Compound Heterozygous Missense Variants Research Articles

Rare CACNA1H and RELN variants interact through mTORC1 pathway in oligogenic autism spectrum disorder

Oligogenic inheritance of autism spectrum disorder (ASD) has been supported by several studies. However, little is known about how the risk variants interact and converge on causative neurobiological pathways. We identified in an ASD proband deleterious compound heterozygous missense variants in the Reelin (RELN) gene, and a de novo splicing variant in the Cav3.2 calcium channel (CACNA1H) gene. Here, by using iPSC-derived neural progenitor cells (NPCs) and a heterologous expression system, we show that the variant in Cav3.2 leads to increased calcium influx into cells, which overactivates mTORC1 pathway and, consequently, further exacerbates the impairment of Reelin signaling. Also, we show that Cav3.2/mTORC1 overactivation induces proliferation of NPCs and that both mutant Cav3.2 and Reelin cause abnormal migration of these cells. Finally, analysis of the sequencing data from two ASD cohorts—a Brazilian cohort of 861 samples, 291 with ASD; the MSSNG cohort of 11,181 samples, 5,102 with ASD—revealed that the co-occurrence of risk variants in both alleles of Reelin pathway genes and in one allele of calcium channel genes confer significant liability for ASD. Our results support the notion that genes with co-occurring deleterious variants tend to have interconnected pathways underlying oligogenic forms of ASD.

Recessive LAMA5 Variants Associated With Partial Epilepsy and Spasms in Infancy.

ObjectiveThe LAMA5 gene encodes the laminin subunit α5, the most abundant laminin α subunit in the human brain. It forms heterotrimers with the subunit β1/β2 and γ1/γ3 and regulates neurodevelopmental processes. Genes encoding subunits of the laminin heterotrimers containing subunit α5 have been reported to be associated with human diseases. Among LAMAs encoding the laminin α subunit, LAMA1-4 have also been reported to be associated with human disease. In this study, we investigated the association between LAMA5 and epilepsy.MethodsTrios-based whole-exome sequencing was performed in a cohort of 118 infants suffering from focal seizures with or without spasms. Protein modeling was used to assess the damaging effects of variations. The LAMAs expression was analyzed with data from the GTEX and VarCards databases.ResultsSix pairs of compound heterozygous missense variants in LAMA5 were identified in six unrelated patients. All affected individuals suffered from focal seizures with mild developmental delay, and three patients presented also spasms. These variants had no or low allele frequencies in controls and presented statistically higher frequency in the case cohort than in controls. The recessive burden analysis showed that recessive LAMA5 variants identified in this cohort were significantly more than the expected number in the East Asian population. Protein modeling showed that at least one variant in each pair of biallelic variants affected hydrogen bonds with surrounding amino acids. Among the biallelic variants in cases with only focal seizures, two variants of each pair were located in different structural domains or domains/links, whereas in the cases with spasms, the biallelic variants were constituted by two variants in the identical functional domains or both with hydrogen bond changes.ConclusionRecessive LAMA5 variants were potentially associated with infant epilepsy. The establishment of the association between LAMA5 and epilepsy will facilitate the genetic diagnosis and management in patients with infant epilepsy.

Recessive PKD1 Mutations Are Associated With Febrile Seizures and Epilepsy With Antecedent Febrile Seizures and the Genotype-Phenotype Correlation

ObjectiveThe PKD1 encodes polycystin-1, a large transmembrane protein that plays important roles in cell proliferation, apoptosis, and cation transport. Previous studies have identified PKD1 mutations in autosomal dominant polycystic kidney disease (ADPKD). However, the expression of PKD1 in the brain is much higher than that in the kidney. This study aimed to explore the association between PKD1 and epilepsy.MethodsTrios-based whole-exome sequencing was performed in a cohort of 314 patients with febrile seizures or epilepsy with antecedent febrile seizures. The damaging effects of variants was predicted by protein modeling and multiple in silico tools. The genotype-phenotype association of PKD1 mutations was systematically reviewed and analyzed.ResultsEight pairs of compound heterozygous missense variants in PKD1 were identified in eight unrelated patients. All patients suffered from febrile seizures or epilepsy with antecedent febrile seizures with favorable prognosis. All of the 16 heterozygous variants presented no or low allele frequencies in the gnomAD database, and presented statistically higher frequency in the case-cohort than that in controls. These missense variants were predicted to be damaging and/or affect hydrogen bonding or free energy stability of amino acids. Five patients showed generalized tonic-clonic seizures (GTCS), who all had one of the paired missense mutations located in the PKD repeat domain, suggesting that mutations in the PKD domains were possibly associated with GTCS. Further analysis demonstrated that monoallelic mutations with haploinsufficiency of PKD1 potentially caused kidney disease, compound heterozygotes with superimposed effects of two missense mutations were associated with epilepsy, whereas the homozygotes with complete loss of PKD1 would be embryonically lethal.ConclusionPKD1 gene was potentially a novel causative gene of epilepsy. The genotype-phenotype relationship of PKD1 mutations suggested a quantitative correlation between genetic impairment and phenotypic variation, which will facilitate the genetic diagnosis and management in patients with PKD1 mutations.

