Bi-allelic variants in CELSR3 are implicated in central nervous system and urinary tract anomalies.

CELSR3 codes for a planar cell polarity protein. We describe twelve affected individuals from eleven independent families with bi-allelic variants in CELSR3. Affected individuals presented with an overlapping phenotypic spectrum comprising central nervous system (CNS) anomalies (7/12), combined CNS anomalies and congenital anomalies of the kidneys and urinary tract (CAKUT) (3/12) and CAKUT only (2/12). Computational simulation of the 3D protein structure suggests the position of the identified variants to be implicated in penetrance and phenotype expression. CELSR3 immunolocalization in human embryonic urinary tract and transient suppression and rescue experiments of Celsr3 in fluorescent zebrafish reporter lines further support an embryonic role of CELSR3 in CNS and urinary tract formation.

Kinesin family member 12-related hepatopathy: A generally indolent disorder with elevated gamma-glutamyl-transferase activity.

Exome sequencing (ES) has identified biallelic kinesin family member 12 (KIF12) mutations as underlying neonatal cholestatic liver disease. We collected information on onset and progression of this entity. Among consecutively referred pediatric patients at our centers, diagnostic ES identified 4 patients with novel, biallelic KIF12 variants using the human GRCh38 reference sequence, as KIF12 remains incompletely annotated in the older reference sequence GRCh37. A review of these and of 21 reported patients with KIF12 variants found that presentation with elevated serum transaminase activity in the context of trivial respiratory infection, without clinical features of liver disease, was more common (n = 18) than manifest cholestatic disease progressing rapidly to liver transplantation (LT; n = 7). Onset of liver disease was at age <1 year in 15 patients; LT was more common in this group. Serum gamma-glutamyl transpeptidase activity (GGT) was elevated in all patients, and total bilirubin was elevated in 15 patients. Liver fibrosis or cirrhosis was present in 14 of 18 patients who were biopsied. The 16 different pathogenic variants and 11 different KIF12 genotypes found were not correlated with age of onset or progression to LT. Identification of biallelic pathogenic KIF12 variants distinguishes KIF12-related disease from other entities with elevated GGT.

Lisch Epithelial Corneal Dystrophy Is Caused by Heterozygous Loss-of-Function Variants in MCOLN1.

PURPOSE: To report the genetic etiology of Lisch epithelial corneal dystrophy (LECD). DESIGN: Multicenter cohort study. METHODS: A discovery cohort of 27 individuals with LECD from 17 families, including 7 affected members from the original LECD family, 6 patients from 2 new families and 14 simplex cases, was recruited. A cohort of 6 individuals carrying a pathogenic MCOLN1 (mucolipin 1) variant was reviewed for signs of LECD. Next-generation sequencing or targeted Sanger sequencing were used in all patients to identify pathogenic or likely pathogenic variants and penetrance of variants. RESULTS: Nine rare heterozygous MCOLN1 variants were identified in 23 of 27 affected individuals from 13 families. The truncating nature of 7 variants and functional testing of 1 missense variant indicated that they result in MCOLN1 haploinsufficiency. Importantly, in the homozygous and compound-heterozygous state, 4 of 9 LECD-associated variants cause the rare lysosomal storage disorder mucolipidosis IV (MLIV). Autosomal recessive MLIV is a systemic disease and comprises neurodegeneration as well as corneal opacity of infantile-onset with epithelial autofluorescent lysosomal inclusions. However, the 6 parents of 3 patients with MLIV confirmed to carry pathogenic MCOLN1 variants did not have the LECD phenotype, suggesting MCOLN1 haploinsufficiency may be associated with reduced penetrance and variable expressivity. CONCLUSIONS: MCOLN1 haploinsufficiency is the major cause of LECD. Based on the overlapping clinical features of corneal epithelial cells with autofluorescent inclusions reported in both LECD and MLIV, it is concluded that some carriers of MCOLN1 haploinsufficiency-causing variants present with LECD.

CUX1-related neurodevelopmental disorder: deep insights into phenotype-genotype spectrum and underlying pathology.

