Expanding the PRAAS spectrum: De novo mutations of immunoproteasome subunit ß-type 10 in six infants with SCID-Omenn syndrome.

Mutations in proteasome ß-subunits or their chaperone and regulatory proteins are associated with proteasome-associated autoinflammatory disorders (PRAAS). We studied six unrelated infants with three de novo heterozygous missense variants in PSMB10, encoding the proteasome ß2i-subunit. Individuals presented with T-B-NK± severe combined immunodeficiency (SCID) and clinical features suggestive of Omenn syndrome, including diarrhea, alopecia, and desquamating erythematous rash. Remaining T cells had limited T cell receptor repertoires, a skewed memory phenotype, and an elevated CD4/CD8 ratio. Bone marrow examination indicated severely impaired B cell maturation with limited V(D)J recombination. All infants received an allogeneic stem cell transplant and exhibited a variety of severe inflammatory complications thereafter, with 2 peri-transplant and 2 delayed deaths. The single long-term transplant survivor showed evidence for genetic rescue through revertant mosaicism overlapping the affected PSMB10 locus. The identified variants (c.166G>C [p.Asp56His] and c.601G>A/c.601G>C [p.Gly201Arg]) were predicted in silico to profoundly disrupt 20S immunoproteasome structure through impaired ß-ring/ß-ring interaction. Our identification of PSMB10 mutations as a cause of SCID-Omenn syndrome reinforces the connection between PRAAS-related diseases and SCID.

An HNRNPK-specific DNA methylation signature makes sense of missense variants and expands the phenotypic spectrum of Au-Kline syndrome.

Au-Kline syndrome (AKS) is a neurodevelopmental disorder associated with multiple malformations and a characteristic facial gestalt. The first individuals ascertained carried de novo loss-of-function (LoF) variants in HNRNPK. Here, we report 32 individuals with AKS (26 previously unpublished), including 13 with de novo missense variants. We propose new clinical diagnostic criteria for AKS that differentiate it from the clinically overlapping Kabuki syndrome and describe a significant phenotypic expansion to include individuals with missense variants who present with subtle facial features and few or no malformations. Many gene-specific DNA methylation (DNAm) signatures have been identified for neurodevelopmental syndromes. Because HNRNPK has roles in chromatin and epigenetic regulation, we hypothesized that pathogenic variants in HNRNPK may be associated with a specific DNAm signature. Here, we report a unique DNAm signature for AKS due to LoF HNRNPK variants, distinct from controls and Kabuki syndrome. This DNAm signature is also identified in some individuals with de novo HNRNPK missense variants, confirming their pathogenicity and the phenotypic expansion of AKS to include more subtle phenotypes. Furthermore, we report that some individuals with missense variants have an "intermediate" DNAm signature that parallels their milder clinical presentation, suggesting the presence of an epi-genotype phenotype correlation. In summary, the AKS DNAm signature may help elucidate the underlying pathophysiology of AKS. This DNAm signature also effectively supported clinical syndrome delineation and is a valuable aid for variant interpretation in individuals where a clinical diagnosis of AKS is unclear, particularly for mild presentations.

Loss-of-function in RBBP5 results in a syndromic neurodevelopmental disorder associated with microcephaly.

PURPOSE: Epigenetic dysregulation has been associated with many inherited disorders. RBBP5 (HGNC:9888) encodes a core member of the protein complex that methylates histone 3 lysine-4 and has not been implicated in human disease. METHODS: We identify 5 unrelated individuals with de novo heterozygous variants in RBBP5. Three nonsense/frameshift and 2 missense variants were identified in probands with neurodevelopmental symptoms, including global developmental delay, intellectual disability, microcephaly, and short stature. Here, we investigate the pathogenicity of the variants through protein structural analysis and transgenic Drosophila models. RESULTS: Both missense p.(T232I) and p.(E296D) variants affect evolutionarily conserved amino acids located at the interface between RBBP5 and the nucleosome. In Drosophila, overexpression analysis identifies partial loss-of-function mechanisms when the variants are expressed using the fly Rbbp5 or human RBBP5 cDNA. Loss of Rbbp5 leads to a reduction in brain size. The human reference or variant transgenes fail to rescue this loss and expression of either missense variant in an Rbbp5 null background results in a less severe microcephaly phenotype than the human reference, indicating both missense variants are partial loss-of-function alleles. CONCLUSION: Haploinsufficiency of RBBP5 observed through de novo null and hypomorphic loss-of-function variants is associated with a syndromic neurodevelopmental disorder.

