Common variable immunodeficiency in two kindreds with heterogeneous phenotypes caused by novel heterozygous NFKB1 mutations.

NFKB1 haploinsufficiengcy was first described in 2015 in three families with common variable immunodeficiency (CVID), presenting heterogeneously with symptoms of increased infectious susceptibility, skin lesions, malignant lymphoproliferation and autoimmunity. The described mutations all led to a rapid degradation of the mutant protein, resulting in a p50 haploinsufficient state. Since then, more than 50 other mutations have been reported, located throughout different domains of NFKB1 with the majority situated in the N-terminal Rel homology domain (RHD). The clinical spectrum has also expanded with possible disease manifestations in almost any organ system. In silico prediction tools are often used to estimate the pathogenicity of NFKB1 variants but to prove causality between disease and genetic findings, further downstream functional validation is required. In this report, we studied 2 families with CVID and two novel variants in NFKB1 (c.1638-2A>G and c.787G>C). Both mutations affected mRNA and/or protein expression of NFKB1 and resulted in excessive NLRP3 inflammasome activation in patient macrophages and upregulated interferon stimulated gene expression. Protein-protein interaction analysis demonstrated a loss of interaction with NFKB1 interaction partners for the p.V263L mutation. In conclusion, we proved pathogenicity of two novel variants in NFKB1 in two families with CVID characterized by variable and incomplete penetrance.

Child Neurology: Familial Hemophagocytic Lymphohistiocytosis Underlying Isolated CNS Inflammation.

Encephalitis and encephalopathy in children represent a diagnostic challenge. We describe a patient with relapsing encephalitis in whom the differential diagnosis included acute disseminated encephalomyelitis, human herpesvirus 6 encephalitis, and hemophagocytic lymphohistiocytosis (HLH). Because of its rarity, HLH is often overlooked as a differential diagnosis in encephalitis, especially in the isolated CNS forms. As this case illustrates, inborn errors of immunity can underlie isolated encephalitis and should be included in the differential diagnosis of these presentations.

Guidelines for Genetic Testing and Management of Alport Syndrome.

Genetic testing for pathogenic COL4A3-5 variants is usually undertaken to investigate the cause of persistent hematuria, especially with a family history of hematuria or kidney function impairment. Alport syndrome experts now advocate genetic testing for persistent hematuria, even when a heterozygous pathogenic COL4A3 or COL4A4 is suspected, and cascade testing of their first-degree family members because of their risk of impaired kidney function. The experts recommend too that COL4A3 or COL4A4 heterozygotes do not act as kidney donors. Testing for variants in the COL4A3-COL4A5 genes should also be performed for persistent proteinuria and steroid-resistant nephrotic syndrome due to suspected inherited FSGS and for familial IgA glomerulonephritis and kidney failure of unknown cause.

Consensus statement on standards and guidelines for the molecular diagnostics of Alport syndrome: refining the ACMG criteria.

The recent Chandos House meeting of the Alport Variant Collaborative extended the indications for screening for pathogenic variants in the COL4A5, COL4A3 and COL4A4 genes beyond the classical Alport phenotype (haematuria, renal failure; family history of haematuria or renal failure) to include persistent proteinuria, steroid-resistant nephrotic syndrome, focal and segmental glomerulosclerosis (FSGS), familial IgA glomerulonephritis and end-stage kidney failure without an obvious cause. The meeting refined the ACMG criteria for variant assessment for the Alport genes (COL4A3-5). It identified 'mutational hotspots' (PM1) in the collagen IV α5, α3 and α4 chains including position 1 Glycine residues in the Gly-X-Y repeats in the intermediate collagenous domains; and Cysteine residues in the carboxy non-collagenous domain (PP3). It considered that 'well-established' functional assays (PS3, BS3) were still mainly research tools but sequencing and minigene assays were commonly used to confirm splicing variants. It was not possible to define the Minor Allele Frequency (MAF) threshold above which variants were considered Benign (BA1, BS1), because of the different modes of inheritances of Alport syndrome, and the occurrence of hypomorphic variants (often Glycine adjacent to a non-collagenous interruption) and local founder effects. Heterozygous COL4A3 and COL4A4 variants were common 'incidental' findings also present in normal reference databases. The recognition and interpretation of hypomorphic variants in the COL4A3-COL4A5 genes remains a challenge.

Mutations in MAGT1 lead to a glycosylation disorder with a variable phenotype.

