Combined Immunodeficiency Caused by a Novel Nonsense Mutation in LCK.

Mutations affecting T-cell receptor (TCR) signaling typically cause combined immunodeficiency (CID) due to varying degrees of disturbed T-cell homeostasis and differentiation. Here, we describe two cousins with CID due to a novel nonsense mutation in LCK and investigate the effect of this novel nonsense mutation on TCR signaling, T-cell function, and differentiation. Patients underwent clinical, genetic, and immunological investigations. The effect was addressed in primary cells and LCK-deficient T-cell lines after expression of mutated LCK. RESULTS: Both patients primarily presented with infections in early infancy. The LCK mutation led to reduced expression of a truncated LCK protein lacking a substantial part of the kinase domain and two critical regulatory tyrosine residues. T cells were oligoclonal, and especially naïve CD4 and CD8 T-cell counts were reduced, but regulatory and memory including circulating follicular helper T cells were less severely affected. A diagnostic hallmark of this immunodeficiency is the reduced surface expression of CD4. Despite severely impaired TCR signaling mTOR activation was partially preserved in patients' T cells. LCK-deficient T-cell lines reconstituted with mutant LCK corroborated partially preserved signaling. Despite detectable differentiation of memory and effector T cells, their function was severely disturbed. NK cell cytotoxicity was unaffected. Residual TCR signaling in LCK deficiency allows for reduced, but detectable T-cell differentiation, while T-cell function is severely disturbed. Our findings expand the previous report on one single patient on the central role of LCK in human T-cell development and function.

A Novel Homozygous Missense Variant in the LRRC32 Gene Is Associated With a New Syndrome of Cleft Palate, Progressive Vitreoretinopathy, Growth Retardation, and Developmental Delay.

Cleft lip and/or cleft palate are a common group of birth defects that further classify into syndromic and non-syndromic forms. The syndromic forms are usually accompanied by additional physical or cognitive abnormalities. Isolated cleft palate syndromes are less common; however, they are associated with a variety of congenital malformations and generally have an underlying genetic etiology. A single report in 2019 described a novel syndrome in three individuals, characterized by cleft palate, developmental delay and proliferative retinopathy due to a homozygous non-sense mutation in the LRRC32 gene encoding glycoprotein A repetitions predominant (GARP), a cell surface polypeptide crucial for the processing and maturation of transforming growth factor ß (TGF-ß). We describe a patient who presented with cleft palate, prenatal and postnatal severe growth retardation, global developmental delay, dysmorphic facial features and progressive vitreoretinopathy. Whole exome sequencing (WES) revealed a very rare homozygous missense variant in the LRRC32 gene, which resulted in substitution of a highly conserved isoleucine to threonine. Protein modeling suggested this variant may negatively affect GARP function on latent TGF-ß activation. In summary, our report further expands the clinical features of cleft palate, proliferative retinopathy and developmental delay syndrome and emphasizes the association of LRRC32 pathogenic variants with this new syndrome.

Orbital nodular fasciitis in child with biallelic germline RBL2 variant.

RBL2/p130 is one of three highly conserved members of the retinoblastoma (RB) protein family. It is strongly upregulated during neuronal differentiation and brain development, and is critical for survival of post-mitotic neurons. Similar to RB1, it has been implicated as a tumor suppressor gene and has been shown to be dysregulated in various types of cancer. Recent publications describe biallelic, germline loss of function variants in RBL2 in individuals with profound developmental delay. We report a child with profound developmental delay, microcephaly, and hypotonia, who developed fulminant exophthalmos at age 6 years. Brain MRI followed by a biopsy of an intra-orbital mass revealed a mesenchymal tumor. Post-surgical histopathologic examination of the resected tumor was compatible with diagnosis of nodular fasciitis. Exome sequencing from peripheral blood identified a biallelic frameshift variant (c.901dupT) in RBL2. Notably, no malignancies were reported in previous cases with RBL2 variants. This case provides a possible association between RBL2 and orbital tumors.

PNC2 (SLC25A36) Deficiency Associated With the Hyperinsulinism/Hyperammonemia Syndrome.

