Hypomorphic mutation of ZAP70 in human results in a late onset immunodeficiency and no autoimmunity

Complete lack of function of the tyrosine kinase ZAP70 in humans results in a severe immunodeficiency, characterized by a lack of mature CD8⁺ T cells and non‐functional CD4⁺ T cells. We report herein an immunodeficiency with an inherited hypomorphic mutation of ZAP70 due to a single G‐to‐A substitution in a non‐coding intron. This mutation introduces a new acceptor splice site and allows low levels of normal alternative splicing and of WT ZAP70 expression. This partial deficiency results in a compromised TCR signaling that was totally restored by increased expression of ZAP70, demonstrating that defective activation of the patient T cells was indeed caused by the low level of ZAP70 expression. This partial ZAP70 deficiency was associated with an attenuated clinical and immunological phenotype as compared with complete ZAP70 deficiency. CD4⁺ helper T‐cell populations including, follicular helper T cells, Th1, Th17 and Treg were detected in the blood. Finally, the patient had no manifestation of autoimmunity suggesting that the T‐cell tolerogenic functions were not compromised, in contrast to what has been observed in mice carrying hypomorphic mutations of Zap70. This report extends the phenotype spectrum of ZAP70 deficiency with a residual function of ZAP70.     

Novel mutations in five Japanese patients with 3-methylcrotonyl-CoA carboxylase deficiency

Isolated 3-methylcrotonyl-CoA carboxylase (MCC) deficiency appears to be the most frequent organic aciduria detected in tandem mass spectrometry (MS/MS) screening programs in the United States, Australia, and Europe. A pilot study of newborn screening using MS/MS has recently been commenced in Japan. Our group detected two asymptomatic MCC deficiency patients by the pilot screening and collected data on another three MCC deficiency patients to study the molecular bases of the MCC deficiency in Japan. Molecular analyses revealed novel mutations in one of the causative genes, MCCA or MCCB, in all five of the patients: nonsense and frameshift mutations in MCCA (c.1750C > T/c.901_902delAA) in patient 1, nonsense and frameshift mutations in MCCB (c.1054_1055delGG/c.592C > T) in patient 2, frameshift and missense mutations in MCCB (c.1625_1626insGG/c.653_654CA > TT) in patient 3, a homozygous missense mutation in MCCA (c.1380T > G/ 1380T > G) in patient 4, and compound heterozygous missense mutations in MCCB (c.569A > G/ c.838G > T) in patient 5. No obvious clinical symptoms were observed in patients 1, 2, and 3. Patient 4 had severe neurological impairment and patient 5 developed Reye-like syndrome. The increasing use of MS/MS newborn screening in Japan will further clarify the clinical and genetic heterogeneity among patients with MCC deficiency in the Japanese population.     

GH-1 gene splicing mutations: Molecular basis of hereditary isolated growth hormone deficiency in children

Children, residents of the Russian Federation, with congenital isolated growth hormone deficiency, were screened for mutations of GH-1 gene, the main gene of this deficiency. Twenty-eight children from 26 families with total congenital isolated growth hormone deficiency were examined. Direct sequencing of GH-1 detected five splicing mutations in intron 2, intron 3, and exon 4, two of them were never described previously. Three dominant negative mutations of GH-1 splicing, the basis for autosomal dominant isolated growth hormone deficiency (type II), are presented: IVS2 −2A>T, IVS3 +2T>C, and IVS3 +1G<A. GH-1 is the main gene of type II isolated growth hormone deficiency in patients living in the Russian Federation. All detected mutations of GH-1 impair splicing processes, which distinguishes them from mutations in other forms of isolated growth hormone deficiency. The detected variety of GH-1 splicing mutations attests to allele genetic heterogeneity of this pathology. The “hot spot” of mutations is 5′-donor splicing site of GH-1 intron 3, while IVS3 +1G>A mutation can be regarded as the most incident in type II isolated growth hormone deficiency in the Russian population. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 141, No. 3, pp. 324–329, March, 2006     