Adult-onset Alexander disease among patients of Jewish Syrian descent.

Alexander disease (AxD) is a rare autosomal dominant leukodystrophy caused by heterozygous mutations in the glial fibrillary acid protein (GFAP) gene. The age of symptoms onset ranges from infancy to adulthood, with variable clinical and radiological manifestations. Adult-onset AxD manifests as a chronic and progressive condition, characterized by bulbar, motor, cerebellar, and other clinical signs and symptoms. Neuroradiological findings typically involve the brainstem and cervical spinal cord. Adult-onset AxD has been described in diverse populations but is rare in Israel. We present a series of patients diagnosed with adult-onset AxD from three families, all of Jewish Syrian descent. Five patients (4 females) were diagnosed with adult-onset AxD due to the heterozygous mutation c.219G > A, p.Met73Ile in GFAP. Age at symptoms onset ranged from 48 to 61 years. Clinical characteristics were typical and involved progressive bulbar and gait disturbance, followed by pyramidal and cerebellar impairment, dysautonomia, and cognitive decline. Imaging findings included medullary and cervical spinal atrophy and mostly infratentorial white matter hyperintensities. A newly recognized cluster of adult-onset AxD in Jews of Syrian origin is presented. This disorder should be considered in differential diagnosis in appropriate circumstances. Genetic counselling for family members is required in order to discuss options for future family planning.

Whole-exome sequencing reveals a monogenic cause in 56% of individuals with laterality disorders and associated congenital heart defects.

BACKGROUND: The molecular basis of heterotaxy and congenital heart malformations associated with disruption of left-right asymmetry is broad and heterogenous, with over 25 genes implicated in its pathogenesis thus far. OBJECTIVE: We sought to elucidate the molecular basis of laterality disorders and associated congenital heart defects in a cohort of 30 unrelated probands of Arab-Muslim descent, using next-generation sequencing techniques. METHODS: Detailed clinical phenotyping followed by whole-exome sequencing (WES) was pursued for each of the probands and their parents (when available). Sanger sequencing was used for segregation analysis of disease-causing mutations in the families. RESULTS: Using WES, we reached a molecular diagnosis for 17 of the 30 probands (56.7%). Genes known to be associated with heterotaxy and/or primary ciliary dyskinesia, in which homozygous pathogenic or likely pathogenic variants were detected, included CFAP53 (CCDC11), CFAP298 (C21orf59), CFAP300, LRRC6, GDF1, DNAAF1, DNAH5, CCDC39, CCDC40, PKD1L1 and TTC25. Additionally, we detected a homozygous disease causing mutation in DAND5, as a novel recessive monogenic cause for heterotaxy in humans. Three additional probands were found to harbour variants of uncertain significance. These included variants in DNAH6, HYDIN, CELSR1 and CFAP46. CONCLUSIONS: Our findings contribute to the current knowledge regarding monogenic causes of heterotaxy and its associated congenital heart defects and underscore the role of next-generation sequencing techniques in the diagnostic workup of such patients, and especially among consanguineous families.

PPP2R1A neurodevelopmental disorder is associated with congenital heart defects.

Protein phosphatase 2A (PP2A) is a heterotrimeric serine/threonine phosphatase that regulates numerous biological processes. PPP2R1A encodes the scaffolding "Aα" subunit of PP2A. To date, nearly 40 patients have been previously reported with 19 different pathogenic PPP2R1A variants, with phenotypes including intellectual disability, developmental delay, epilepsy, infant agenesis/dysgenesis of the corpus callosum, and dysmorphic features. Apart from a single case, severe congenital heart defects (CHD) have not been described. We report four new unrelated individuals with pathogenic heterozygous PPP2R1A variants and CHD and model the crystal structure of several variants to investigate mechanisms of phenotype disparity. Individuals 1 and 2 have a previously described variant (c.548G>A, p.R183Q) and similar phenotypes with severe ventriculomegaly, agenesis/dysgenesis of the corpus callosum, and severe CHD. Individual 3 also has a recurrent variant (c.544C>T, p.R182W) and presented with agenesis of corpus callosum, ventriculomegaly, mild pulmonic stenosis, and small patent foramen ovale. Individual 4 has a novel variant (c.536C>A, p.P179H), ventriculomegaly, and atrial septal defect. To conclude, we propose expansion of the phenotype of PPP2R1A neurodevelopmental disorder to include CHD. Further, the R183Q variant has now been described in three individuals, all with severe neurologic abnormalities, severe CHD, and early death suggesting that this variant may be particularly deleterious.