Congenital disorders of glycosylation (CDG) are a group of rare metabolic diseases, due to impaired protein and lipid glycosylation. We identified two patients with defective serum transferrin glycosylation and mutations in the MAGT1 gene. These patients present with a phenotype that is mainly characterized by intellectual and developmental disability. MAGT1 has been described to be a subunit of the oligosaccharyltransferase (OST) complex and more specifically of the STT3B complex. However, it was also claimed that MAGT1 is a magnesium (Mg2+) transporter. So far, patients with mutations in MAGT1 were linked to a primary immunodeficiency, characterized by chronic EBV infections attributed to a Mg2+ homeostasis defect (XMEN). We compared the clinical and cellular phenotype of our two patients to that of an XMEN patient that we recently identified. All three patients have an N-glycosylation defect, as was shown by the study of different substrates, such as GLUT1 and SHBG, demonstrating that the posttranslational glycosylation carried out by the STT3B complex is dysfunctional in all three patients. Moreover, MAGT1 deficiency is associated with an enhanced expression of TUSC3, the homolog protein of MAGT1, pointing toward a compensatory mechanism. Hence, we delineate MAGT1-CDG as a disorder associated with two different clinical phenotypes caused by defects in glycosylation.

Discordance for placental mesenchymal dysplasia in a monochorionic diamniotic twin pregnancy: A case report.

Placental mesenchymal dysplasia (PMD) occurs in about 1 in 5000 pregnancies. The differential diagnosis between PMD and partial mole is difficult on ultrasound scan, and karyotyping plays a key role in distinguishing PMD from partial mole. Our report is the first to report on the discordancy for PMD in a monochorionic setting.

Inherited p40phox deficiency differs from classic chronic granulomatous disease.

Biallelic loss-of-function (LOF) mutations of the NCF4 gene, encoding the p40phox subunit of the phagocyte NADPH oxidase, have been described in only 1 patient. We report on 24 p40phox-deficient patients from 12 additional families in 8 countries. These patients display 8 different in-frame or out-of-frame mutations of NCF4 that are homozygous in 11 of the families and compound heterozygous in another. When overexpressed in NB4 neutrophil-like cells and EBV-transformed B cells in vitro, the mutant alleles were found to be LOF, with the exception of the p.R58C and c.120_134del alleles, which were hypomorphic. Particle-induced NADPH oxidase activity was severely impaired in the patients' neutrophils, whereas PMA-induced dihydrorhodamine-1,2,3 (DHR) oxidation, which is widely used as a diagnostic test for chronic granulomatous disease (CGD), was normal or mildly impaired in the patients. Moreover, the NADPH oxidase activity of EBV-transformed B cells was also severely impaired, whereas that of mononuclear phagocytes was normal. Finally, the killing of Candida albicans and Aspergillus fumigatus hyphae by neutrophils was conserved in these patients, unlike in patients with CGD. The patients suffer from hyperinflammation and peripheral infections, but they do not have any of the invasive bacterial or fungal infections seen in CGD. Inherited p40phox deficiency underlies a distinctive condition, resembling a mild, atypical form of CGD.

Targeted capture sequencing in a large LQTS family reveals a new pathogenic mutation c.2038delG in KCNH2 initially missed due to allelic dropout.

We present a new mutation in KCNH2 (c.2038delG) resulting in a frameshift and premature truncation of the IKr channel protein in a large LQTS family with several sudden death cases. This mutation was initially missed by mutation scanning with DHPLC due to allelic dropout and only retrieved after repeat genetic testing with targeted capture and massive parallel sequencing. There was full penetrance of this mutation, only if an individualized QT correction derived from 24-hour Holter data was used. This case again underscores the importance of repeat genetic testing in robust cases of LQTS that remained genotype negative with mutation scanning techniques.

PID in Disguise: Molecular Diagnosis of IRAK-4 Deficiency in an Adult Previously Misdiagnosed With Autosomal Dominant Hyper IgE Syndrome.

Autosomal recessive IL-1R-associated kinase 4 (IRAK-4) deficiency is a rare cause of recurrent pyogenic infections with limited inflammatory responses. We describe an adult female patient with severe lung disease who was phenotypically diagnosed as suffering from autosomal dominant Hyper IgE syndrome (AD HIES) because of recurrent skin infections with Staphylococcus aureus, recurrent pneumonia and elevated serum IgE levels. In contrast to findings in AD HIES patients, no abnormalities were found in the Th17 and circulating follicular helper T cell subsets. A panel-based sequencing approach led to the identification of a homozygous IRAK4 stop mutation (c.877C > T, p.Gln293*).

Criteria for HNF1B analysis in patients with congenital abnormalities of kidney and urinary tract.