Clinical spectrum of BCS1L Mitopathies and their underlying structural relationships

The most frequent cause of isolated complex III deficits is mutations to the nuclear‐encoded ATPase BCS1L. Disease phenotypes are varied and can be as mild as Björnstad syndrome, characterized by pili torti and sensorineural hearing loss, or as severe as GRACILE syndrome, characterized by growth restriction, aminoaciduria, cholestasis, iron overload, lactic acidosis, and early death. BCS1L mutations are also linked to an undefined complex III deficiency, a heterogeneous condition generally involving low birth weight, renal and hepatic pathologies, hypotonia, and developmental delays. We analyzed all published patient cases of mutations to BCS1L and modeled the tertiary and quaternary structure of the BCS1L protein to map the location of disease‐causing BCS1L mutations. We show that higher order structural analysis can be used to understand the phenotype observed in a patient with the novel compound heterozygous c.550C>T(p.Arg184Cys) and c.838C>T(p.Leu280Phe) mutations. More broadly, higher order structural analysis reveals genotype–phenotype relationships within the intermediate complex III deficiency category that help to make sense of the spectrum of observed phenotypes. We propose a change in nomenclature that unifies the intermediate phenotype under “BCS1L Mitopathies”. Patterns in genotype–phenotype correlations within these BCS1L Mitopathies are evident in the context of the tertiary and quaternary structure of BCS1L.     

Activating PI3Kδ mutations in a cohort of 669 patients with primary immunodeficiency

The gene PIK3CD codes for the catalytic subunit of phosphoinositide 3‐kinase δ (PI3K δ ), and is expressed solely in leucocytes. Activating mutations of PIK3CD have been described to cause an autosomal dominant immunodeficiency that shares clinical features with common variable immunodeficiency (CVID). We screened a cohort of 669 molecularly undefined primary immunodeficiency patients for five reported mutations (four gain‐of‐function mutations in PIK3CD and a loss of function mutation in PIK3R1) using pyrosequencing. PIK3CD mutations were identified in three siblings diagnosed with CVID and two sporadic cases with a combined immunodeficiency (CID). The PIK3R1 mutation was not identified in the cohort. Our patients with activated PI3Kδ syndrome (APDS) showed a range of clinical and immunological findings, even within a single family, but shared a reduction in naive T cells. PIK3CD gain of function mutations are more likely to occur in patients with defective B and T cell responses and should be screened for in CVID and CID, but are less likely in patients with a pure B cell/hypogammaglobulinaemia phenotype.     

Novel mutations causing biotinidase deficiency in individuals identified by the newborn screening program in Minas Gerais,Brazil

Biotinidase deficiency is an autosomal recessive inherited metabolic disorder caused by mutations in the BTD gene. Clinical manifestations can be treated and effectively prevented with pharmacological doses of biotin. Nine novel mutations in BTD are reported in 14 children diagnosed by the newborn screening program in Minas Gerais, Brazil, from June 2013 to December 2017. Serum BTD enzyme activity was determined for all cases and some parents. Two of the mutations are deletions and seven missense mutations located in the exonic region of the BTD gene, mostly in exon 4. Two newborns were profoundly biotinidase‐deficient (one homozygous p.A534V [c.1601C > T] and another, double heterozygous for a novel mutation p.R211S [c.631C > A] co‐inherited with an already described mutation p.T532 M [c.1595C > T]). Two mutations were associated with a partial deficiency of biotinidase (p.F361 V [c.1081 T > G] in two homozygous children, and p.S311 T [c.932G > C] in a compound heterozygous child who co‐inherited a known severe mutation p.Y438X [c.1314 T > A]). The remaining five mutations were found in compound heterozygous children. Hence, a definitive conclusion about the degree of biotinidase deficiency is not possible yet. These results emphasize the importance of sequencing the BTD gene as an important tool to gain a better understanding of the correlation between biochemical phenotype and genotype.     