Kidney and urinary tract findings among patients with Kabuki (make-up) syndrome.

BACKGROUND: Kabuki syndrome (KS) is a genetic disorder caused mainly by de novo pathogenic variants in KMT2D or KDM6A, characterized by recognizable facial features, intellectual disability, and multi-systemic involvement, including short stature, microcephaly, hearing loss, cardiac defects, and additional congenital anomalies. While congenital anomalies of the kidneys and urinary tract (CAKUT) are known manifestations of this disorder, studies focused solely on kidney involvement are scarce, and its prevalence is most likely underestimated. This study aimed to describe the prevalence and nature of CAKUT and other renal manifestations, in a cohort of KS patients followed at a single tertiary center. METHODS: All patients who were evaluated at the Sheba Medical Center and received a clinical and/or molecular diagnosis of KS, over a 16-year period (2004-2020), were included. Digital medical records, including ultrasound studies, were reviewed by a team of pediatric nephrologists. RESULTS: Thirteen patients were included in the study, at ages ranging from the neonatal period to 20 years. In eight patients, a pathogenic variant in KMT2D was established. CAKUT were detected in 8/13 (61.5%) of patients and varied from hypospadias, hydronephrosis, or double collecting systems to pelvic kidney, kidney asymmetry, horseshoe kidney, or kidney agenesis. One patient experienced kidney failure necessitating transplantation at 20 years of age. CONCLUSIONS: Our findings underscore the high prevalence of CAKUT and genitourinary involvement in patients with KS and suggest that assessment by pediatric nephrology specialists is warranted as part of the routine multidisciplinary evaluation of newly diagnosed patients. A higher resolution version of the Graphical abstract is available as Supplementary information.

Atopic Predilection among Kawasaki Disease Patients: A Cross-Sectional Study of 1,187,757 Teenagers.

BACKGROUND: Kawasaki disease (KD) is an acute, systemic vasculitis in children, with an etiology that is not completely understood. It is assumed that the development of KD is mediated by an immunologic response. Several reports from East Asia have found a higher prevalence of atopic diseases among patients with KD, but a large-scale study of a non-Asian population regarding this correlation is still lacking. The purpose of this article was to achieve this goal. METHODS: We conducted a cross-sectional, large-scale study to estimate the correlation of KD with allergic diseases. The medical history of 1,187,757 Israeli teenagers (aged 16-20 years during the years 1998-2013) was retrieved. The study population was divided into 3 groups according to a past history of noncomplicated and complicated KD and a control group. The prevalence of allergic diseases among these groups was further investigated. RESULTS: The prevalence of atopic diseases in the 3 study groups was presented (asthma in 11.4, 8.1 and 3.5%, respectively; angioedema/urticaria in 7.1, 0 and 0.46%, respectively; allergic rhinitis in 20, 12.1 and 6.7%, respectively). In noncomplicated KD, a statistically significant link to asthma [odds ratio (OR) 2.4; p = 0.048] and a borderline significant link to allergic rhinitis (OR 1.9; p = 0.06) were found. In KD complicated with cardiac disease, statistically significant links were found for all the allergic conditions, asthma (OR 3.5; p = 0.003), allergic rhinitis (OR 3.5; p < 0.001) and angioedema/urticaria (OR 16.48; p < 0.001). CONCLUSION: KD is associated with allergic diseases. This association increases with the severity of the disease.

Very Early Levodopa May Prevent Self-Injury in Lesch-Nyhan Disease.