Disruption of cellular iron homeostasis by IREB2 missense variants causes severe neurodevelopmental delay, dystonia and seizures.

Altered brain iron homeostasis can contribute to neurodegeneration by interfering with the delivery of the iron needed to support key cellular processes, including mitochondrial respiration, synthesis of myelin and essential neurotransmitters. Intracellular iron homeostasis in mammals is maintained by two homologous ubiquitously expressed iron-responsive element-binding proteins (IRP1 and IRP2). Using exome sequencing, two patients with severe neurodegenerative disease and bi-allelic mutations in the gene IREB2 were first identified and clinically characterized in 2019. Here, we report the case of a 7-year-old male patient with compound heterozygous missense variants in IREB2, whose neurological features resembled those of the two previously reported IRP2-deficient patients, including a profound global neurodevelopmental delay and dystonia. Biochemical characterization of a lymphoblast cell line derived from the patient revealed functional iron deficiency, altered post-transcriptional regulation of iron metabolism genes and mitochondrial dysfunction. The iron metabolism abnormalities of the patient cell line were reversed by lentiviral-mediated restoration of IREB2 expression. These results, in addition to confirming the essential role of IRP2 in the regulation of iron metabolism in humans, expand the scope of the known IRP2-related neurodegenerative disorders and underscore that IREB2 pathological variants may impact the iron-responsive element-binding activity of IRP2 with varying degrees of severity. The three severely affected patients identified so far all suffered from complete loss of function of IRP2, raising the possibility that individuals with significant but incomplete loss of IRP2 function may develop less severe forms of the disease, analogous to other human conditions that present with a wide range of phenotypic manifestations.

Late Onset Pompe Disease with Novel Mutations and Atypical Phenotypes.

Late onset Pompe disease (LOPD) is rare and generally manifests predominantly as progressive limb girdle muscle weakness. It is linked to the pathogenic mutations in GAA gene, which leads to glycogen accumulation in various tissues. We describe the unusual clinical, biochemical, histopathological and genetic characteristics of 5 cases of LOPD. The first case had progressive anterior horn cell like disease (AHCD) that evolved later to classical limb girdle syndrome and respiratory failure, the second patient had rigid spine syndrome with gastrointestinal manifestations, the third had limb girdle weakness superimposed with episodic prolonged worsening and respiratory failure, the fourth had large fibre sensory neuropathy without primary muscle involvement and the fifth presented with classical limb girdle muscle weakness. Two homozygous missense mutations c.1461C > A (p.Phe487Leu) and c.1082C > T (p.Pro361Leu) in the GAA gene were identified in case 1 and 2 respectively. Case 3 was compound heterozygous with inframe c.1935_1940del (p.Val646_Cys647del) and an intronic splice effecting variant c.-32-13T > G. Compound heterozygous missense variants c.971C > T (p.Pro324Leu) and c.794G > A (p.Ser265Asn) were identified in case 4. Case 5 had a frameshift insertion c.1396dupG (p.Val466GlyfsTer40) and a synonymous splice affecting variant c.546G > T(p.Thr182=). We are describing for the first time from India on LOPD with unusual phenotypes identified. A high degree of clinical suspicion and diagnosing rare phenotypes of Pompe disease is imperative to consider early initiation of Enzyme Replacement Therapy (ERT).