CONTEXT: The hyperinsulinism/hyperammonemia (HI/HA) syndrome, the second-most common form of congenital hyperinsulinism, has been associated with dominant mutations in GLUD1, coding for the mitochondrial enzyme glutamate dehydrogenase, that increase enzyme activity by reducing its sensitivity to allosteric inhibition by GTP. OBJECTIVE: To identify the underlying genetic etiology in 2 siblings who presented with the biochemical features of HI/HA syndrome but did not carry pathogenic variants in GLUD1, and to determine the functional impact of the newly identified mutation. METHODS: The patients were investigated by whole exome sequencing. Yeast complementation studies and biochemical assays on the recombinant mutated protein were performed. The consequences of stable slc25a36 silencing in HeLa cells were also investigated. RESULTS: A homozygous splice site variant was identified in solute carrier family 25, member 36 (SLC25A36), encoding the pyrimidine nucleotide carrier 2 (PNC2), a mitochondrial nucleotide carrier that transports pyrimidine as well as guanine nucleotides across the inner mitochondrial membrane. The mutation leads to a 26-aa in-frame deletion in the first repeat domain of the protein, which abolishes transport activity. Furthermore, knockdown of slc25a36 expression in HeLa cells caused a marked reduction in the mitochondrial GTP content, which likely leads to a hyperactivation of glutamate dehydrogenase in our patients. CONCLUSION: We report for the first time a mutation in PNC2/SLC25A36 leading to HI/HA and provide functional evidence of the molecular mechanism responsible for this phenotype. Our findings underscore the importance of mitochondrial nucleotide metabolism and expand the role of mitochondrial transporters in insulin secretion.

Biallelic TMEM260 variants cause truncus arteriosus, with or without renal defects.

Only two families have been reported with biallelic TMEM260 variants segregating with structural heart defects and renal anomalies syndrome (SHDRA). With a combination of genome, exome sequencing and RNA studies, we identified eight individuals from five families with biallelic TMEM260 variants. Variants included one multi-exon deletion, four nonsense/frameshifts, two splicing changes and one missense change. Together with the published cases, analysis of clinical data revealed ventricular septal defects (12/12), mostly secondary to truncus arteriosus (10/12), elevated creatinine levels (6/12), horse-shoe kidneys (1/12) and renal cysts (1/12) in patients. Three pregnancies were terminated on detection of severe congenital anomalies. Six patients died between the ages of 6 weeks and 5 years. Using a range of stringencies, carrier frequency for SHDRA was estimated at 0.0007-0.007 across ancestries. In conclusion, this study confirms the genetic basis of SHDRA, expands its known mutational spectrum and clarifies its clinical features. We demonstrate that SHDRA is a severe condition associated with substantial mortality in early childhood and characterised by congenital cardiac malformations with a variable renal phenotype.

Clinical presentation and analysis of genotype-phenotype correlations in patients with malignant infantile osteopetrosis.

Malignant infantile osteopetrosis (MIOP) is the autosomal recessive, severe form of osteopetrosis. This rare genetic syndrome usually presents soon after birth and is often fatal if left untreated. Early diagnosis is key for proper management but clinical presentation is diverse, and oftentimes diagnosis may be challenging. In this study, we retrospectively collected data of genetic mutations and phenotypic characteristics at the initial presentation of 81 MIOP patients and analyzed genotype-phenotype correlations. The most common genetic mutation was in the TCIRG1 gene (n = 46, 56.8%), followed by SNX10 (n = 20, 25%). Other genetic mutations included RANK (n = 7, 8.7%), CLCN7 (n = 5, 6.2%) and CA2 (n = 3, 3.7%). More than half of the patients presented with growth retardation (n = 46, 56.8%). Twenty-one of the patients were blind (26%) and thirty-seven patients had other neurological deficits (45.7%) at the time of initial presentation. Most patients presented with hematological signs of bone marrow failure including anemia (n = 69, 85.2%) and thrombocytopenia (n = 33, 40.7%). Thrombocytopenia at initial presentation was significantly more prevalent in patients with mutations in the TCIRG1 gene (p = 0.036). Other phenotypic presenting features were not found to be significantly correlated to specific gene mutations. In conclusion, the initial presentation of MIOP is variable, but some features are common such as growth retardation, visual impairment, and cytopenias. High awareness of MIOP presenting signs is essential for prompt diagnosis of this challenging disease.