BACKGROUND: In Lesch-Nyhan disease (LND), early dopamine deficiency is thought to contribute to dystonia and self-injury, gradually developing over the first years of life. Previous attempts to restore dopamine levels in older patients have been unsuccessful. Based on the hypothesis that very early dopamine replacement can prevent full phenotypic development, we treated three patients with LND from infancy with levodopa. METHODS: Levodopa/carbidopa (4:1) was started at age 11 to 13 months, aiming at escalating to 5 to 6 mg/kg levodopa per day. Follow-up focused on dystonia severity and whether self-injury occurred. In addition, the literature was reviewed to delineate the age at onset of self-injury for all reported cases to date. RESULTS: During long-term follow-up, self-injury appears to have been prevented in two patients (now aged 14 and 15.5 years), as their HPRT1 gene mutations had been invariably associated with self-injury before. Future self-injury is unlikely, as only 1.1% of 264 published cases had self-injury onset later in life than these patients' current ages. The third patient started self-injury at age 1.5 years, while on a substantially lower levodopa dose. A clear effect of levodopa on dystonia could not be determined. CONCLUSIONS: Our observations suggest that levodopa, given early enough and sufficiently dosed, might be able to prevent self-injury in LND. Therefore, levodopa could be considered in patients with LND as early as possible, at least before the self-injury appears. Further research is needed to establish very early levodopa as an effective treatment strategy in LND, and to optimize timing and dosing.

Muscular dystrophy patients show low exercise-induced blood flow in muscles with normal strength.

OBJECTIVE: Neuromuscular evaluation increasingly employs muscle ultrasonography to determine muscle thickness, mean grayscale echointensity, and visual semiquantitative echotexture attenuation. However, these measures provide low sensitivity for detection of mild muscle abnormality. Exercise-induced intramuscular blood flow is a physiologic phenomenon, which may be impaired in mildly affected muscles, particularly in dystrophinopathies, and may indicate functional muscle ischemia. We aimed to determine if muscle blood flow is reduced in patients with neuromuscular disorders and preserved muscle strength, and if it correlates with echointensity and digital echotexture measurements. METHODS: Peak exercise-induced blood flow, echointensity, and echotexture were quantified in the elbow flexor muscles of 15 adult patients with Becker muscular dystrophy (BMD) and 13 patients with other muscular dystrophies (OMD). These were compared to 17 patients with Charcot-Marie-Tooth type 1 (CMT1) neuropathy and 21 healthy adults from a previous study. RESULTS: Muscle blood flow was reduced in all patient groups compared to controls, most prominently in BMD patients (p < 0.0001). Echointensity was similarly increased in all patient groups (p < 0.05), while echotexture was reduced only in muscular dystrophy patients (p ≤ 0.002). In BMD, blood flow correlated with echotexture (Pearson r = 0.6098, p = 0.0158) and strength (Spearman r = 0.5471; p = 0.0370). In patients with normal muscle strength, reduced muscle blood flow was evident in all patient groups (p < 0.001), echotexture was reduced in BMD and OMD (p < 0.01), and echointensity was increased in CMT (p < 0.05). INTERPRETATION: Muscle blood flow is a sensitive measure to detect abnormality, even in muscles with normal strength. Increased echointensity may indicate a neurogenic disorder when strength is preserved, while low echotexture suggests a dystrophic disease.

The yield of genetic workup for middle-aged and elderly patients with neurological disorders in a real-world setting.

Genetic workup is becoming increasingly common in the clinical assessment of neurological disorders. We evaluated its yield among middle-aged and elderly neurological patients, in a real-world context. This retrospective study included 368 consecutive Israeli patients aged 50 years and older (202 [54.9%] males), who were referred to a single neurogenetics clinic between 2017 and mid-2023. All had neurological disorders, without a previous molecular diagnosis. Demographic, clinical and genetic data were collected from medical records. The mean age at first genetic counseling at the clinic was 62.3 ± 7.8 years (range 50-85 years), and the main indications for referral were neuromuscular, movement and cerebrovascular disorders, as well as cognitive impairment and dementia. Out of the 368 patients, 245 (66.6%) underwent genetic testing that included exome sequencing (ES), analysis of nucleotide repeat expansions, detection of specific mutations, targeted gene panel sequencing or chromosomal microarray analysis. Overall, 80 patients (21.7%) received a molecular diagnosis due to 36 conditions, accounting for 32.7% of the patients who performed genetic testing. The diagnostic rates were highest for neuromuscular (58/186 patients [31.2%] in this group, 39.2% of 148 tested individuals) and movement disorders (14/79 [17.7%] patients, 29.2% of 48 tested), but lower for other disorders. Testing of nucleotide repeat expansions and ES provided a diagnosis to 28/73 (38.4%) and 19/132 (14.4%) individuals, respectively. Based on our findings, genetic workup and testing are useful in the diagnostic process of neurological patients aged ≥50 years, in particular for those with neuromuscular and movement disorders.