Hyper immunoglobulin M syndrome due to CD40 deficiency: clinical, molecular, and immunological features

Summary: CD40 is a member of the tumor necrosis factor receptor family, which is expressed by a variety of cells including B cells, macrophages, dendritic cells, and other nonimmune cell types. CD40 activation is critical for B‐cell proliferation, immunoglobulin (Ig)‐isotype switching, and germinal center formation. In physiological conditions, the activation of CD40 occurs by binding to its natural ligand, CD154, which is expressed on activated T cells. The in vivo critical role of CD40–CD154 interaction on B‐cell differentiation and isotype switching is provided by the discovery that mutations in either CD40 or CD154 gene cause the hyper IgM syndrome, termed HIGM3 or HIGM1, respectively, characterized by very low levels of serum IgG, IgA, and IgE, with normal or elevated IgM, associated with a defective germinal center formation. Originally considered humoral primary immunodeficiencies, the clinical features and the defect of T‐cell priming, resulting from a defective T–B cell or dendritic cell interaction, is now considered as combined immunodeficiencies. In this article, we present a comprehensive overview of the clinical, genetic, and immunological features of patients with hyper IgM syndrome due to CD40 mutations.     

Molecular characterization of galactokinase deficiency in Japanese patients

Galactokinase (GALK) deficiency is an autosomal recessive disorder, which causes cataract formation in children not maintained on a lactose-free diet. We characterized the human GALK gene by screening a Japanese genomic DNA phage library, and found that several nucleotides in the 5′-untranslated region and introns 1, 2, and 5 in our GALK genomic analysis differed from published data. A 20-bp tandem repeat was found in three places in intron 5, which were considered insertion sequences. We identified five novel mutations in seven unrelated Japanese patients with GALK deficiency. There were three missense mutations and two deletions. All three missense mutations (R256W, T344M, and G349S) occurred at CpG dinucleotides, and the T344M and G349S mutations occurred in the conserved region. The three missense mutations led to a drastic reduction in GALK activity when individual mutant cDNAs were expressed in a mammalian cell system. These findings indicated that these missense mutations caused GALK deficiency. The two deletions, of 410delG and 509–510delGT, occurred at the nucleotide repeats GGGGGG and GTGTGT, respectively, and resulted in in-frame nonsense codons at amino acids 163 and 201. These mutations arose by slipped strand mispairing. All five mutations occurred at hot spots in the CpG dinucleotide for missense mutations and in short direct repeats for deletions. These five mutations in Japanese have not yet been identified in Caucasians. We speculate that the origin of GALK mutations in Japanese is different from that in Caucasians. Received: May 19, 1999 / Accepted: August 21, 1999     

T1 mutants with increased transduction frequency are defective in host chromosome degradation

Mutants of T1 with enhanced transduction ability (tar mutants) were characterized by experiments designed to identify the cause for enhancement. Several bacterial markers and Mu PFU located in separate operons were transduced at different efficiencies. Infectious particles were produced in relatively normal amounts. Sensitivity to hydroxyurea (which inhibits de novo synthesis of nucleotides by the host) by a majority of the mutants suggested that they were defective in degradation of the host chromosome. One of the mutants was unable to incorporate host cell nucleotides, confirming its ability to degrade the host chromosome. Complementation experiments suggested that the tar mutations define a new T1 gene.     

Fertility defects revealing germline biallelic nonsense NBN mutations

Biallelic mutations in the NBN/NBS1 gene are the cause of Nijmegen breakage syndrome (NBS), a severe pediatric disease characterized by dysmorphy with a bird‐like face, microcephaly, growth retardation, immune deficiency, and proneness to cancer. We here report two adult siblings that are compound heterozygotes for two previously unreported NBN nonsense mutations. These patients presented with the unique clinical symptom of fertility defects. Contrasting with the absence of any developmental abnormality, biological analyses revealed defects similar to those observed in NBS patients, including chromosomal instability, cellular hyperradiosensitivity and checkpoint defects as measured by radioresistant DNA synthesis (RDS). NBN mutations should thus be considered a new cause of infertility, and should be searched for if associated with the biological abnormalities of NBS. Hum Mutat 0, 1–7, 2008. © 2008 Wiley‐Liss, Inc.     