Characterization of a L136P mutation in Formin-like 2 (FMNL2) from a patient with chronic inflammatory bowel disease.

Diaphanous related formins are highly conserved proteins regulated by Rho-GTPases that act as actin nucleation and assembly factors. Here we report the functional characterization of a non-inherited heterozygous FMNL2 p.L136P mutation carried by a patient who presented with severe very early onset inflammatory bowel disease (IBD). We found that the FMNL2 L136P protein displayed subcellular mislocalization and deregulated protein autoinhibition indicating gain-of-function mechanism. Expression of FMNL2 L136P impaired cell spreading as well as filopodia formation. THP-1 macrophages expressing FMNL2 L136P revealed dysregulated podosome formation and a defect in matrix degradation. Our data indicate that the L136P mutation affects cellular actin dynamics in fibroblasts and immune cells such as macrophages.

Biallelic deletion in a minimal CAPN15 intron in siblings with a recognizable syndrome of congenital malformations and developmental delay.

Calpainopathies constitute a heterogeneous group of disorders resulting from deficiencies in calpains, calcium-specific proteases that modulate substrates by limited proteolysis. Clinical manifestations depend on tissue-specific expression of the defective calpain and substrate specificity. CAPN15, encoding the Drosophila small optic lobes (sol) homolog, was recently found to cause various eye defects in individuals carrying bi-allelic missense variants. Here we report on two siblings with manifestations reminiscent of Johanson-Blizzard syndrome including failure to thrive, microcephaly, global developmental delay, dysmorphic features, endocrine abnormalities and congenital malformations, in addition to eye abnormalities. Exome sequencing identified a homozygous 47 base-pair deletion in a minimal intron of CAPN15, including the splice donor site. Sequencing of cDNA revealed single exon skipping, resulting in an out-of-frame deletion with a predicted premature termination codon. These findings expand the phenotypic spectrum associated with CAPN15 variants, and suggest that complete loss-of-function is associated with a recognizable syndrome of congenital malformations and developmental delay, overlapping Johanson-Blizzard syndrome and the recently observed brain defects in Capn15 knockout (KO) mice. Moreover, the data highlight the unique opportunity for indel detection in minimal introns.

Bacillus Calmette-Guerin (BCG) Vaccine-associated Complications in Immunodeficient Patients Following Stem Cell Transplantation.

PURPOSE: Bacillus Calmette-Guerin (BCG) is a live attenuated vaccine with the potential of causing severe iatrogenic complications in patients with primary immunodeficiency diseases (PID) before and after hematopoietic stem cell transplantation (HSCT). We aim to investigate risk factors of post-HSCT BCG-related complications in PID patients. METHODS: A retrospective analysis of pediatric PID patients who had received the BCG vaccine and underwent HSCT at Hadassah-Hebrew University Medical Center, between 2007 and 2019. RESULTS: We found 15/36 (41.67%) patients who developed post-HSCT BCG-related complications. The most significant risk factor for developing BCG-related complications was T cell deficiency (47.6% of the non-complicated vs 83.3% of the BCGitis and 100% of the BCGosis groups had T cell lymphopenia, p = 0.013). None of the chronic granulomatous patients developed BCG-related manifestation post-transplant. Among T cell-deficient patients, lower NK (127 vs 698 cells/µl, p = 0.04) cell counts and NK-SCID were risk factors for ongoing post-HSCT BCGosis, as was pretransplant disseminated BCGosis (33.3% of patients with BCGosis vs none of the non-BCGosis patients, p = 0.04). Immune reconstitution inflammatory syndrome (IRIS) was observed in 3/5 patients with Omenn syndrome. Prophylactic antimycobacterial treatment was not proven effective. CONCLUSION: BCG vaccination can cause significant morbidity and mortality in the post-transplant T cell-deficient patient, especially in the presence of pre-transplant disease. Taking a detailed medical history prior to administering, the BCG vaccine is crucial for prevention of this complication.