A novel 1-bp deletion variant in DAG1 in Japanese familial asymptomatic hyper-CK-emia

Asymptomatic hyper-CK-emia (ASCK) is characterized by persistent elevation of creatine kinase (CK) in serum without any neurological symptoms. We ascertained a two-generation family of ASCK patients without clear neurological abnormalities except for the high levels of serum CK (810.5 ± 522.4 U/L). We identified a novel 1-bp deletion variant in the DAG1 gene shared by the patients in the family (NM_001177639: exon 3: c.930delC:p.R311Gfs*70). The variant causes premature termination of translation at codon 477, resulting in a protein product completely devoid of the essential DAG1 domain. Since ASCK has been associated with DAG1 in only one case carrying compound heterozygous missense variants, our new finding of a novel 1-bp deletion revealed the previously unknown dominant effect of DAG1 on ASCK.

Clinical characteristics and genetic analysis of a Chinese pedigree affected with mitochondrial DNA depletion syndrome due to compound heterozygous variants of RRM2B gene

To analyze the clinical characteristics and pathogenic gene in a Chinese pedigree affected with mitochondrial DNA depletion syndrome 8A (MTDPS8A). Whole exome sequencing was carried out for the patient. Sanger sequencing was used to verify the results, and PolyPhen-2 and PROVEAN software were used to predict the impact of amino acid changes on the function of the protein. The patient, a two-month-old female, was admitted to the hospital for poor milk intake and poor mental response. Her clinical manifestations included feeding difficulty, shortness of breath and low muscle tone. Auxiliary laboratory test indicated that the infant was underdeveloped with abnormal liver, kidney, and heart functions accompanied by hyperlacticacidemia. She responded poorly to treatment and eventually died. Sequencing revealed that the child has carried compound heterozygous missense variants of the RRM2B gene, namely c.16delA (p.R6Gfs*22) and c.175G>C (p.A59P), which were respectively inherited from her father and mother, and both were newly discovered pathologic variants. The c.16delA and c.175G>C compound heterozygous variants of the RRM2B gene probably underlay the pathogenesis of MTDPS8A. Above finding has strengthened the understanding of the clinical feature and genetic etiology of this disease and expanded the mutation spectrum of the RRM2B gene.

The longest reported sibling survivors of a severe form of congenital myasthenic syndrome with the ALG14 pathogenic variant

Congenital myasthenic syndromes (CMS) is a group of diseases that causes abnormalities at the neuromuscular junction owing to genetic anomalies. The pathogenic variant in ALG14 results in a severe pathological form of CMS causing end-plate acetylcholine receptor deficiency. Here, we report the cases of two siblings with CMS associated with a novel variant in ALG14. Immediately after birth, they showed hypotonia and multiple joint contractures with low Apgar scores. Ptosis, low-set ears, and high-arched palate were noted. Deep tendon reflexes were symmetrical. They showed worsening swallowing and respiratory problems; hence, nasal feeding and tracheotomy were performed. Cranial magnetic resonance imaging scans revealed delayed myelination and cerebral atrophy. Exome sequencing indicated that the siblings had novel compound heterozygous missense variants, c.590T>G (p.Val197Gly) and c.433G>A (p.Gly145Arg), in exon 4 of ALG14. Repetitive nerve stimulation test showed an abnormal decrease in compound muscle action potential. After treatment with pyridostigmine, the time off the respirator increased. Their epileptic seizures were well controlled by anti-epileptic drugs. Their clinical course is stable even now at the ages of 5 and 2 years, making them the longest reported survivors of a severe form of CMS with the ALG14 variant thus far.

Clear Evidence of LAMA5 Gene Biallelic Truncating Variants Causing Infantile Nephrotic Syndrome.

Pathogenic variants in single genes encoding podocyte-associated proteins have been implicated in about 30% of steroid-resistant nephrotic syndrome (SRNS) patients in children. However, LAMA5 gene biallelic variants have been identified in only seven patients so far, and most are missense variants of unknown significance. Furthermore, no functional analysis had been conducted for all but one of these variants. Here, we report three patients with LAMA5 gene biallelic truncating variants manifesting infantile nephrotic syndrome, and one patient with SRNS with biallelic LAMA5 missense variants. We conducted comprehensive gene screening of Japanese patients with severe proteinuria. With the use of targeted next-generation sequencing, 62 podocyte-related genes were screened in 407 unrelated patients with proteinuria. For the newly discovered LAMA5 variants, we conducted in vitro heterotrimer formation assays. Biallelic truncating variants in the LAMA5 gene (NM_005560) were detected in three patients from two families. All patients presented with proteinuria within 6 months of age. Patients 1 and 2 were siblings possessing a nonsense variant (c.9232C>T, p.[Arg3078*]) and a splice site variant (c.1282 + 1G>A) that led to exon 9 skipping and a frameshift. Patient 3 had a remarkable irregular contour of the glomerular basement membrane. She was subsequently found to have a nonsense variant (c.8185C>T, p.[Arg2720*]) and the same splice site variant in patients 1 and 2. By in vitro heterotrimer formation assays, both truncating variants produced smaller laminin α5 proteins that nevertheless formed trimers with laminin β1 and γ1 chains. Patient 4 showed SRNS at the age of 8 years, and carried compound heterozygous missense variants (c.1493C>T, p.[Ala498Val] and c.8399G>A, p.[Arg2800His]). Our patients showed clear evidence of biallelic LAMA5 truncating variants causing infantile nephrotic syndrome. We also discerned the clinical and pathologic characteristics observed in LAMA5-related nephropathy. LAMA5 variant screening should be performed in patients with congenital/infantile nephrotic syndrome.

