the importance of an accurate genetic diagnosis. Also, it is a prerequisite for enrollment in clinical trials, and to date, no other treatment has been approved for STGD1.

The paired-domain transcription factor paired box gene 6 (PAX6) causes a wide spectrum of ocular developmental anomalies, including congenital aniridia, Peters anomaly and microphthalmia. Here, we aimed to functionally assess the involvement of seven potentially non-canonical splicing variants on missplicing of exon 6, which represents the main hotspot region for loss-of-functionBy locus-specific analysis ofFive out seven variants, including three synonymous changes, one small exonic deletion and one non-canonical splice variant, showed anomalous splicing patterns yielding partial exon skipping and/or elongation.We describe new spliceogenic mechanisms for

The frequency of mitochondrial diseases (MD) has been scarcely documented, and only a few studies have reported data in certain specific geographical areas. In this study, we arranged a nationwide call in Spain to obtain a global estimate of the number of cases. A total of 3274 cases from 49 Spanish provinces were reported by 39 centres. Excluding duplicated and unsolved cases, 2761 patients harbouring pathogenic mutations in 140 genes were recruited between 1990 and 2020. A total of 508 patients exhibited mutations in nuclear DNA genes (75% paediatric patients) and 1105 in mitochondrial DNA genes (33% paediatric patients). A further 1148 cases harboured mutations in the MT-RNR1 gene (56% paediatric patients). The number of reported cases secondary to nuclear DNA mutations increased in 2014, owing to the implementation of next-generation sequencing technologies. Between 2014 and 2020, excepting MT-RNR1 cases, the incidence was 6.34 (95% CI: 5.71–6.97) cases per million inhabitants at the paediatric age and 1.36 (95% CI: 1.22–1.50) for adults. In conclusion, this is the first study to report nationwide epidemiological data for MD in Spain. The lack of identification of a remarkable number of mitochondrial genes necessitates the systematic application of high-throughput technologies in the routine diagnosis of MD.

Background Retinitis pigmentosa (RP) is the most common form of inherited retinal dystrophy, with a worldwide prevalence of 1 in 4000 persons. While in most cases of RP, the disease is limited to the eye (non-syndromic), over 40 forms of syndromic RP have been described. Objectives To identify the genetic basis for syndromic RP in three unrelated families from Israel and Spain. Methods Whole exome sequencing was conducted in one Israeli and two Spanish families segregating autosomal recessive RP with intellectual disability. Complete ophthalmic examination included best-corrected visual acuity, funduscopy, optical coherence tomography, fluorescein angiography, flash visual evoked potentials, and electroretinography. Reverse transcription (RT)-PCR and immunostaining were used to examine the spatial and temporal expression pattern of SCAPER. Results In all patients, biallelic SCAPER mutations were observed. Clinically, patients with SCAPER mutations show signs of typical RP. In addition, they have mild to moderate intellectual disability and attention-deficit/hyperactivity disorder. SCAPER was found to be ubiquitously expressed in a wide range of human tissues, including retina and brain. Furthermore, RT-PCR analysis revealed that in both mouse eye and brain, Scaper is expressed as early as embryonic day 14. In the mouse retina, SCAPER is located in multiple layers, including the retinal pigment epithelium, photoreceptor outer and inner segments, the inner plexiform layer and the ganglion cell layer. Conclusions Deleterious SCAPER mutations were identified in four patients from three unrelated families of different ethnic backgrounds, thereby confirming the involvement of this gene in the aetiology of autosomal recessive syndromic RP.

We aimed to unravel the molecular basis of sporadic retinitis pigmentosa (sRP) in the largest cohort reported to date.Case series.A cohort of 877 unrelated Spanish sporadic cases with a clinical diagnosis of retinitis pigmentosa (RP) and negative family history.The cohort was studied by classic genotyping or targeted next-generation sequencing (NGS). Multiplex ligation-dependent probe amplification (MLPA) and array-based comparative genomic hybridization were performed to confirm copy number variations detected by NGS. Quantitative fluorescent polymerase chain reaction was assessed in sRP cases carrying de novo variants to confirm paternity.The study of the sRP cohort showed a high proportion of causal autosomal dominant (AD) and X-linked (XL) variants, most of them being de novo.Causative variants were identified in 38% of the patients studied, segregating recessively in 84.5% of the solved cases. Biallelic variants detected in only 6 different autosomal recessive genes explained 50% of the cases characterized. Causal AD and XL variants were found in 7.6% and 7.9% of cases, respectively. Remarkably, 20 de novo variants were confirmed after trio analysis, explaining 6% of the cases. In addition, 17% of the solved sRP cases were reclassified to a different retinopathy phenotype.This study highlights the clinical utility of NGS testing for sRP cases, expands the mutational spectrum, and provides accurate prevalence of mutated genes. Our findings evidence the underestimated role of de novo variants in the etiology of RP, emphasizing the importance of segregation analysis as well as comprehensive screening of genes carrying XL and AD variants in sporadic cases. Such in-depth study is essential for accurate family counseling and future enrollment in gene therapy-based treatments.