CFAP45 deficiency causes situs abnormalities and asthenospermia by disrupting an axonemal adenine nucleotide homeostasis module.

Axonemal dynein ATPases direct ciliary and flagellar beating via adenosine triphosphate (ATP) hydrolysis. The modulatory effect of adenosine monophosphate (AMP) and adenosine diphosphate (ADP) on flagellar beating is not fully understood. Here, we describe a deficiency of cilia and flagella associated protein 45 (CFAP45) in humans and mice that presents a motile ciliopathy featuring situs inversus totalis and asthenospermia. CFAP45-deficient cilia and flagella show normal morphology and axonemal ultrastructure. Proteomic profiling links CFAP45 to an axonemal module including dynein ATPases and adenylate kinase as well as CFAP52, whose mutations cause a similar ciliopathy. CFAP45 binds AMP in vitro, consistent with structural modelling that identifies an AMP-binding interface between CFAP45 and AK8. Microtubule sliding of dyskinetic sperm from Cfap45-/- mice is rescued with the addition of either AMP or ADP with ATP, compared to ATP alone. We propose that CFAP45 supports mammalian ciliary and flagellar beating via an adenine nucleotide homeostasis module.

Loss of ADAMTS19 causes progressive non-syndromic heart valve disease.

Valvular heart disease is observed in approximately 2% of the general population1. Although the initial observation is often localized (for example, to the aortic or mitral valve), disease manifestations are regularly observed in the other valves and patients frequently require surgery. Despite the high frequency of heart valve disease, only a handful of genes have so far been identified as the monogenic causes of disease2-7. Here we identify two consanguineous families, each with two affected family members presenting with progressive heart valve disease early in life. Whole-exome sequencing revealed homozygous, truncating nonsense alleles in ADAMTS19 in all four affected individuals. Homozygous knockout mice for Adamts19 show aortic valve dysfunction, recapitulating aspects of the human phenotype. Expression analysis using a lacZ reporter and single-cell RNA sequencing highlight Adamts19 as a novel marker for valvular interstitial cells; inference of gene regulatory networks in valvular interstitial cells positions Adamts19 in a highly discriminatory network driven by the transcription factor lymphoid enhancer-binding factor 1 downstream of the Wnt signaling pathway. Upregulation of endocardial Krüppel-like factor 2 in Adamts19 knockout mice precedes hemodynamic perturbation, showing that a tight balance in the Wnt-Adamts19-Klf2 axis is required for proper valve maturation and maintenance.

Stem cell transplantation for osteopetrosis in patients beyond the age of 5 years.

Osteopetrosis (OP) is a rare disease caused by defective osteoclast differentiation or function. Hematopoietic stem cell transplantation (HSCT) is the only curative treatment available in the infantile "malignant" form of OP. Improved clinical and genetic diagnosis of OP has seen the emergence of a cohort of patients with less severe and heterogeneous clinical presentations. This intermediate form of OP does not call for urgent intervention, but patients accumulate debilitating skeletal complications over years and decades, which are severe enough to require curative treatment and may also require intermittent transfusion of blood products. Here we present data from 7 patients with intermediate OP caused by mutations in TCIRG1 (n = 2), CLCN7 (n = 2), RANK (n = 1), SNX10 (n = 1), and CA2 (n = 1), who were transplanted between the ages of 5 to 30 years (mean, 15; median, 12). Donors were matched siblings or family (n = 4), matched unrelated (n = 2), or HLA haploidentical family donors (n = 1). Conditioning was fludarabine and treosulfan based. All 6 patients transplanted from matched donors are currently alive with a follow-up period between 1 and 8 years at time of publication (median, 4 years) and have demonstrated a significant improvement in symptoms and quality of life. Patients with intermediate OP should be considered for HSCT.

OTULIN deficiency in ORAS causes cell type-specific LUBAC degradation, dysregulated TNF signalling and cell death.