Triheptanoin – Novel therapeutic approach for the ultra-rare disease mitochondrial malate dehydrogenase deficiency

Mitochondrial malate dehydrogenase (MDH2) deficiency (MDH2D) is an ultra-rare disease with only three patients described in literature to date. MDH2D leads to an interruption of the tricarboxylic acid (TCA) cycle and malate-aspartate shuttle (MAS) and results in severe early onset encephalopathy. Affected infants suffer from psychomotor delay, muscular hypotonia and frequent seizures. Laboratory findings are unspecific, including elevated lactate in blood and cerebrospinal fluid. Brain magnetic resonance imaging reveals delayed myelination and brain atrophy. Currently there is no curative therapy to treat this devastating disease.Here, we present a female patient diagnosed with MDH2D after a stroke-like episode at 18 months. Trio-whole exome sequencing revealed compound heterozygous missense variants in the MDH2 gene: c.398C>T, p.(Pro133Leu) and c.445delinsACA, p.(Pro149Hisfs*22). MDH2 activity assay and oxygraphic analysis in patient's fibroblasts confirmed the variants were pathogenic. At the age of 36 months, a drug trial with triheptanoin was initiated and well tolerated. The patient's neurologic and biochemical phenotype improved and she had no further metabolic decompensations during the treatment period suggesting a beneficial effect of triheptanoin on MDH2D.Further preclinical and clinical studies are required to evaluate triheptanoin treatment for MDH2D and other TCA cycle and MAS defects.

Overexpression of the PLK4 Gene as a Novel Strategy for the Treatment of Autosomal Recessive Microcephaly by Improving Centrosomal Dysfunction.

Autosomal recessive microcephaly and chorioretinopathy (MCCRP) is a neurodevelopmental disorder characterized by delayed psychomotor development, growth retardation with dwarfism, and ocular abnormalities, and its occurrence has been found to be closely related to variants of the gene encoding centrosomes. However, the association between centrosomal duplication defects and the etiology of microcephaly syndromes is poorly understood. It is well known that polo-like kinase 4 (PLK4) is a key regulator of centriole duplication, and the abnormalities of centrosomal function caused by its protein variation need to be further explored in the pathogenesis of microcephaly. In our study, we found that a patient with microcephaly and chorioretinopathy harbored compound heterozygous missense variants NM_014264.4: c.2221C > T (p.Gln741*) and NM_014264.4: c.2062T > C (p.Tyr688His) in the PLK4 gene. Overexpression experiments of the variant PLK4 proteins then showed that the G741 variant rather than the T688H variant had lost centrosomal amplification ability, and the G741 variant but not the T688H variant induced centrosomal replication disorder, which further inhibited cell proliferation, cycle division and cytoskeleton morphology in HeLa cells. Moreover, the overexpression of the two variant proteins had inconsistent effects on the target protein PLK4 by western blot analysis, also indicating that T688H variant overexpression is not functionally equivalent to WT-PLK4 overexpression. Therefore, all data support the idea that the PLK4 mutation induces centriolar duplication disorder and reduces the efficiency of mitosis inducing cell death or cell proliferation in the etiology of microcephaly disorder.

Biallelic ADGRV1 variants are associated with Rolandic epilepsy.