BACKGROUND: Congenital anomalies of kidneys and urinary tract (CAKUT) are the most predominant developmental disorders comprising ∼20-30% of all anomalies identified in the prenatal period. Mutations in hepatocyte nuclear factor 1-beta (HNF-1ß) involved in the development of kidneys, liver, pancreas and urogenital tract are currently the most frequent monogenetic cause of CAKUT found in 10-30% of patients depending on screening policy and study design. We aimed to validate criteria for analysis of HNF1B in a prospective cohort of paediatric and adult CAKUT patients. METHODS: We included CAKUT patients diagnosed in our paediatric and adult nephrology departments from January 2010 until April 2013 based on predefined screening criteria. Subjects presenting with at least one major renal criterion or one minor renal criterion combined with one or more extra-renal criteria in the personal history or a familial history of renal or extra-renal manifestations were considered eligible. RESULTS: We prospectively screened 205 patients and detected HNF1B mutations in 10% [n = 20, 12 children, median age 4.2 (range 0-13.1) years and 8 adults, median age 34.8 (range 16.6-62) years]. We observed that bilateral renal anomaly, renal cysts from unknown origin, a combination of two major renal anomalies and hypomagnesaemia were predictive for finding HNF1B mutations (P < 0.001; P < 0.001; P = 0.004; P = 0.008, respectively). CONCLUSIONS: We demonstrated that HNF1B mutations are responsible for ∼10% of CAKUT cases, both in children and in adults. Based on our results we propose adapted criteria for HNF1B analysis to reduce the screening costs without missing affected patients. These criteria should be reaffirmed in a larger validation cohort.

A novel heterozygous mutation of three consecutive nucleotides causing Apert syndrome in a Congolese family.

Apert syndrome (OMIM 101200) is a rare genetic condition characterized by craniosynostosis and syndactyly of hands and feet with clinical variability. Two single nucleotides mutations in the linker region between the immunoglobulin-like domains II and IIIa of the ectodomainin the Fibroblast Growth Factor Receptor 2 gene (FGFR2, OMIM 176943) are responsible of the vast majority of cases: c.755C > G; p.Ser252Trp (65%) and c.758C > G; p.Pro253Arg (34%. Three exceptional cases carry multiple substitutions of adjacent nucleotides in the linker region. Here we present a Congolese male patient and his mother, both affected with Apert syndrome of variable severity, carrying a previously undescribed heterozygous mutation of three consecutive nucleotides (c.756_758delGCCinsCTT) in the IgII-IgIIIa linker region. This is the fourth live-born patient to carry a multiple nucleotide substitution in the linker region and is the second alternative amino acid substitutions of the Pro253. Remarkably, this novel mutation was detected in the first Central African patient ever to be tested molecularly for the Apert syndrome. To discriminate between a hitherto unreported mutation and an ethnic specific polymorphism, we tested 105 Congolese controls, and no variation was detected.

Heterozygous α1-antitrypsin Z allele mutation in presumed healthy donor livers used for transplantation.

OBJECTIVES: The Z allele (Glu342Lys) in α1-antitrypsin (AAT) deficiency is a combined deficiency and dysfunctional allele. Carrying one Z allele induces a risk of a more aggressive evolution in patients with a chronic liver disease. As most of the carriers of Z allele do not have overt liver disease, it is likely that Z allele-containing livers have been used previously for liver transplantation. We analyzed the incidence, epidemiology, and clinical features of AAT accumulation in the hepatocytes after liver transplantation. METHODS: Follow-up biopsies of liver transplant recipients were analyzed with periodic acid Schiff staining until 2006 (n=486); from 2006 on (n=303), all biopsies were stained with a specific monoclonal antibody against mutated AATZ protein. Genotyping of both recipient and donor was performed in the case of positive staining. RESULTS: Of 789 liver transplantation patients, six patients (0.8%) showed mutated AATZ accumulation in the transplanted liver. Mutation analysis confirmed the presence of the Z allele in all donor organs including one transplanted organ with the SZ phenotype. There was a clear concordance between the isoelectrical focusing of the recipient AAT after transplantation and the genotype of the donor. CONCLUSION: Presumed healthy donor organs containing the Z allele were used for transplantation in 0.8% of cases in our series. As the presence of a Z allele is an independent risk factor of aggravation of chronic liver disease, AATZ accumulation in biopsies after liver transplantation should be actively looked for.

Misdiagnosis as asphyxiating thoracic dystrophy and CMV-associated haemophagocytic lymphohistiocytosis in Shwachman-Diamond syndrome.