The deubiquitinase OTULIN removes methionine-1 (M1)-linked polyubiquitin signals conjugated by the linear ubiquitin chain assembly complex (LUBAC) and is critical for preventing TNF-driven inflammation in OTULIN-related autoinflammatory syndrome (ORAS). Five ORAS patients have been reported, but how dysregulated M1-linked polyubiquitin signalling causes their symptoms is unclear. Here, we report a new case of ORAS in which an OTULIN-Gly281Arg mutation leads to reduced activity and stability in vitro and in cells. In contrast to OTULIN-deficient monocytes, in which TNF signalling and NF-κB activation are increased, loss of OTULIN in patient-derived fibroblasts leads to a reduction in LUBAC levels and an impaired response to TNF Interestingly, both patient-derived fibroblasts and OTULIN-deficient monocytes are sensitised to certain types of TNF-induced death, and apoptotic cells are evident in ORAS patient skin lesions. Remarkably, haematopoietic stem cell transplantation leads to complete resolution of inflammatory symptoms, including fevers, panniculitis and diarrhoea. Therefore, haematopoietic cells are necessary for clinical manifestation of ORAS Together, our data suggest that ORAS pathogenesis involves hyper-inflammatory immune cells and TNF-induced death of both leukocytes and non-haematopoietic cells.

Combined loss of LAP1B and LAP1C results in an early onset multisystemic nuclear envelopathy.

Nuclear envelopathies comprise a heterogeneous group of diseases caused by mutations in genes encoding nuclear envelope proteins. Mutations affecting lamina-associated polypeptide 1 (LAP1) result in two discrete phenotypes of muscular dystrophy and progressive dystonia with cerebellar atrophy. We report 7 patients presenting at birth with severe progressive neurological impairment, bilateral cataract, growth retardation and early lethality. All the patients are homozygous for a nonsense mutation in the TOR1AIP1 gene resulting in the loss of both protein isoforms LAP1B and LAP1C. Patient-derived fibroblasts exhibit changes in nuclear envelope morphology and large nuclear-spanning channels containing trapped cytoplasmic organelles. Decreased and inefficient cellular motility is also observed in these fibroblasts. Our study describes the complete absence of both major human LAP1 isoforms, underscoring their crucial role in early development and organogenesis. LAP1-associated defects may thus comprise a broad clinical spectrum depending on the availability of both isoforms in the nuclear envelope throughout life.

Two separate functions of NME3 critical for cell survival underlie a neurodegenerative disorder.

We report a patient who presented with congenital hypotonia, hypoventilation, and cerebellar histopathological alterations. Exome analysis revealed a homozygous mutation in the initiation codon of the NME3 gene, which encodes an NDP kinase. The initiation-codon mutation leads to deficiency in NME3 protein expression. NME3 is a mitochondrial outer-membrane protein capable of interacting with MFN1/2, and its depletion causes dysfunction in mitochondrial dynamics. Consistently, the patient's fibroblasts were characterized by a slow rate of mitochondrial dynamics, which was reversed by expression of wild-type or catalytic-dead NME3. Moreover, glucose starvation caused mitochondrial fragmentation and cell death in the patient's cells. The expression of wild-type and catalytic-dead but not oligomerization-attenuated NME3 restored mitochondrial elongation. However, only wild-type NME3 sustained ATP production and viability. Thus, the separate functions of NME3 in mitochondrial fusion and NDP kinase cooperate in metabolic adaptation for cell survival in response to glucose starvation. Given the critical role of mitochondrial dynamics and energy requirements in neuronal development, the homozygous mutation in NME3 is linked to a fatal mitochondrial neurodegenerative disorder.

Homozygous mutation in MFSD2A, encoding a lysolipid transporter for docosahexanoic acid, is associated with microcephaly and hypomyelination.