Rolandic epilepsy (RE) is among the most common focal epilepsies in childhood. For the majority of patients with RE and atypical RE (ARE), the etiology remains elusive. We thus screened patients with RE/ARE in order to detect disease-causing variants.. A trios-based whole-exome sequencing approach was performed in a cohort of 28 patients with RE/ARE. Clinical data and EEGs were reviewed. Variants were validated by Sanger sequencing. Two compound heterozygous missense variants p.Val272Ile/p.Asn3028Ser and p.Ala3657Val/p.Met4419Val of ADGRV1 were identified in two unrelated familial cases of RE/ARE. All the variants were in the calcium exchanger β domain and were suggested to be damaging by at least one web-based prediction tool. These variants are not present or are present at a very low minor allele frequency in the gnomAD database. Previously, biallelic ADGRV1 variants (p.Gly2756Arg and p.Glu4410Lys) have been observed in RE, consistent with the observation in this study and supporting the association between ADGRV1 variants and RE. Additionally, a de novo mutation, p.Asp668Asn, in GRIN2B was identified in a sporadic case of ARE, and a missense variant, p.Asn1551Ser, in RyR2 was identified in a family with RE with incomplete penetrance. These genes are all calcium homeostasis associated genes, suggesting the potential effect of calcium homeostasis in RE/ARE. The results from the present study suggest that the genes ADGRV1, GRIN2B, and RyR2 are associated with RE/ARE. These data link defects in neuronal intracellular calcium homeostasis to RE/ARE pathogenesis implicating that these defects plays an important role in the development of these conditions.

Bi-allelic variants in MTMR5/SBF1 cause Charcot-Marie-Tooth type 4B3 featuring mitochondrial dysfunction

BackgroundCharcot-Marie-Tooth disease (CMT) type 4B3 (CMT4B3) is a rare form of genetic neuropathy associated with variants in the MTMR5/SBF1 gene. MTMR5/SBF1 is a pseudophosphatase predicted to regulate endo-lysosomal trafficking in tandem with other MTMRs. Although almost ubiquitously expressed, pathogenic variants primarily impact on the peripheral nervous system, corroborating the involvement of MTMR5/SBF1 and its molecular partners in Schwann cells-mediated myelinization.Case presentationWe report a case of severe CMT4B3 characterized by early-onset motor and axonal polyneuropathy in an Italian child in absence of any evidence of brain and spine MRI abnormalities or intellectual disability and with a biochemical profile suggestive of mitochondrial disease. Using an integrated approach combining both NGS gene panels and WES analysis, we identified two novel compound heterozygous missense variants in MTMR5/SBF1 gene, p.R763H (c.2291G > A) and p.G1064E (c.3194G > A). Studies in muscle identified partial defects of oxidative metabolism.ConclusionWe describe the first case of an early onset severe polyneuropathy with motor and axonal involvement, due to recessive variants in the MTMR5/SBF1 gene, with no evidence of brain and spine MRI abnormalities, intellectual disability, no clinical and neurophysiological evidences of distal sensory impairment, and rapid neuromuscular deterioration. This report suggests that MTMR5/SBF1 should be considered in cases of infantile-onset CMT with secondary mitochondrial dysfunction.

Functionally confirmed compound heterozygous ADAM17 missense loss-of-function variants cause neonatal inflammatory skin and bowel disease 1

A disintegrin and metalloprotease 17 (ADAM17) is the major sheddase that processes more than 80 substrates, including tumour necrosis factor-α (TNFα). The homozygous genetic deficiency of ADAM17 causing a complete loss of ADAM17 expression was reported to be linked to neonatal inflammatory skin and bowel disease 1 (NISBD1). Here we report for the first time, a family with NISBD1 caused by functionally confirmed compound heterozygous missense variants of ADAM17, namely c.1699T>C (p.Cys567Arg) and c.1799G>A (p.Cys600Tyr). Both variants were detected in two siblings with clinical features of NISBD1, such as erythroderma with exudate in whole body, recurrent skin infection and sepsis and prolonged diarrhoea. In a cell-based assay using Adam10/17 double-knockout mouse embryonic fibroblasts (Adam10/17−/− mEFs) exogenously expressing each of these mutants, phorbol 12-myristate 13-acetate-stimulated shedding was strongly reduced compared with wild-type ADAM17. Thus, in vitro functional assays demonstrated that both missense variants cause the loss-of-function of ADAM17, resulting in the development of NISBD1. Our study further expands the spectrum of genetic pathology underlying ADAM17 in NISBD1 and establishes functional assay systems for its missense variants.