Heterozygous, pathogenic CUX1 variants are associated with global developmental delay or intellectual disability. This study delineates the clinical presentation in an extended cohort and investigates the molecular mechanism underlying the disorder in a Cux1+/- mouse model. Through international collaboration, we assembled the phenotypic and molecular information for 34 individuals (23 unpublished individuals). We analyze brain CUX1 expression and susceptibility to epilepsy in Cux1+/- mice. We describe 34 individuals, from which 30 were unrelated, with 26 different null and four missense variants. The leading symptoms were mild to moderate delayed speech and motor development and borderline to moderate intellectual disability. Additional symptoms were muscular hypotonia, seizures, joint laxity, and abnormalities of the forehead. In Cux1+/- mice, we found delayed growth, histologically normal brains, and increased susceptibility to seizures. In Cux1+/- brains, the expression of Cux1 transcripts was half of WT animals. Expression of CUX1 proteins was reduced, although in early postnatal animals significantly more than in adults. In summary, disease-causing CUX1 variants result in a non-syndromic phenotype of developmental delay and intellectual disability. In some individuals, this phenotype ameliorates with age, resulting in a clinical catch-up and normal IQ in adulthood. The post-transcriptional balance of CUX1 expression in the heterozygous brain at late developmental stages appears important for this favorable clinical course.

SLC5A1 Variants in Turkish Patients with Congenital Glucose-Galactose Malabsorption.

Congenital glucose-galactose malabsorption is a rare autosomal recessive disorder caused by mutations in SLC5A1 encoding the apical sodium/glucose cotransporter SGLT1. We present clinical and molecular data from eleven affected individuals with congenital glucose-galactose malabsorption from four unrelated, consanguineous Turkish families. Early recognition and timely management by eliminating glucose and galactose from the diet are fundamental for affected individuals to survive and develop normally. We identified novel SLC5A1 missense variants, p.Gly43Arg and p.Ala92Val, which were linked to disease in two families. Stable expression in CaCo-2 cells showed that the p.Ala92Val variant did not reach the plasma membrane, but was retained in the endoplasmic reticulum. The p.Gly43Arg variant, however, displayed processing and plasma membrane localization comparable to wild-type SGLT1. Glycine-43 displays nearly invariant conservation in the relevant structural family of cotransporters and exchangers, and localizes to SGLT1 transmembrane domain TM0. p.Gly43Arg represents the first disease-associated variant in TM0; however, the role of TM0 in the SGLT1 function has not been established. In summary, we are expanding the mutational spectrum of this rare disorder.

Leri-Weill Dyschondrosteosis Caused by a Leaky Homozygous SHOX Splice-Site Variant.

SHOX deficiency is a common genetic cause of short stature of variable degree. SHOX haploinsufficiency causes Leri-Weill dyschondrosteosis (LWD) as well as nonspecific short stature. SHOX haploinsufficiency is known to result from heterozygous loss-of-function variants with pseudo-autosomal dominant inheritance, while biallelic SHOX loss-of-function variants cause the more severe skeletal dysplasia, Langer mesomelic dyschondrosteosis (LMD). Here we report for the first time the pseudo-autosomal recessive inheritance of LWD in two siblings caused by a novel homozygous non-canonical, leaky splice-site variant in intron 3 of SHOX: c.544+5G>C. Transcript analyses in patient-derived fibroblasts showed homozygous patients to produce approximately equal amounts of normally spliced mRNA and mRNA with the abnormal retention of intron 3 and containing a premature stop codon (p.Val183Glyfs*31). The aberrant transcript was shown to undergo nonsense-mediated mRNA decay, and thus resulting in SHOX haploinsufficiency in the homozygous patient. Six healthy relatives who are of normal height are heterozygous for this variant and fibroblasts from a heterozygote for the c.544+5G>C variant produced wild-type transcript amounts comparable to healthy control. The unique situation reported here highlights the fact that the dosage of SHOX determines the clinical phenotype rather than the Mendelian inheritance pattern of SHOX variants. This study extends the molecular and inheritance spectrum of SHOX deficiency disorder and highlights the importance of functional testing of SHOX variants of unknown significance in order to allow appropriate counseling and precision medicine for each family individual.