The major facilitator superfamily domain-containing protein 2A (MFSD2A) is a constituent of the blood-brain barrier and functions to transport lysophosphatidylcholines (LPCs) into the central nervous system. LPCs such as that derived from docosahexanoic acid (DHA) are indispensable to neurogenesis and maintenance of neurons, yet cannot be synthesized within the brain and are dependent on MFSD2A for brain uptake. Recent studies have implicated MFSD2A mutations in lethal and non-lethal microcephaly syndromes, with the severity correlating to the residual activity of the transporter. We describe two siblings with shared parental ancestry, in whom we identified a homozygous missense mutation (c.1205C > A; p.Pro402His) in MFSD2A. Both affected individuals had microcephaly, hypotonia, appendicular spasticity, dystonia, strabismus, and global developmental delay. Neuroimaging revealed paucity of white matter with enlarged lateral ventricles. Plasma lysophosphatidylcholine (LPC) levels were elevated, reflecting reduced brain transport. Cell-based studies of the p.Pro402His mutant protein indicated complete loss of activity of the transporter despite the non-lethal, attenuated phenotype. The aggregate data of MFSD2A-associated genotypes and phenotypes suggest that additional factors, such as nutritional supplementation or modifying genetic factors, may modulate the severity of disease and call for consideration of treatment options for affected individuals.

T+ NK+ IL-2 Receptor γ Chain Mutation: a Challenging Diagnosis of Atypical Severe Combined Immunodeficiency.

PURPOSE: All reported patients with hypomorphic X-linked severe combined immunodeficiency (X-SCID) due to c.664C>T (p.R222C) mutations in the gene (IL2RG) encoding the common γ chain (γc) have presented with opportunistic infections within the first year of life, despite the presence of nearly normal NK and T cell numbers. Reporting five children of one extended family with hemizygous mutations in IL2RG, we explore potential diagnostic clues and extend our comprehension of the functional impact of this mutation. METHODS: Whole exome sequencing (WES); detailed immune phenotyping; cytokine-induced STAT phosphorylation; B, T, and NK cell activation; and quantification of sjTRECs in five Arab children with c.664C>T (p.R222C) IL2RG mutation. RESULTS: The mean age at clinical presentation with respiratory tract infection or diarrhea was 6.8 (range: 2-12) months. None of the children presented with opportunistic infections. Diagnostic clues were early onset in the first year of life, and a suggestive family history associated with reduced naïve CD4 T cells and absent switched memory B cells. Number and phenotype of NK cells and innate-like lymphocytes were normal. The diagnosis was made by WES and corroborated by absent STAT phosphorylation and reduced functional response after IL-2 and IL-21 stimulation. Four patients underwent successful hematopoietic stem cell transplantation. CONCLUSIONS: As early diagnosis and treatment are important, a high index of suspicion in the diagnosis of c.664C>T (p.R222C) X-SCID is needed. This requires prompt genetic testing by next generation sequencing in order to avoid unnecessary delays in the definite diagnosis since immunological work up may not be discriminating. Assays directly testing cytokine signaling or cytokine-dependent functions are helpful in confirming the functional impact of the identified hypomorphic variants.

Respiratory manifestations in LPS-responsive beige-like anchor (LRBA) protein-deficient patients.

Lipopolysaccharide (LPS)-responsive beige-like anchor (LRBA) protein deficiency is a rare syndrome of primary immune deficiency and immune dysregulation. In this study, we sought to summarize our experience with respiratory manifestations in LRBA-deficient patients. We conducted a retrospective analysis of the medical records of LRBA-deficient patients treated at Hadassah-Hebrew University Medical Center, Jerusalem, Israel. Data retrieved included pulmonary workup, disease course, treatment, and outcome. Ten patients were included. Mean age at presentation of LRBA deficiency-related symptoms was 4.65 years (range 3 months-14 years). Respiratory symptoms were noted in six patients and consisted of chronic cough. Computed tomography revealed consolidation in five patients, atelectasis and bronchiectasis in two patients each, and diffuse interstitial lung disease in two additional patients. Respiratory tract cultures yielded a bacterial pathogen in five patients. Seven patients required active therapy: intravenous immunoglobulins (six patients), immunosuppressive drugs (five patients), and one was successfully treated with abatacept. Two patients underwent successful bone marrow transplantation. Mean follow-up period was 4.5 (range 0.4-14.4) years. On their latest examination, seven patients had no respiratory symptoms. CONCLUSION: Pulmonary manifestations are common in LRBA deficiency. Respiratory characteristics in LRBA-deficient patients should be investigated, monitored, and treated from the time of diagnosis. What is Known: • Lipopolysaccharide-responsive beige-like anchor (LRBA) deficiency is a syndrome of primary immune deficiency and immune dysregulation. • Studies concerning the pulmonary characteristics of LRBA-deficient patients are lacking. What is New: • Respiratory manifestations include infections, bronchiectasis, interstitial lung disease, thoracic lymphadenopathy, and clubbing. • Awareness to pulmonary morbidity in LRBA-deficient patients and involvement of a pulmonologist in the workup and clinical decision-making is important. • Respiratory characteristics in LRBA-deficient patients should be investigated, monitored, and treated from a young age.