Mitochondrial aspartate glutamate carrier (citrin) deficiency as the cause of adult-onset type II citrullinemia (CTLN2) and idiopathic neonatal hepatitis (NICCD)

 By using homozygosity mapping and positional cloning, we have shown that adult-onset type II citrullinemia (CTLN2) is caused by mutations of the SLC25A13 gene, which is localized on chromosome 7q21.3 and encodes a mitochondrial solute carrier protein named citrin. So far, we have reported nine mutations, most of which cause loss of citrin, and we have established several methods for DNA diagnosis. These methods have shown that more than 90% of the patients diagnosed as suffering from CTLN2 by enzymatic analysis carry SLC25A13 mutations in both alleles, indicating that CTLN2 is caused by citrin deficiency. Furthermore, by using the same DNA diagnosis methods, we discovered that 70 neonates or infants suffering from a particular type of neonatal hepatitis carry the same SLC25A13 mutations. Since the symptoms of the neonates are different from those of the more severe CTLN2 and usually ameliorate without special treatment, we designated the neonatal disease neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD). We conclude that citrin deficiency causes NICCD in neonates and CTLN2 in adults through the additional effects of genetic or environmental modifiers. Since the function of citrin, together with that of an isoform, aralar, was found to be as a mitochondrial aspartate glutamate carrier, the various symptoms of NICCD and CTLN2 may be understood as caused by defective aspartate export from the mitochondria to the cytosol and defects in the malate aspartate shuttle. It is, however, still difficult to understand the cause of the hepatic deficiency of argininosuccinate synthetase protein in CTLN2. Received: March 20, 2002 / Accepted: March 28, 2002     

Signaling pathways involved in the T‐cell‐mediated immunity against Epstein‐Barr virus: Lessons from genetic diseases

Primary immunodeficiencies (PIDs) provide researchers with unique models to understand in vivo immune responses in general and immunity to infections in particular. In humans, impaired immune control of Epstein‐Barr virus (EBV) infection is associated with the occurrence of several different immunopathologic conditions; these include non‐malignant and malignant B‐cell lymphoproliferative disorders, hemophagocytic lymphohistiocytosis (HLH), a severe inflammatory condition, and a chronic acute EBV infection of T cells. Studies of PIDs associated with a predisposition to develop severe, chronic EBV infections have led to the identification of key components of immunity to EBV – notably the central role of T‐cell expansion and its regulation in the pathophysiology of EBV‐associated diseases. On one hand, the defective expansion of EBV‐specific CD8 T cells results from mutations in genes involved in T‐cell activation (such as RASGRP1, MAGT1, and ITK), DNA metabolism (CTPS1) or co‐stimulatory pathways (CD70, CD27, and TNFSFR9 (also known as CD137/4‐1BB)) leads to impaired elimination of proliferating EBV‐infected B cells and the occurrence of lymphoma. On the other hand, protracted T‐cell expansion and activation after the defective killing of EBV‐infected B cells is caused by genetic defects in the components of the lytic granule exocytosis pathway or in the small adapter protein SH2D1A (also known as SAP), a key activator of T‐ and NK cell‐cytotoxicity. In this setting, the persistence of EBV‐infected cells results in HLH, a condition characterized by unleashed T‐cell and macrophage activation. Moreover, genetic defects causing selective vulnerability to EBV infection have highlighted the role of co‐receptor molecules (CD27, CD137, and SLAM‐R) selectively involved in immune responses against infected B cells via specific T‐B cell interactions.     