Internal Ileal Diversion as Treatment for Progressive Familial Intrahepatic Cholestasis Type 1-Associated Graft Inflammation and Steatosis after Liver Transplantation.

BACKGROUND: Progressive Familial Intrahepatic cholestasis type I (PFIC1) is a rare congenital hepatopathy causing cholestasis with progressive liver disease. Surgical interruption of the enterohepatic circulation, e.g., surgical biliary diversion (SBD) can slow down development of liver cirrhosis. Eventually, end stage liver disease necessitates liver transplantation (LT). PFIC1 patients might develop diarrhea, graft steatosis and inflammation after LT. SBD after LT was shown to be effective in the alleviation of liver steatosis and graft injury. CASE REPORT: Three PFIC1 patients received LT at the ages of two, two and a half and five years. Shortly after LT diarrhea and graft steatosis was recognized, SBD to the terminal ileum was opted to prevent risk for ascending cholangitis. After SBD, inflammation and steatosis was found to be reduced to resolved, as seen by liver biochemistry and ultrasounds. Diarrhea was reported unchanged. CONCLUSION: We present three PFIC1 cases for whom SBD to the terminal ileum successfully helped to resolve graft inflammation and steatosis.

Further delineation of SLC9A3-related congenital sodium diarrhea.

BACKGROUND: Congenital sodium diarrhea (CSD) is a rare enteropathy displaying both broad variability in clinical severity and genetic locus and allelic heterogeneity. Eleven CSD patients were reported so far with SLC9A3 variants that impair the function of the encoded intestinal sodium-proton exchanger 3 (NHE3). METHODS: We report a 4-year-old patient, born prematurely in the 35th week of gestation, with antenatal polyhydramnios and dilated intestinal loops, and with diarrhea of congenital onset, 2-6 times a day, and with polydipsia. She thrived age-appropriately under a normal family diet. Serum sodium levels were repeatedly normal but urinary sodium excretion was low. Exome sequencing revealed compound heterozygous variants in SLC9A3 as the likely cause of the congenital diarrhea. RESULTS: While exome sequencing did not reveal pathogenic or likely pathogenic variants in other genes that cause syndromic or non-syndromic forms of congenital and intractable diarrheas, we identified novel compound heterozygous variants in SLC9A3, a complex allele with two missense changes, NP_004165.2:p.[Ser331Leu;Val449Ile] and in-trans the missense variant p.(Phe451Ser). CONCLUSION: The clinical phenotype here appears to localize to the milder end of the known CSD spectrum, and the identified variants suggest that this is the twelfth patient reported to date with CSD due to mutations in SLC9A3.

UNC45A deficiency causes microvillus inclusion disease-like phenotype by impairing myosin VB-dependent apical trafficking.

Variants in the UNC45A cochaperone have been recently associated with a syndrome combining diarrhea, cholestasis, deafness, and bone fragility. Yet the mechanism underlying intestinal failure in UNC45A deficiency remains unclear. Here, biallelic variants in UNC45A were identified by next-generation sequencing in 6 patients with congenital diarrhea. Corroborating in silico prediction, variants either abolished UNC45A expression or altered protein conformation. Myosin VB was identified by mass spectrometry as client of the UNC45A chaperone and was found misfolded in UNC45AKO Caco-2 cells. In keeping with impaired myosin VB function, UNC45AKO Caco-2 cells showed abnormal epithelial morphogenesis that was restored by full-length UNC45A, but not by mutant alleles. Patients and UNC45AKO 3D organoids displayed altered luminal development and microvillus inclusions, while 2D cultures revealed Rab11 and apical transporter mislocalization as well as sparse and disorganized microvilli. All those features resembled the subcellular abnormalities observed in duodenal biopsies from patients with microvillus inclusion disease. Finally, microvillus inclusions and shortened microvilli were evidenced in enterocytes from unc45a-deficient zebrafish. Taken together, our results provide evidence that UNC45A plays an essential role in epithelial morphogenesis through its cochaperone function of myosin VB and that UNC45A loss causes a variant of microvillus inclusion disease.

