BACKGROUND Filament aggregating protein (Filaggrin) is a skeletal cell component that provides a protective function for the epidermis. Mutations of the filaggrin gene (FLG) cause a loss of filaggrin protein. These mutations are seen in 50% of atopic dermatitis (AD). The aim of the study was to investigate the polymorphisms and mutations of the FLG in Iranian children with AD. MATERIALS AND METHODS This project was a case-controlled study with 25 children diagnosed with AD as the case group and 25 healthy children as the control group. Demographic data, clinical manifestations, and filaggrin single nucleotide polymorphisms (SNPs) and mutations were recorded. Blood samples were collected for the immunoglobulin E (IgE) assay and complete blood count tests. RESULTS We found a significant association between the presence of polymorphism (rs66831674) and patients' age, and polymorphism (rs41267154) and early onset of AD. We found no significant differences between the FLG polymorphisms with respect to the severity of AD, ethnicity, concurrent allergic diseases, eosinophilia, and IgE serum levels. CONCLUSION Interestingly, FLG variants (rs66831674 and rs41267154) were associated with age and early onset of AD. However, additional studies are required to confirm these results on a large scale of Iranian population. Moreover, establishing a cohort prospective study is suggested to assess the progression of other atopic disorders based on FLG polymorphisms.

BACKGROUND AND AIM:Leukocyte adhesion deficiency type 1 is a rare, autosomal recessive disorder that results from mutations in the ITGB2 gene. This gene encodes the CD18 subunit of β2 integrin leukocyte adhesion cell molecules. Leukocyte adhesion deficiency type 1 is characterized by recurrent bacterial infections, impaired wound healing, inadequate pus formation, and delayed separation of the umbilical cord.MATERIALS AND METHODS:Blood samples were taken from 13 patients after written consent had been obtained. Genomic DNA was extracted, and ITGB2 exons and exon-intron boundaries were amplified by polymerase chain reaction. The products were examined by Sanger sequencing.RESULTS:In this study, 8 different previously reported mutations (intron7+1G>A, c.715G>A, c.1777 C>T, c.843del C, c.1768T>C, c.1821C>A, Intron7+1G>A, c.1885G>A) and 2 novel mutations (c.1821C>A; p.Tyr607Ter and c.1822C>T; p.Gln608Ter) were found.CONCLUSIONS:c.1821C>A (p.Tyr607Ter) and c.1822C>T (p.Gln608Ter) mutations should be included in the panel of carrier detection and prenatal diagnosis.

Mucopolysaccharidosis VI is a rare autosomal recessive disorder caused by the deficiency of enzyme Arylsulfatase B. The enzyme deficiency leads to the accumulation of dermatan sulfate in connective tissue which causes manifestations related to MPS VI. Up to now, three different disease causing variants are reported in Iranian patients. In this study, we scanned ARSB gene of 13 Iranian patients from 12 families in whom all parents were consanguineous and from the same ethnicity except one family that were not consanguineous but co-ethnic. We found six not previously reported disease causing variants. We extracted DNA from peripheral blood samples of patients that were previously confirmed as MPS VI by clinical, biochemical and enzymatic assays including berry-spot test and fluorimetry, followed by PCR and direct sequencing. Computational approaches were used to analyze novel variants in terms of their impact on the protein structure. 11 disease causing variants and 15 polymorphisms were found. Six disease causing variants were novel and five were previously reported of which three were in Iranian population. Four of patients, who were unrelated, two by two had the same disease causing variant and polymorphisms, which indicates a possible founder effect. Our study also implicates genotype–phenotype correlation. Computational structural modeling indicated these disease causing variants might affect structural stability and function of the protein. Data of this study confirms the existence of mutational heterogeneity in the ARSB between Iranian patients. Disease causing variants with high frequency can be used in the prenatal diagnosis and genetic counseling. Also, the existence of the same variants and polymorphisms in some of the unrelated patients indicates a possible founder effect.