Clinical and experimental evidence suggest a link between KIF7 and C5orf42-related ciliopathies through Sonic Hedgehog signaling.

Acrocallosal syndrome (ACLS) is an autosomal recessive neurodevelopmental disorder caused by KIF7 defects and belongs to the heterogeneous group of ciliopathies related to Joubert syndrome (JBTS). While ACLS is characterized by macrocephaly, prominent forehead, depressed nasal bridge, and hypertelorism, facial dysmorphism has not been emphasized in JBTS cohorts with molecular diagnosis. To evaluate the specificity and etiology of ACLS craniofacial features, we performed whole exome or targeted Sanger sequencing in patients with the aforementioned overlapping craniofacial appearance but variable additional ciliopathy features followed by functional studies. We found (likely) pathogenic variants of KIF7 in 5 out of 9 families, including the original ACLS patients, and delineated 1000 to 4000-year-old Swiss founder alleles. Three of the remaining families had (likely) pathogenic variants in the JBTS gene C5orf42, and one patient had a novel de novo frameshift variant in SHH known to cause autosomal dominant holoprosencephaly. In accordance with the patients' craniofacial anomalies, we showed facial midline widening after silencing of C5orf42 in chicken embryos. We further supported the link between KIF7, SHH, and C5orf42 by demonstrating abnormal primary cilia and diminished response to a SHH agonist in fibroblasts of C5orf42-mutated patients, as well as axonal pathfinding errors in C5orf42-silenced chicken embryos similar to those observed after perturbation of Shh signaling. Our findings, therefore, suggest that beside the neurodevelopmental features, macrocephaly and facial widening are likely more general signs of disturbed SHH signaling. Nevertheless, long-term follow-up revealed that C5orf42-mutated patients showed catch-up development and fainting of facial features contrary to KIF7-mutated patients.

A homozygous deleterious CDK10 mutation in a patient with agenesis of corpus callosum, retinopathy, and deafness.

The primary cilium is a key organelle in numerous physiological and developmental processes. Genetic defects in the formation of this non-motile structure, in its maintenance and function, underlie a wide array of ciliopathies in human, including craniofacial, brain and heart malformations, and retinal and hearing defects. We used exome sequencing to study the molecular basis of disease in an 11-year-old female patient who suffered from growth retardation, global developmental delay with absent speech acquisition, agenesis of corpus callosum and paucity of white matter, sensorineural deafness, retinitis pigmentosa, vertebral anomalies, patent ductus arteriosus, and facial dysmorphism reminiscent of STAR syndrome, a suspected ciliopathy. A homozygous variant, c.870_871del, was identified in the CDK10 gene, predicted to cause a frameshift, p.Trp291Alafs*18, in the cyclin-dependent kinase 10 protein. CDK10 mRNAs were detected in patient cells and do not seem to undergo non-sense mediated decay. CDK10 is the binding partner of Cyclin M (CycM) and CDK10/CycM protein kinase regulates ciliogenesis and primary cilium elongation. Notably, CycM gene is mutated in patients with STAR syndrome. Following incubation, the patient cells appeared less elongated and more densely populated than the control cells suggesting that the CDK10 mutation affects the cytoskeleton. Upon starvation and staining with acetylated-tubulin, γ-tubulin, and Arl13b, the patient cells exhibited fewer and shorter cilia than control cells. These findings underscore the importance of CDK10 for the regulation of ciliogenesis. CDK10 defect is likely associated with a new form of ciliopathy phenotype; additional patients may further validate this association.