Shwachman-Diamond syndrome (SDS) is an autosomal recessive disorder characterised by skeletal dysplasia, exocrine pancreatic insufficiency and bone marrow failure. Various other conditions, such as hepatopathy and failure to thrive have been associated with SDS. A retrospective study was conducted to describe mutations, clinical features, and the immunological profile of 11 Belgian patients with genetically confirmed diagnosis of SDS. This study confirms the existing understanding of the classical features of SDS although the typical triad was present in only six out of nine fully studied patients. The following important observations are made in this cohort. Four out of eleven patients were misdiagnosed as having Asphyxiating Thoracic Dystrophy (Jeune syndrome) because of severe thoracic dystrophy. Another two patients presented with unexplained episodes of symptomatic hypoglycaemia. The immunological phenotype was heterogeneous although laboratory abnormalities were noticed in eight out of ten patients assessed. Three patients experienced a life threatening viral infection (respiratory syncytial virus, cytomegalovirus (CMV) and rotavirus). In one patient, CMV infection caused an episode of haemophagocytic lymphohistiocytosis. One patient has bronchiectasis at the age of 3 years due to recurrent respiratory tract infections. These findings strengthen the suspicion of an abnormal immune system in SDS. Liver anomalies, usually described as benign and transitory in SDS patients, were severe in two patients of the cohort. One patient developed hepatopulmonary syndrome. The findings in this national cohort of SDS patients could contribute to the prevention of misdiagnosis in the future and enable more rapid recognition of certain severe complications.

The der(12)t(12;16) breakpoint in an acute leukaemia case targets a Sec7 domain containing protein.

A balanced translocation t(12;16)(p13;p13) in a patient with AML-M1 and bone marrow eosinophilia was previously analysed by FISH. The 16p13 breakpoint was shown to occur in the MYH11 gene, the fusion partner of CBFbeta in leukaemia patients with an inv(16) or a t(16;16), whereas the 12p13 breakpoint was shown to be present in cosmid c4H9. We present the molecular analysis of c4H9, resulting in the identification of a novel gene, SLAG. At the N-terminus, SLAG contains a Sec7 domain also found in proteins of the cytohesin family. In contrast to the cytohesins, no pleckstrin homology domain is present in SLAG. database searches identified several homologues, suggesting that SLAG defines a new subfamily of Sec7 proteins, characterised by an N-terminal Sec7 domain and a new C-terminal pleckstrin homology-like domain. The FISH data led to the hypothesis that rearrangement of SLAG might be involved in the pathogenesis of AML through the generation of a new fusion gene with MYH11. However, the putative SLAG-MYH11 fusions showed only weak transforming properties in NIH3T3 cells in a focus formation assay.

Cellular transformation of NIH3T3 fibroblasts by CIZ/NMP4 fusions.

Molecular cloning of the translocations t(12;22)(p13;q12) and t(12;17)(p13;q11) in acute leukaemia showed that either EWSR1 or its homologue TAF15 are fused to the transcription factor CIZ. EWSR1 and TAF15 belong to the TET family (TLS/FUS, EWSR1 and TAF15) of proteins. TET fusions have been identified in both solid tumours and acute myeloid leukaemia. The novel 12p translocations directly implicated TET fusions in acute lymphoblastic leukaemia as well, and demonstrated the involvement of CIZ in haematopoietic malignancies. In addition, a new fusion E2A-CIZ was recently cloned as a result of a t(12;19)(p13;p13) in a patient with acute lymphoblastic leukaemia. NIH3T3 cells stably expressing TET-CIZ fusions display a transformed phenotype in a focus formation assay. We show here that E2A-CIZ also transforms 3T3 fibroblasts, suggesting that the addition of a transactivation domain to the CIZ protein is involved in this phenotype. An artificial VP16-CIZ construct reveals similar transforming properties, supporting this. We have then analysed the domains within TAF15-CIZ that are necessary for 3T3 fibroblast transformation. Deletion of the zinc fingers of CIZ resulted in loss of both DNA-binding and transforming properties of TAF15-CIZ, whereas deletion of the other functional domains of CIZ had no effect. Fusion of a transactivation domain to CIZ is suggestive for a transactivating function in transformation. Luciferase experiments indeed showed that E2A-CIZ as well as VP16-CIZ transactivates the MMP7 promoter. Taken together, our results reported here suggest that transformation of 3T3 fibroblasts by CIZ fusions is dependent on DNA-binding and might involve transactivation of CIZ target genes.

Recurrent rearrangement of the Ewing's sarcoma gene, EWSR1, or its homologue, TAF15, with the transcription factor CIZ/NMP4 in acute leukemia.

Fusions of the TET-proteins (TLS/FUS, EWSR1, and TAF15/RBP56) to different transcription factors are involved in various malignancies including Ewing's sarcoma, primitive neuroectodermal tumors, and acute myeloid leukemia. These are thought to arise through transcriptional deregulation, with the transcription factor defining the tumor phenotype. We show that, as result of a t(12;17)(p13;q11) or its variant t(12;22)(p13;q12), the transcription factor gene CIZ/NMP4 is recurrently involved in acute leukemia through fusion with either EWSR1 or TAF15. The fusions possess transforming properties in NIH3T3 cells but do not affect the expression of CIZ target genes, suggesting a contribution to oncogenesis that is independent of the transactivating properties of the fusion protein. These results also extend the involvement of TET-protein fusions to acute lymphoblastic leukemia and suggest a role for CIZ/NMP4 in lymphoid and myeloid development.