Novel compound heterozygous missense variants (c.G955A and c.A1822C) of CACNA2D4 likely causing autosomal recessive retinitis pigmentosa in a Chinese patient.

Retinitis pigmentosa (RP) is a rare and heterogeneous group of inherited ocular diseases. However, the relationship between CACNA2D4 mutations and RP is not well understood. In this study, a Chinese autosomal recessive retinitis pigmentosa (arRP) pedigree was enrolled and targeted next-generation sequencing was employed for identifying the causative gene in the proband. These steps were followed by confirmatory Sanger sequencing and segregation analysis. RNA-sequencing (RNA-seq) data and semi-quantitative reverse transcription polymerase chain reaction analysis were then applied to examine the expressions in the human and mouse tissues. Novel compound heterozygous, deleterious missense variants of the CACNA2D4 gene, NM_172364.4: c.G955A (p.D319N) and c.A1822C (p.I608L), were identified in the arRP pedigree, co-segregating with the clinical phenotype in the patient. The CACNA2D4 protein is highly conserved among species. The CACNA2D4 mRNA expression showed the highest expression in the retina of humans and in the later four developmental stages/times of retinal tissues in mice, indicating its role in retina/eye functions and developments. This study is the first to identify novel compound heterozygous mutations c.G955A (p.D319N) and c.A1822C (p.I608L) in the CACNA2D4 gene. These might be disease-causing mutations, thereby extending the mutational spectra. The identification of pathogenic CACNA2D4 variants is expected to enhance our understanding of the genotype-phenotype correlations of arRP for disease diagnosis and genetic counseling. The relationship between the CACNA2D4 variants and diseases/phenotypes other than RP has also been reviewed and discussed in this paper.

Biallelic hypomorphic variants in ALDH1A2 cause a novel lethal human multiple congenital anomaly syndrome encompassing diaphragmatic, pulmonary, and cardiovascular defects.

This study shows a causal association between ALDH1A2 variants and a novel, severe multiple congenital anomaly syndrome in humans that is neonatally lethal due to associated pulmonary hypoplasia and respiratory failure. In two families, exome sequencing identified compound heterozygous missense variants in ALDH1A2. ALDH1A2 is involved in the conversion of retinol (vitamin A) into retinoic acid (RA), which is an essential regulator of diaphragm and cardiovascular formation during embryogenesis. Reduced RA causes cardiovascular, diaphragmatic, and associated pulmonary defects in several animal models, matching the phenotype observed in our patients. In silico protein modeling showed probable impairment of ALDH1A2 for three of the four substitutions. In vitro studies show a reduction of RA. Few pathogenic variants in genes encoding components of the retinoic signaling pathway have been described to date, likely due to embryonic lethality. Thus, this study contributes significantly to knowledge of the role of this pathway in human diaphragm and cardiovascular development and disease. Some clinical features in our patients are also observed in Fryns syndrome (MIM# 229850), syndromic microphthalmia 9 (MIM# 601186), and DiGeorge syndrome (MIM# 188400). Patients with similar clinical features who are genetically undiagnosed should be tested for recessive ALDH1A2-deficient malformation syndrome.

EP127 - Evidence that FGFRL1 contributes to congenital diaphragmatic hernia development in humans

Fibroblast growth factor receptor-like 1 (FGFRL1) encodes a transmembrane protein that is related to fibroblast growth factor receptors but lacks an intercellular tyrosine kinase domain. in vitro studies suggest that FGFRL1 inhibits cell proliferation and promotes cell differentiation and cell adhesion. Mice that lack FGFRL1 die shortly after birth from respiratory distress and have abnormally thin diaphragms whose muscular hypoplasia allows the liver to protrude into the thoracic cavity. Haploinsufficiency of FGFRL1 has been hypothesized to contribute to the development of congenital diaphragmatic hernia (CDH) associated with Wolf-Hirschhorn syndrome. However, data from both humans and mice suggest that disruption of one copy of FGFRL1 alone is insufficient to cause diaphragm defects. Here we report a female fetus with CDH whose 4p16.3 deletion allows us to refine the Wolf-Hirschhorn syndrome CDH critical region to an approximately 1.9 Mb region that contains FGFRL1. We also report a male infant with isolated left-sided diaphragm agenesis who carried compound heterozygous missense variants in FGFRL1. These cases provide additional evidence that deleterious FGFRL1 variants may contribute to the development of CDH in humans.