Background Filaggrin is a key structural epidermal protein in terminal differentiation and formation of skin barrier. The important role of filaggrin and its effects in various cutaneous and noncutaneous disorders initiated a cascade of considerable research in recent years. Loss-of-function mutations in FLG, the human gene encoding profilaggrin/filaggrin, is the cause of the common skin condition ichthyosis vulgaris (IV) and major genetic predisposing factor for atopic dermatitis (AD). Several null mutations in the FLG gene that lead to a decrease or absence of filaggrin in skin and predispose these conditions have been described. Objective Methods The aim of this study was to investigative genetic polymorphism of FLG in Iranian patients with IV and AD. In the current study, we carried out full sequencing of the entire FLG coding region in 30 IV patients and 30 AD patients, and also 60 healthy controls. Results Conclusion In our research, we identified 43 variants reported previously and two novel variants. In our study, in the AD and IV patients, loss-of-function FLG mutation was not found. This means that another mechanism other than FLG nonsense mutation is involved in the pathogenesis of these patients.

•We found a new compound heterozygote mutation in ARSB gene.•This can be important for prenatal diagnosis.•This is the first study and the first compound heterozygote that we report in this region of Asia

Chronic granulomatous disease (CGD) is an inherited disease of the innate immune system that results from defects in 1 of the 5 subunits of nicotinamide adenine dinucleotide phosphate oxidase complex and leads to life-threatening infections with granuloma formation. During 3 years of study, we recognized 10 male patients with X-linked CGD from a tertiary referral center for immune deficiencies in Iran. The CGD patients were diagnosed according to clinical features and biochemical tests, including nitroblue tetrazolium and dihydrorhodamine-1, 2, 3 tests, performed on patients and their mothers. In all patients, Western blot analysis showed a gp91(0) phenotype. Mutation screening by single strand conformation polymorphism and multiplex ligation-dependent probe amplification analysis of the CYBB gene encoding gp91(phox), followed by sequencing, showed 9 different mutations, 4 of them novel as far as we know.

BACKGROUND AND AIM:Leukocyte adhesion deficiency type 1 (LAD-I) is a rare, autosomal recessive disorder of neutrophil migration, characterized by severe, recurrent bacterial infections, inadequate pus formation and impaired wound healing. The ITGB2 gene encodes the β2 integrin subunit (CD18) of the leukocyte adhesion cell molecules, and mutations in this gene cause LAD-I. The aim of the current study was to investigate the mutations in patients diagnosed with LAD-I and functional studies of the impact of two previously reported and a novel mutation on the expression of the CD18/CD11a heterodimer.MATERIALS AND METHODS:Blood samples were taken from three patients who had signed the consent form. Genomic DNA was extracted and ITGB2 exons and flanking intronic regions were amplified by polymerase chain reaction. Mutation screening was performed after Sanger sequencing of PCR products. For functional studies, COS-7 cells were co-transfected with an expression vector containing cDNA encoding mutant CD18 proteins and normal CD11a. Flow cytometry analysis of CD18/CD11a expression was assessed by dimer-specific IB4 monoclonal antibody.RESULTS:Two previously reported mutations and one novel mutation,p. Cys562Tyr, were found. All mutations reduced CD18/CD11 heterodimer expression.CONCLUSION:Our strategy recognized the p.Cys562Tyr mutation as a pathogenic alteration that does not support CD18 heterodimer formation. Therefore, it can be put into a panel of carrier and prenatal diagnosis programs.