Comprehensive variant spectrum of the CNGA3 gene in patients affected by achromatopsia.

Achromatopsia (ACHM) is a congenital cone photoreceptor disorder characterized by impaired color discrimination, low visual acuity, photosensitivity, and nystagmus. To date, six genes have been associated with ACHM (CNGA3, CNGB3, GNAT2, PDE6C, PDE6H, and ATF6), the majority of these being implicated in the cone phototransduction cascade. CNGA3 encodes the CNGA3 subunit of the cyclic nucleotide-gated ion channel in cone photoreceptors and is one of the major disease-associated genes for ACHM. Herein, we provide a comprehensive overview of the CNGA3 variant spectrum in a cohort of 1060 genetically confirmed ACHM patients, 385 (36.3%) of these carrying "likely disease-causing" variants in CNGA3. Compiling our own genetic data with those reported in the literature and in public databases, we further extend the CNGA3 variant spectrum to a total of 316 variants, 244 of which we interpreted as "likely disease-causing" according to ACMG/AMP criteria. We report 48 novel "likely disease-causing" variants, 24 of which are missense substitutions underlining the predominant role of this mutation class in the CNGA3 variant spectrum. In addition, we provide extensive in silico analyses and summarize reported functional data of previously analyzed missense, nonsense and splicing variants to further advance the pathogenicity assessment of the identified variants.

Clinical and molecular features of 66 patients with musculocontractural Ehlers-Danlos syndrome caused by pathogenic variants in CHST14 (mcEDS-CHST14).

BACKGROUND: Musculocontractural Ehlers-Danlos syndrome is caused by biallelic loss-of-function variants in CHST14 (mcEDS-CHST14) or DSE (mcEDS-DSE). Although 48 patients in 33 families with mcEDS-CHST14 have been reported, the spectrum of pathogenic variants, accurate prevalence of various manifestations and detailed natural history have not been systematically investigated. METHODS: We collected detailed and comprehensive clinical and molecular information regarding previously reported and newly identified patients with mcEDS-CHST14 through international collaborations. RESULTS: Sixty-six patients in 48 families (33 males/females; 0-59 years), including 18 newly reported patients, were evaluated. Japanese was the predominant ethnicity (27 families), associated with three recurrent variants. No apparent genotype-phenotype correlation was noted. Specific craniofacial (large fontanelle with delayed closure, downslanting palpebral fissures and hypertelorism), skeletal (characteristic finger morphologies, joint hypermobility, multiple congenital contractures, progressive talipes deformities and recurrent joint dislocation), cutaneous (hyperextensibility, fine/acrogeria-like/wrinkling palmar creases and bruisability) and ocular (refractive errors) features were observed in most patients (>90%). Large subcutaneous haematomas, constipation, cryptorchidism, hypotonia and motor developmental delay were also common (>80%). Median ages at the initial episode of dislocation or large subcutaneous haematoma were both 6 years. Nine patients died; their median age was 12 years. Several features, including joint and skin characteristics (hypermobility/extensibility and fragility), were significantly more frequent in patients with mcEDS-CHST14 than in eight reported patients with mcEDS-DSE. CONCLUSION: This first international collaborative study of mcEDS-CHST14 demonstrated that the subtype represents a multisystem disorder with unique set of clinical phenotypes consisting of multiple malformations and progressive fragility-related manifestations; these require lifelong, multidisciplinary healthcare approaches.

A Potential Treatment of Congenital Sodium Diarrhea in Patients With Activating GUCY2C Mutations.