Oral and fecal polio vaccine excretion following bOPV vaccination among Israeli infants.

INTRODUCTION: Inactivated polio virus (IPV) vaccinations are a mainstay of immunization schedules in developed countries, while oral polio vaccine (OPV) is administered in developing countries and is the main vaccine in outbreaks. Due to circulating wild poliovirus (WPV1) detection in Israel (2013), oral bivalent polio vaccination (bOPV) was administered to IPV primed children and incorporated into the vaccination regimen. OBJECTIVES: We aimed to determine the extent and timeframe of fecal and salivary polio vaccine virus (Sabin strains) shedding following bOPV vaccination among IPV primed children. METHODS: Fecal samples were collected from a convenience sample of infants and toddlers attending 11 Israeli daycare centers. Salivary samples were collected from infants and toddlers following bOPV vaccination. RESULTS: 398 fecal samples were collected from 251 children (ages: 6-32 months), 168 received bOPV vaccination 4-55 days prior to sample collection. Fecal excretion continued among 80 %, 50 %, and 20 %, 2, 3, and 7 weeks following vaccination. There were no significant differences in the rate and duration of positive samples among children immunized with 3 or 4 IPV doses. Boys were 2.3-fold more likely to excrete the virus (p = 0.006). Salivary shedding of Sabin strains occurred in 1/47 (2 %) and 1/49 (2 %) samples 4, and 6 days following vaccination respectively. CONCLUSIONS: Fecal detection of Sabin strains among IPV-primed children continues for 7 weeks; additional doses of IPV do not augment intestinal immunity; limited salivary shedding occurs for up to a week. This data can enhance understanding of intestinal immunity achieved by different vaccination schedules and guide recommendations for contact precautions of children following bOPV vaccination.

What Can We Learn from the Parents of Children Affected with Mucopolysaccharidosis Type III-A in Israel?

Sanfilippo Syndrome, or mucopolysaccharidosis type III (MPS III), is a group of autosomal-recessive lysosomal storage disorders leading to tissue accumulation of heparan sulfate. MPS III is caused by deficiency in one of 4 enzymes involved in lysosomal degradation of heparan sulfate. Based on the relevant enzyme deficiency, 4 types have been recognized. MPS III constitutes a progressive neurodegenerative and systemic disorder. Parents of children diagnosed with MPS III were interviewed using a retrospective questionnaire based on the known clinical manifestations of MPS III. Eight patients from 4 unrelated families of varied ethnic origin were included. All children were diagnosed with MPS type III-A. Average age at diagnosis was 6.1 years. The most common early clinical manifestations leading to parental suspicion of illness were speech delay and coarse facial features. All children were reported to have global developmental delay, sleep disorders, recurrent infections, hyperactivity, and decreased hearing. The time from first medical inquiry until diagnosis was over 2 years on average, consistent with the delay in diagnosis described in the literature. MPS III children frequently undergo early and repeated ear, nose and throat surgeries, thus we suggest that a high index of suspicion is warranted in relevant clinical circumstances.

Broadening the phenotype of LRRK1 mutations - Features of malignant osteopetrosis and optic nerve atrophy with intrafamilial variable expressivity.

Osteosclerotic metaphyseal dysplasia is a rare disorder which features osteosclerosis involving long bones, vertebrae, ribs, clavicles and the iliac crests. Additional features which have variably been reported include developmental delay, short stature, hypotonia and seizures. The disease is caused by pathogenic variants in the LRRK1 gene, and inherited in an autosomal recessive manner. We report three siblings (ages 14 years, 11.5 years and 0.9 years), born to consanguineous parents of Arab-Muslim descent, harboring a homozygous pathogenic variant in the LRRK1 gene (Chr15:101068759 AGGGGCT>A, c.5965_5970del TGGGGC, p.Trp1989Gly1990del). The patients displayed variable degrees of skeletal dysplasia, with the oldest sibling most severely affected, and the youngest infant with minor skeletal involvement. Two of the siblings exhibited normal neurological development, while the youngest sibling exhibited global developmental delay. None of the siblings had seizures; however, two of them exhibited nystagmus. Optic nerve involvement has not previously been reported to be part of the clinical spectrum of this disease. The degree of optic nerve involvement did not correlate with the degree of skeletal involvement. This indicates both intra-familial variable expressivity along with a broadening of the spectrum of LRRK1-associated disease. These findings warrant reconsideration of therapeutic strategies, including the possibility of hematopoietic stem cell transplantation (HSCT) as is performed in cases of malignant and intermediate forms of osteopetrosis.