Epidermolysis bullosa simplex (EBS), which shows blister formation within the basal layer of the epidermis, is primarily caused by mutations in KRT5 and KRT14 (Coulombe et al., 2009Coulombe P.A. Kerns M.L. Fuchs E. Epidermolysis bullosa simplex: a paradigm for disorders of tissue fragility.J Clin Invest. 2009; 119: 1784-1793Crossref PubMed Scopus (153) Google Scholar, Fine et al., 2014Fine J.D. Bruckner-Tuderman L. Eady R.A. Bauer E.A. Bauer J.W. Has C. et al.Inherited epidermolysis bullosa: Updated recommendations on diagnosis and classification.J Am Acad Dermatol. 2014; 70: 1103-1126Abstract Full Text Full Text PDF PubMed Scopus (624) Google Scholar). Most cases result from dominant negative missense mutations in either one of these genes, with an autosomal dominant inheritance. Most mutations are in KRT14 compared with KRT5, reflecting, at least in part, a high frequency of hotspot mutations, such as p.Arg125His and p.Arg125Cys, in KRT14 (Coulombe and Lee, 2012Coulombe P.A. Lee C.H. Defining keratin protein function in skin epithelia: epidermolysis bullosa simplex and its aftermath.J Invest Dermatol. 2012; 132: 763-775Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Only 16 keratin mutations, have been shown to cause the autosomal recessive form of EBS, all of them in KRT14 (Garcia et al., 2011Garcia M. Santiago J.L. Terron A. Hernandez-Martin A. Vicente A. Fortuny C. et al.Two novel recessive mutations in KRT14 identified in a cohort of 21 Spanish families with epidermolysis bullosa simplex.Br J Dermatol. 2011; 165: 683-692Crossref PubMed Scopus (21) Google Scholar). In addition to mutations in KRT5 and KRT14, up to approximately 10% of autosomal recessive cases of EBS have been shown to result from mutations in TGM5 encoding transglutaminase 5, whereas relatively few cases, mostly autosomal recessive, have been shown to result from mutations in other genes, including JUP, DSP, PKP1, EXPH5, PLEC, and DST-e (Uitto et al., 2016Uitto J. Bruckner-Tuderman L. Christiano A.M. McGrath J.A. Has C. South A.P. et al.Progress towards treatment and cure of epidermolysis bullosa: Summary of the DEBRA International Research Symposium EB2015.J Invest Dermatol. 2016; 136: 352-358Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, Uitto et al., 2016Uitto J. Vahidnezhad H. Youssefian L. Genotypic heterogeneity and the mode of inheritance in epidermolysis bullosa.JAMA Dermatol. 2016; 152: 517-520Crossref PubMed Scopus (8) Google Scholar). As part of an effort to characterize a large cohort of epidermolysis bullosa patients in Iran, a country of close to 80 million inhabitants in which approximately 38% of marriages are consanguineous, patients with clinical presentation and immunoepitope mapping suggestive of EBS (see Supplementary Figure S1 online) were studied by whole-genome homozygosity mapping (see Supplementary Figure S2a and b online), followed by Sanger sequencing (Barzegar et al., 2015Barzegar M. Asadi-Kani Z. Mozafari N. Vahidnezhad H. Kariminejad A. Toossi P. Using immunofluorescence (antigen) mapping in the diagnosis and classification of epidermolysis bullosa: a first report from Iran.Int J Dermatol. 2015; 54: e416-e423Crossref PubMed Scopus (10) Google Scholar) (Table 1). This study was approved by the institutional review board of the Pasteur Institute of Iran, and the patients gave written informed consent to participate in research and to have their images and medical histories published. Eleven families out of 12 were found to harbor mutations in either KRT5 or KRT14 (Figure 1). In seven families, a heterozygous missense mutation, three of them previously unreported, was found to affect a fully conserved amino acid residue (Figure 1a–c and h–k). In four families, there were multiple affected individuals, and one of the parents and/or other affected family members were shown to harbor the same mutation as found in the proband, consistent with autosomal dominant inheritance. In three cases with a heterozygous missense mutation, the parents were clinically normal and had the wild-type KRT14 sequence, suggesting de novo origin of the mutation (families 4, 7, and 11; Table 1). In addition, one of these patients with severe generalized blistering with p.Arg125Cys mutation in KRT14 (family 7) was found to be compound heterozygous with a p.Gly183Glu substitution in KRT5; the latter mutation has been previously suggested to serve as a modifier of the severity of EBS phenotype (Kowalewski et al., 