Mutations in PAX6 are involved in several developmental eye disorders. These disorders have considerable phenotypic variability, ranging from panocular forms of congenital aniridia and microphthalmia to isolated anomalies of the anterior or posterior segment. Here, we describe 3 families with variable inter-generational ocular expression of aniridia, iris coloboma, or microphthalmia, and an unusual transmission of PAX6 mutations from an unaffected or mildly affected parent; all of which raised suspicion of gonosomal mosaicism. We first identified two previously known nonsense mutations and one novel likely pathogenic missense variant in PAX6 in probands by means of targeted NGS. The subsequent segregation analysis by Sanger sequencing evidenced the presence of highly probable mosaic events in paternal blood samples. Mosaicism was further confirmed by droplet digital PCR analysis in several somatic tissues of mosaic fathers. Quantification of the mutant allele fraction in parental samples showed a marked deviation from 50%, with a range between 12 and 29% depending on cell type. Gonosomal mosaicsm was definitively confirmed in one of the families thanks to the availability of a sperm sample from the mosaic father. Thus, the recurrence risk in this family was estimated to be about one-third. This is the first report confirming parental PAX6 mosaicism as a cause of disease recurrence in aniridia and other related phenotypes. In addition, we demonstrated that post-zygotic mosaicism is a frequent and underestimated pathogenic mechanism in aniridia, explaining intra-familial phenotypic variability in many cases. Our findings may have substantial implications for genetic counseling in congenital aniridia. Thus, we also highlight the importance of comprehensive genetic screening of parents for new sporadic cases with aniridia or related developmental eye disease to more accurately assess recurrence risk. In conclusion, somatic and/or gonosomal mosaicism should be taken into consideration as a genetic factor to explain not only families with unaffected parents despite multiple affected children but also variable expressivity, apparent de novo cases, and even uncharacterized cases of aniridia and related developmental eye disorders, apparently lacking PAX6 mutations.

Inherited syndromic retinopathies are a highly heterogeneous group of diseases that involve retinal anomalies and systemic manifestations. They include retinal ciliopathies, other well-defined clinical syndromes presenting with retinal alterations and cases of non-specific multisystemic diseases. The heterogeneity of these conditions makes molecular and clinical characterization of patients challenging in daily clinical practice. We explored the capacity of targeted resequencing and copy-number variation analysis to improve diagnosis of a heterogeneous cohort of 47 patients mainly comprising atypical cases that did not clearly fit a specific clinical diagnosis. Thirty-three likely pathogenic variants were identified in 18 genes (ABCC6, ALMS1, BBS1, BBS2, BBS12, CEP41, CEP290, IFT172, IFT27, MKKS, MYO7A, OTX2, PDZD7, PEX1, RPGRIP1, USH2A, VPS13B, and WDPCP). Molecular findings and additional clinical reassessments made it possible to accurately characterize 14 probands (30% of the total). Notably, clinical refinement of complex phenotypes was achieved in 4 cases, including 2 de novo OTX2-related syndromes, a novel phenotypic association for the ciliary CEP41 gene, and the co-existence of biallelic USH2A variants and a Koolen-de-Vries syndrome-related 17q21.31 microdeletion. We demonstrate that combining next-generation sequencing and CNV analysis is a comprehensive and useful approach to unravel the extensive phenotypic and genotypic complexity of inherited syndromic retinopathies, This study was funded by several grants from the Instituto de Salud Carlos III (ISCIII) from the Spanish Ministry of Health, including CIBERER (06/07/0036), FIS - FEDER (European Regional Development Fund) (PI016/00425), IIS-FJD Biobank PT13/0010/0012. In addition, sponsored chair HU-FJD-UAM “Cátedra de Patrocinio Medicina Genómica”, the Spanish National Organization of the Blind (ONCE) and the Spanish Fighting Blindness Foundation (FUNDALUCE) also supported our work. ISN and RSA are sponsored by Sara Borrell Postdoctoral Program (CD13-00085 and CD12-00676) from ISCIII/FEDER. MC is supported by the Miguel Servet Program (CP12/03256) from ISCIII/FEDER. RPC is supported by Fundación Conchita Rábago and LRJS is supported by CAPES Foundation, Ministry of Education of Brazil

Retinitis pigmentosa (RP) is the most common form of inherited retinal dystrophy, with a worldwide prevalence of 1 in 4000 persons. While in most cases of RP, the disease is limited to the eye (non-syndromic), over 40 forms of syndromic RP have been described.To identify the genetic basis for syndromic RP in three unrelated families from Israel and Spain.Whole exome sequencing was conducted in one Israeli and two Spanish families segregating autosomal recessive RP with intellectual disability. Complete ophthalmic examination included best-corrected visual acuity, funduscopy, optical coherence tomography, fluorescein angiography, flash visual evoked potentials, and electroretinography. Reverse transcription (RT)-PCR and immunostaining were used to examine the spatial and temporal expression pattern of SCAPER.In all patients, biallelicDeleterious