Functional and structural analysis of cytokine-selective IL6ST defects that cause recessive hyper-IgE syndrome

Biallelic variants in IL6ST, encoding GP130, cause a recessive form of hyper-IgE syndrome (HIES) characterized by high IgE level, eosinophilia, defective acute phase response, susceptibility to bacterial infections, and skeletal abnormalities due to cytokine-selective loss of function in GP130, with defective IL-6and IL-11 and variable oncostatin M (OSM) and IL-27levels but sparing leukemia inhibitory factor (LIF) signaling. Our aim was to understand the functional and structural impact of recessive HIES-associated IL6ST variants. We investigated a patient with HIES by using exome, genome, and RNA sequencing. Functional assays assessed IL-6, IL-11, IL-27, OSM, LIF, CT-1, CLC, and CNTF signaling. Molecular dynamics simulations and structural modeling of GP130 cytokine receptor complexes were performed. We identified a patient with compound heterozygous novel missense variants in IL6ST (p.Ala517Pro and the exon-skipping null variant p.Gly484_Pro518delinsArg). The p.Ala517Pro variant resulted in a more profound IL-6- and IL-11-dominated signaling defect than did the previously identified recessive HIES IL6ST variants p.Asn404Tyr and p.Pro498Leu. Molecular dynamics simulations suggested that the p.Ala517Pro and p.Asn404Tyr variants result in increased flexibility of the extracellular membrane-proximal domains of GP130. We propose a structural model that explains the cytokine selectivity of pathogenic IL6ST variants that result in recessive HIES. The variants destabilized the conformation of the hexameric cytokine receptor complexes, whereas the trimeric LIF-GP130-LIFR complex remained stable through an additional membrane-proximal interaction. Deletion of this membrane-proximal interaction site in GP130 consequently caused additional defective LIF signaling and Stüve-Wiedemann syndrome. Our data provide a structural basis to understand clinical phenotypes in patients with IL6ST variants.

Genetic and phenotypic analysis of a patient with phosphogylcerate dehydrogenase deficiency

To explore the genetic basis for a child with ocular anomaly, microcephaly, growth retardation and intrauterine growth restriction. The patient underwent ophthalmologic examinations including anterior segment photography, fundus color photography, and fundus fluorescein angiography. The patient and her parents were subjected to whole exome sequencing. Candidate variants were verified by Sanger sequencing and bioinformatic analysis. The patient was found to have bilateral persistent pupillary membrane and coloboma of inferior iris, in addition with macular dysplasia and radial pigmentation near the hemal arch of the temporal retina. She was found to have carried compound heterozygous missense variants of the PHGDH gene, namely c.196G>A and c.1177G>A, which were respectively inherited from her father and mother. Bioinformatic analysis suggested both variants to be pathogenic. The patient was diagnosed with phosphoglycerate dehydrogenase deficiency. Above finding has enriched the phenotypic spectrum of the disease with ocular manifestations.

Expanding the Spectrum of FAT1 Nephropathies by Novel Mutations That Affect Hippo Signaling

IntroductionDisease-causing mutations in the protocadherin FAT1 have been recently described both in patients with a glomerulotubular nephropathy and in patients with a syndromic nephropathy.MethodsWe identified 4 patients with FAT1-associated disease, performed clinical and genetic characterization, and compared our findings to the previously published patients. Patient-derived primary urinary epithelial cells were analyzed by quantitative polymerase chain reaction (qPCR) and immunoblotting to identify possible alterations in Hippo signaling.ResultsHere we expand the spectrum of FAT1-associated disease with the identification of novel FAT1 mutations in 4 patients from 3 families (homozygous truncating variants in 3, compound heterozygous missense variants in 1 patient). All patients show an ophthalmologic phenotype together with heterogeneous renal phenotypes ranging from normal renal function to early-onset end-stage kidney failure. Molecular analysis of primary urine-derived urinary renal epithelial cells revealed alterations in the Hippo signaling cascade with a decreased phosphorylation of both the core kinase MST and the downstream effector YAP. Consistently, we found a transcriptional upregulation of bona fide YAP target genes.ConclusionA comprehensive review of the here identified patients and those previously published indicates a highly diverse phenotype in patients with missense mutations but a more uniform and better recognizable phenotype in the patients with truncating mutations. Altered Hippo signaling and de-repressed YAP activity might be novel contributing factors to the pathomechanism in FAT1-associated renal disease.