Identification of two novel and four uncommon missense mutations among Chinese Gaucher disease patients

Gaucher disease is the most prevalent lysosomal storage disease. It is panethnic and results from an inherited deficiency of glucocerebrosidase. Most mutations to date have been identified among Jewish and non-Jewish Caucasian patients; mutations in Chinese patients are largely unknown. We have performed nucleotide sequence analysis of PCR-amplified glucocerebrosidase genomic DNA from five unrelated Chinese patients affected with type 1 (non-neuropathic) Gaucher disease. A novel heterozygous C → T mutation at cDNA nucleotide position 475 (R120W) was detected in a patient who is also heterozygous for a C → T transition at cDNA nucleotide position 259 (R48W). In a second patient, a novel, heterozygous T → G transversion at cDNA 226 (F37V) was detected. Mutation 1448 (L444P), the most prevalent mutation among non-Jewish Caucasian Gaucher patients, was found in the heterozygous form in four patients. The mutations in the second Gaucher allele in the other three patients are mutations 254 (G46E), 680 (N188S), and 754 (F213I), which were recently reported in Korean, Arab, and Chinese (Taiwanese) patients. We have developed screening methods that utilize PCR amplification of glucocerebrosidase genomic DNA and Eco571, Nci1, Hinc11, BsaJ1, and Bsr1 restriction endonuclease analyses for the detection of each of these mutations. The population genetics of some of these Gaucher alleles and their implications in genotype/phenotype correlation are discussed. Am. J. Med. Genet. 71:172–178, 1997. © 1997 Wiley-Liss, Inc.     

Mutational Spectrum of the C1 Inhibitor Gene in a Cohort of Italian Patients with Hereditary Angioedema: Description of Nine Novel Mutations

Hereditary angioedema (HAE) is an autosomal dominant disease due to mutations in the C1 inhibitor gene (C1NH) that affects protein synthesis (HAE type I) or function (HAE type II). In 45 subjects affected by HAE diagnosed through clinical features and C1 inhibitor deficiency from the south of Italy (38 with type I and 7 with type II HAE), the whole C1NH coding region was screened for mutations by direct DNA sequencing. A severity score based on clinical manifestation, age at disease onset, and need for long‐term prophylaxis was used to investigate possible genotype‐phenotype correlations. A series of 22 different mutations was identified: nine missense (40.9%), five nonsense (22.7%), six frameshift (27.3), one small deletion (4.5%), and one splicing defect (4.5%). Nine C1NH mutations have not been previously described. No correlation was found between C1 inhibitor function level and severity score or age at first attack. Moreover, there was no correlation between different types of mutations and clinical phenotype. The number of different mutations identified highlights the heterogeneity of C1 inhibitor deficiency and supports the hypothesis that HAE clinical phenotype is not strictly related to the type of mutation but rather depends on unknown factors.     

Mutations in FASTKD2 are associated with mitochondrial disease with multi‐OXPHOS deficiency

Mutations in FASTKD2, a mitochondrial RNA binding protein, have been associated with mitochondrial encephalomyopathy with isolated complex IV deficiency. However, deficiencies related to other oxidative phosphorylation system (OXPHOS) complexes have not been reported. Here, we identified three novel FASTKD2 mutations, c.808_809insTTTCAGTTTTG, homoplasmic mutation c.868C>T, and heteroplasmic mutation c.1859delT/c.868C>T, in patients with mitochondrial encephalomyopathy. Cell‐based complementation assay revealed that these three FASTKD2 mutations were pathogenic. Mitochondrial functional analysis revealed that mutations in FASTKD2 impaired the mitochondrial function in patient‐derived lymphocytes due to the deficiency in multi‐OXPHOS complexes, whereas mitochondrial complex II remained unaffected. Consistent results were also found in human primary muscle cell and zebrafish with knockdown of FASTKD2. Furthermore, we discovered that FASTKD2 mutation is not inherently associated with epileptic seizures, optic atrophy, and loss of visual function. Alternatively, a patient with FASTKD2 mutation can show sinus tachycardia and hypertrophic cardiomyopathy, which was partially confirmed in zebrafish with knockdown of FASTKD2. In conclusion, both in vivo and in vitro studies suggest that loss of function mutation in FASTKD2 is responsible for multi‐OXPHOS complexes deficiency, and FASTKD2‐associated mitochondrial disease has a high degree of clinical heterogenicity.