INTRODUCTION: Gain-of-function mutations in guanylyl cyclase C (GCC) result in persistent diarrhea with perinatal onset. We investigated a specific GCC inhibitor, SSP2518, for its potential to treat this disorder. METHODS: We investigated the effect of SSP2518 on GCC-mediated intracellular cyclic guanosine monophosphate (cGMP) levels and on GCC-mediated chloride secretion in intestinal organoids from 3 patients with distinct activating GCC mutations and from controls, with and without stimulation of GCC with heat-stable enterotoxin. RESULTS: Patient-derived organoids had significantly higher basal cGMP levels than control organoids, which were lowered by SSP2518 to levels found in control organoids. In addition, SSP2518 significantly reduced cGMP levels and chloride secretion in patient-derived and control organoids (P < 0.05 for all comparisons) after heat-stable enterotoxin stimulation. DISCUSSION: We reported in this study that the GCC inhibitor SSP2518 normalizes cGMP levels in intestinal organoids derived from patients with GCC gain-of-function mutations and markedly reduces cystic fibrosis transmembrane conductance regulator-dependent chloride secretion, the driver of persistent diarrhea.

Expanding the Phenotype of the FAM149B1-Related Ciliopathy and Identification of Three Neurogenetic Disorders in a Single Family.

Biallelic truncating FAM149B1 variants result in cilia dysfunction and have been reported in four infants with Joubert syndrome and orofaciodigital syndrome type VI, respectively. We report here on three adult siblings, 18 to 40 years of age, homozygous for the known FAM149B1 c.354_357delinsCACTC (p.Gln118Hisfs*20) variant. Detailed clinical examinations were performed including ocular and gait analyses, skeletal- and neuroimaging. All three patients presented with neurological and oculomotor symptoms since birth and mild skeletal dysplasia in infancy resulting in characteristic gait abnormalities. We document mild skeletal dysplasia, abnormal gait with increased hip rotation and increased external foot rotation, ataxia, variable polydactyly, ocular Duane syndrome, progressive ophthalmoplegia, nystagmus, situs inversus of the retinal vessels, olfactory bulb aplasia, and corpus callosal dysgenesis as novel features in FAM149B1-ciliopathy. We show that intellectual disability is mild to moderate and retinal, renal and liver function is normal in these affected adults. Our study thus expands the FAM149B1-related Joubert syndrome to a mainly neurological and skeletal ciliopathy phenotype with predominant oculomotor dysfunction but otherwise stable outcome in adults. Diagnosis of FAM149B1-related disorder was impeded by segregation of multiple neurogenetic disorders in the same family, highlighting the importance of extended clinical and genetic studies in families with complex phenotypes.

Three Novel EPCAM Variants Causing Tufting Enteropathy in Three Families.

Tufting enteropathy (TE) is caused by recessive EPCAM mutations, and is characterized by intractable diarrhea of congenital onset and disorganization of enterocytes. TE generally requires parenteral nutrition (PN) during childhood or intestinal bowel transplantation. We report three unrelated families with six children with TE. We highlight the high rate of disease-related mortality. We observe adequate weight gain with PN, but low to normal and stunted body length, supporting the recent notion that a short stature might be intrinsic to TE. The diagnosis of TE in the index patients from each family was delayed for months to years, even when clinical data, duodenal biopsies, or exome sequencing data were obtained early on. We identified three novel pathogenic EPCAM variants: a deletion of exon 1 that removes the ATG initiation codon, a missense variant c.326A > G (p.Gln109Arg), and nonsense mutation c.429G > A (p.Trp143*) in a compound heterozygous state with the Mediterranean splice site variant c.556-14A > G (Tyr186Phefs*6). Homozygosity for p.Gln109Arg was associated with absent EPCAM staining, and compound heterozygosity for p.Trp143*/Tyr186Phefs*6 was associated with reduced EPCAM staining in duodenal biopsies; such observations might contribute to a genotype-phenotype correlation in larger cohorts of TE patients. This study extends the clinical and molecular spectrum of TE.

Early onset congenital diarrheas; single center experience.