Vici syndrome in Israel: Clinical and molecular insights.

Introduction: Vici Syndrome is a rare, severe, neurodevelopmental/neurodegenerative disorder with multi-systemic manifestations presenting in infancy. It is mainly characterized by global developmental delay, seizures, agenesis of the corpus callosum, hair and skin hypopigmentation, bilateral cataract, and varying degrees of immunodeficiency, among other features. Vici Syndrome is caused by biallelic pathogenic variants in EPG5, resulting in impaired autophagy. Thus far, the condition has been reported in less than a hundred individuals. Objective and Methods: We aimed to characterize the clinical and molecular findings in individuals harboring biallelic EPG5 variants, recruited from four medical centers in Israel. Furthermore, we aimed to utilize a machine learning-based tool to assess facial features of Vici syndrome. Results: Eleven cases of Vici Syndrome from five unrelated families, one of which was diagnosed prenatally with subsequent termination of pregnancy, were recruited. A total of five disease causing variants were detected in EPG5: two novel: c.2554-5A>G and c.1461delC; and 3 previously reported: c.3447G>A, c.5993C>G, and c.1007A>G, the latter previously identified in several patients of Ashkenazi-Jewish (AJ) descent. Amongst 140,491 individuals screened by the Dor Yeshorim Program, we show that the c.1007A>G variant has an overall carrier frequency of 0.45% (1 in 224) among AJ individuals. Finally, based on two-dimensional facial photographs of individuals with Vici syndrome (n = 19), a composite facial mask was created using the DeepGestalt algorithm, illustrating facial features typical of this disorder. Conclusion: We report on ten children and one fetus from five unrelated families, affected with Vici syndrome, and describe prenatal and postnatal characteristics. Our findings contribute to the current knowledge regarding the molecular basis and phenotypic features of this rare syndrome. Additionally, the deep learning-based facial gestalt adds to the clinician's diagnostic toolbox and may aid in facilitating identification of affected individuals.

Clinical impact of exome sequencing in the setting of a general pediatric ward for hospitalized children with suspected genetic disorders.

Background: Genetic conditions contribute a significant portion of disease etiologies in children admitted to general pediatric wards worldwide. While exome sequencing (ES) has improved clinical diagnosis and management over a variety of pediatric subspecialties, it is not yet routinely used by general pediatric hospitalists. We aim to investigate the impact of exome sequencing in sequencing-naive children suspected of having monogenic disorders while receiving inpatient care. Methods: We prospectively employed exome sequencing in children admitted to the general pediatric inpatient service at a large tertiary medical center in Israel. Genetic analysis was triggered by general and/or subspecialist pediatricians who were part of the primary inpatient team. We determined the diagnostic yield among children who were referred for exome sequencing and observed the effects of genetic diagnosis on medical care. Results: A total of fifty probands were evaluated and exome sequenced during the study period. The most common phenotypes included were neurodevelopmental (56%), gastrointestinal (34%), and congenital cardiac anomalies (24%). A molecular diagnosis was reached in 38% of patients. Among seven patients (37%), the molecular genetic diagnosis influenced subsequent clinical management already during admission or shortly following discharge. Conclusion: We identified a significant fraction of genetic etiologies among undiagnosed children admitted to the general pediatric ward. Our results support that early application of exome sequencing may be maximized by pediatric hospitalists' high index of suspicion for an underlying genetic etiology, prompting an in-house genetic evaluation. This framework should include a multidisciplinary co-management approach of the primary care team working alongside with subspecialties, geneticists and bioinformaticians.

Ophthalmic manifestations in Kabuki (make-up) syndrome: A single-center pediatric cohort and systematic review of the literature.