Background: Pseudohypoparathyroidism (PHP) is a rare disease whose phenotypic features are rather difficult to identify in some cases. Thus, although these patients may present with the Albright's hereditary osteodystrophy (AHO) phenotype, which is characterized by small stature, obesity with a rounded face, subcutaneous ossifications, mental retardation and brachydactyly, its manifestations are somewhat variable. Indeed, some of them present with a complete phenotype, whereas others show only subtle manifestations. In addition, the features of the AHO phenotype are not specific to it and a similar phenotype is also commonly observed in other syndromes. Brachydactyly type E (BDE) is the most specific and objective feature of the AHO phenotype, and several genes have been associated with syndromic BDE in the past few years. Moreover, these syndromes have a skeletal and endocrinological phenotype that overlaps with AHO/PHP. In light of the above, we have developed an algorithm to aid in genetic testing of patients with clinical features of AHO but with no causative molecular defect at the GNAS locus. Starting with the feature of brachydactyly, this algorithm allows the differential diagnosis to be broadened and, with the addition of other clinical features, can guide genetic testing. Methods: We reviewed our series of patients (n = 23) with a clinical diagnosis of AHO and with brachydactyly type E or similar pattern, who were negative for GNAS anomalies, and classify them according to the diagnosis algorithm to finally propose and analyse the most probable gene(s) in each case. Results: A review of the clinical data for our series of patients, and subsequent analysis of the candidate gene(s), allowed detection of the underlying molecular defect in 12 out of 23 patients: five patients harboured a mutation in PRKAR1A, one in PDE4D, four in TRPS1 and two in PTHLH. Conclusions: This study confirmed that the screening of other genes implicated in syndromes with BDE and AHO or a similar phenotype is very helpful for establishing a correct genetic diagnosis for those patients who have been misdiagnosed with "AHO-like phenotype" with an unknown genetic cause, and also for better describing the characteristic and differential features of these less common syndromes. The study was supported by funding from a research project grant (PI13/00467) from the Instituto de Salud Carlos III (Carlos III Institute of Health) of the Ministry of Economy and Competitiveness (Spain), co-financed by the European Regional Development Fund, and the Department of Health of the Basque Government (GV2014111017). AP is partly supported by the University of the Basque Country (Ref: 48198). GPN is partly supported by the I3SNS Program of the Spanish Ministry of Health (CP03/0064; SIVI 1395/09).

Summary Background & aim Vitamin D deficiency is an important concern in clinical settings and recently, international agencies have recognised the importance of 25-OHD assays in defining vitamin D status. Thus, our aim was to assess the consistency of different vitamin D assays in clinical practice. Methods: 25-OH-vitamin D was measured in 332 patients by ultra-fast liquid chromatography (UHPLC) and two immunoassays (Liaison Total 25(OH) and ADVIA Centaur Vitamin D Total Assay). Samples from the Vitamin D External Quality Survey (DEQAS) and the Standard Reference Material SRM 972 were used for analytical quality control. Results: All methods displayed an acceptable performance with DEQAS samples but immunoassays showed a significant bias against certified materials. Compared to UHPLC, differences were significant for both immunoassays in the deficiency interval but the systematic bias was higher for the ADVIA assay throughout the whole range of concentrations. Conclusion: The prevalence of vitamin D deficiency in clinical practice is assay-dependent and physicians should be aware of the uncertainty associated with vitamin D assessment.

Biallelic pathogenic RPE65 variants are related to a spectrum of clinically overlapping inherited retinal dystrophies (IRD). Most affected individuals progress to severe disease, with 50% of patients becoming legally blind by 20 years of age. Deeper knowledge of the mutational spectrum and the phenotype-genotype correlation in RPE65-related IRD is needed.Forty-five affected subjects from 27 unrelated families with a clinical diagnosis of RPE65-related IRD were included. Clinical evaluation consisted of self-reported ophthalmological history and objective ophthalmological examination. Patients' genotype was classified according to variant class (truncating or missense) or to variant location at different protein domains. The main phenotypic outcome measure was age at onset (AAO) of symptomatic disease and a Kaplan-Meier analysis of disease symptom event-free survival was performed.Twenty-nine different RPE65 variants were identified in our cohort, 7 of them novel. Patients carrying two missense alleles showed a later disease onset than those with 1 or 2 truncating variants (log-rank test p 0.05). While 60% of patients carrying a missense/missense genotype presented symptoms before or during the first year of life, almost all patients with at least 1 truncating allele (91%) had an AAO ≤1 year (p 0.05).Our findings suggest an association between the type of RPE65 variant carried and AAO. These findings provide useful data on RPE65-associated IRD phenotypes and may help improve clinical and therapeutic management of these patients.