BACKGROUND: Congenital diarrheal disorders (CDDs) are a rare group of enteropathies that typically present in the early few months of life and pose a diagnostic challenge. We aimed to analyze the clinical findings and outcome of infants with CDDs and share experience about genetic testing. METHODS: Demographic, clinical and genetic findings, and outcome of the patients (n = 24) with CDDs were recorded from hospital files. RESULTS: The onset of diarrhea was within the neonatal period in 45.8% of the patients. The most frequent causes of CDDs were defects in digestion, absorption and transport of nutrients and electrolytes (DATN) (n = 11, 45.8%) and defects in intestinal immune-related homeostasis (IIH) (n = 6, 25%). Fat malabsorption (n = 6) was the leading cause of defects in DATN. Extraintestinal manifestations including neurological involvement (25%) and renal involvement (20.8%) were common among the patients. Genetic analyses were performed for 16 patients (targeted gene analysis in 9, congenital diarrhea panel in 3, immune deficiency panel in 1 and whole-exome sequencing in 3 patients). Genetic diagnosis was achieved in 14 of 16 patients (87.5%) with therapeutic consequences in 8 of 16 patients (50%). During the follow-up, 6 patients (25%) died. CONCLUSION: The percentage of undefined etiology decreased, and treatment of the patients improved with the increased number of genetic testing in patients with CDDs.

Biallelic variants in VPS50 cause a neurodevelopmental disorder with neonatal cholestasis.

Golgi-associated retrograde protein (GARP) and endosome-associated recycling protein (EARP) complexes are membrane-tethering heterotetramers located at the trans-Golgi network and recycling endosomes, respectively. GARP and EARP share the three subunits VPS51, VPS52 and VPS53, while VPS50 is unique to EARP and VPS54 to GARP. Retrograde transport of endosomal cargos to the trans-Golgi network is mediated by GARP and endocytic recycling by EARP. Here we report two unrelated individuals with homozygous variants in VPS50, a splice variant (c.1978-1G>T) and an in-frame deletion (p.Thr608del). Both patients had severe developmental delay, postnatal microcephaly, corpus callosum hypoplasia, seizures and irritability, transient neonatal cholestasis and failure to thrive. Light and transmission electron microscopy of liver from one revealed the absence of gamma-glutamyltransferase at bile canaliculi, with mislocalization to basolateral membranes and abnormal tight junctions. Using patient-derived fibroblasts, we identified reduced VPS50 protein accompanied by reduced levels of VPS52 and VPS53. While the transferrin receptor internalization rate was normal in cells of both patients, recycling of the receptor to the plasma membrane was significantly delayed. These data underscore the importance of VPS50 and/or the EARP complex in endocytic recycling and suggest an additional function in establishing cell polarity and trafficking between basolateral and apical membranes in hepatocytes. Individuals with biallelic hypomorphic variants in VPS50, VPS51 or VPS53 show an overarching neurodegenerative disorder with severe developmental delay, intellectual disability, microcephaly, early-onset epilepsy and variable atrophy of the cerebellum, cerebrum and/or brainstem. The term 'GARP/EARP deficiency' designates disorders in such individuals.

Pathogenic STX3 variants affecting the retinal and intestinal transcripts cause an early-onset severe retinal dystrophy in microvillus inclusion disease subjects.

Biallelic STX3 variants were previously reported in five individuals with the severe congenital enteropathy, microvillus inclusion disease (MVID). Here, we provide a significant extension of the phenotypic spectrum caused by STX3 variants. We report ten individuals of diverse geographic origin with biallelic STX3 loss-of-function variants, identified through exome sequencing, single-nucleotide polymorphism array-based homozygosity mapping, and international collaboration. The evaluated individuals all presented with MVID. Eight individuals also displayed early-onset severe retinal dystrophy, i.e., syndromic-intestinal and retinal-disease. These individuals harbored STX3 variants that affected both the retinal and intestinal STX3 transcripts, whereas STX3 variants affected only the intestinal transcript in individuals with solitary MVID. That STX3 is essential for retinal photoreceptor survival was confirmed by the creation of a rod photoreceptor-specific STX3 knockout mouse model which revealed a time-dependent reduction in the number of rod photoreceptors, thinning of the outer nuclear layer, and the eventual loss of both rod and cone photoreceptors. Together, our results provide a link between STX3 loss-of-function variants and a human retinal dystrophy. Depending on the genomic site of a human loss-of-function STX3 variant, it can cause MVID, the novel intestinal-retinal syndrome reported here or, hypothetically, an isolated retinal dystrophy.