Kabuki syndrome (KS) is a genetic disorder caused by pathogenic variants in KMT2D or KDM6A, and manifesting with multi-systemic involvement, including recognizable facial features, developmental delay and multiple congenital anomalies. Ophthalmological involvement has been described in varying rates in several studies. We aimed to evaluate the prevalence and nature of ophthalmological findings in a cohort of KS patients in Israel. Medical records of all patients diagnosed with KS in our tertiary center between 2004 and 2020 were retrospectively reviewed. Data collected included physical examination findings, molecular analysis as well as comprehensive ophthalmic characteristics including visual acuity, ocular alignment and motility, ocular adnexa, anterior segments and dilated fundus exams. Finally, an updated systematic review of the literature was performed. Thirteen unrelated patients were included in the study, diagnosed at an age raging from the first months of life to 20 years. Of these, three (23%) showed significant ophthalmological abnormalities, beyond the characteristic structural findings of long palpebral fissures and lower eyelid eversion. These included bilateral posterior colobomata in the first patient; bilateral ptosis, hypermetropia, esotropia, blue sclera and anisocoria in the second; and bilateral congenital cataracts in the third. To conclude, our findings underscore the importance of a comprehensive ophthalmological evaluation as part of the routine multidisciplinary assessment of children suspected/diagnosed with KS.

A single center experience with publicly funded clinical exome sequencing for neurodevelopmental disorders or multiple congenital anomalies.

Exome sequencing (ES) is an important diagnostic tool for individuals with neurodevelopmental disorders (NDD) and/or multiple congenital anomalies (MCA). However, the cost of ES limits the test's accessibility for many patients. We evaluated the yield of publicly funded clinical ES, performed at a tertiary center in Israel, over a 3-year period (2018-2020). Probands presented with (1) moderate-to-profound global developmental delay (GDD)/intellectual disability (ID); or (2) mild GDD/ID with epilepsy or congenital anomaly; and/or (3) MCA. Subjects with normal chromosomal microarray analysis who met inclusion criteria were included, totaling 280 consecutive cases. Trio ES (proband and parents) was the default option. In 252 cases (90.0%), indication of NDD was noted. Most probands were males (62.9%), and their mean age at ES submission was 9.3 years (range 1 month to 51 years). Molecular diagnosis was reached in 109 probands (38.9%), mainly due to de novo variants (91/109, 83.5%). Disease-causing variants were identified in 92 genes, 15 of which were implicated in more than a single case. Male sex, families with multiple-affected members and premature birth were significantly associated with lower ES yield (p < 0.05). Other factors, including MCA and coexistence of epilepsy, autism spectrum disorder, microcephaly or abnormal brain magnetic resonance imaging findings, were not associated with the yield. To conclude, our findings support the utility of clinical ES in a real-world setting, as part of a publicly funded genetic workup for individuals with GDD/ID and/or MCA.

Transcriptome sequencing identifies a noncoding, deep intronic variant in CLCN7 causing autosomal recessive osteopetrosis.

BACKGROUND: Over half of children with rare genetic diseases remain undiagnosed despite maximal clinical evaluation and DNA-based genetic testing. As part of an Undiagnosed Diseases Program applying transcriptome (RNA) sequencing to identify the causes of these unsolved cases, we studied a child with severe infantile osteopetrosis leading to cranial nerve palsies, bone deformities, and bone marrow failure, for whom whole-genome sequencing was nondiagnostic. METHODS: We performed transcriptome (RNA) sequencing of whole blood followed by analysis of aberrant transcript isoforms and osteoclast functional studies. RESULTS: We identified a pathogenic deep intronic variant in CLCN7 creating an unexpected, frameshifting pseudoexon causing complete loss of function. Functional studies, including osteoclastogenesis and bone resorption assays, confirmed normal osteoclast differentiation but loss of osteoclast function. CONCLUSION: This is the first report of a pathogenic deep intronic variant in CLCN7, and our approach provides a model for systematic identification of noncoding variants causing osteopetrosis-a disease for which molecular-genetic diagnosis can be pivotal for potentially curative hematopoietic stem cell transplantation. Our work illustrates that cryptic splice variants may elude DNA-only sequencing and supports broad first-line use of transcriptome sequencing for children with undiagnosed diseases.