2007Kowalewski C. Hamada T. Wozniak K. Kawano Y. Szczecinska W. Yasumoto S. et al.A novel autosomal partially dominant mutation designated G476D in the keratin 5 gene causing epidermolysis bullosa simplex Weber-Cockayne type: a family study with a genetic twist.Int J Mol Med. 2007; 20: 75-78PubMed Google Scholar). In one family (family 10), a previously unreported 18-base pair de novo in-frame deletion in exon 6 of KRT14 was identified in a patient with generalized severe blistering and extensive palmoplantar keratoderma (see Supplementary Figure S1c). This mutation results in deletion of six amino acids from the keratin 14 polypeptide in the 2B domain critical for keratin filament assembly. Because the association of palmoplantar keratoderma with EBS is rare, this finding, together with previous observations (Livingston et al., 2001Livingston R.J. Sybert V.P. Smith L.T. Dale B.A. Presland R.B. Stephens K. Expression of a truncated keratin 5 may contribute to severe palmar—plantar hyperkeratosis in epidermolysis bullosa simplex patients.J Invest Dermatol. 2001; 116: 970-974Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, Titeux et al., 2006Titeux M. Mazereeuw-Hautier J. Hadj-Rabia S. Prost C. Tonasso L. Fraitag S. et al.Three severe cases of EBS Dowling-Meara caused by missense and frameshift mutations in the keratin 14 gene.J Invest Dermatol. 2006; 126: 773-776Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar), suggests that mutations altering the sequence of this domain in keratin 14 can result in palmoplantar keratoderma. Thus, most patients studied here harbored heterozygous mutations, apparently resulting in dominant negative interference of the association of keratin 5 and keratin 14 to form heterodimers during intracellular assembly of intermediate filaments in basal keratinocytes.Table 1Mutation analysis of KRT5 and KRT14 in Iranian families affected by epidermolysis bullosa simplexFamilyGeneExonMutation at DNA LevelInheritance1Epidermolysis bullosa simplex.Homozygous/HeterozygousMutation at Protein LevelPredicted ConsequencePhenotype1Epidermolysis bullosa simplex.PolyPhen2 score2http://genetics.bwh.harvard.edu/pph2/. The values range from 0–1.000, the latter being most damaging.Score of PROVEAN (cutoff = –2.5)3http://provean.jcvi.org/index.php. The values below the cutoff score –2.5 indicate deleterious mutations.Mutation First Reported1KRT51c.530A>GADHeterozygousp.Asn177SerMissenseEBS, localized1.000 (Probably damaging)–4.373 (Deleterious)Liovic et al., 2004Liovic M. Bowden P.E. Marks R. Komel R. A mutation (N177S) in the structurally conserved helix initiation peptide motif of keratin 5 causes a mild EBS phenotype.Exp Dermatol. 2004; 13: 332-334Crossref PubMed Scopus (13) Google Scholar2KRT52c.557T>CADHeterozygousp.Leu189ProMissenseEBS, localized0.939 (Possibly damaging)–6.095 (Deleterious)This study3KRT57c.1283C>AADHeterozygousp.Ala428AspMissenseEBS, localized1.000 (Probably damaging)–5.670 (Deleterious)This study4KRT57c.1424A>GADHeterozygousp.Glu475GlyMissenseEBS, generalized severe0.969 (Probably damaging)–6.367 (Deleterious)Lane et al., 1992Lane E.B. Rugg E.L. Navsaria H. Leigh I.M. Heagerty A.H. Ishida-Yamamoto A. et al.A mutation in the conserved helix termination peptide of keratin 5 in hereditary skin blistering.Nature. 1992; 356: 244-246Crossref PubMed Scopus (341) Google Scholar5KRT141c.92delTARHomozygousp.Ile31ThrfsX39FrameshiftEBS, generalized intermediateN/AN/ABatta et al., 2000Batta K. Rugg E.L. Wilson N.J. West N. Goodyear H. Lane E.B. et al.A keratin 14 'knockout' mutation in recessive epidermolysis bullosa simplex resulting in less severe disease.Br J Dermatol. 2000; 143: 621-627Crossref PubMed Google Scholar6KRT141c.200delGARHomozygousp.G67AfsX51FrameshiftEBS, generalized severeN/AN/AThis study7KRT141c.373C>TADHeterozygousp.Arg125CysMissenseEBS, generalized severe0.946 (Possibly damaging)–6.729 (Deleterious)Coulombe et al., 1991Coulombe P.A. Hutton M.E. Letai A. Hebert A. Paller A.S. Fuchs E. Point mutations in human keratin 14 genes of epidermolysis bullosa simplex patients: genetic and functional analyses.Cell. 1991; 66: 1301-1311Abstract Full Text PDF PubMed Scopus (533) Google Scholar8KRT144c.915G>AARHomozygousp.Trp305TerNonsenseEBS, generalized severeN/AN/ACorden et al., 1998Corden L.D. Mellerio J.E. Gratian M.J. Eady R.A. Harper J.I. Lacour M. et al.Homozygous nonsense mutation in helix 2 of K14 causes severe recessive epidermolysis bullosa simplex.Hum Mutat. 1998; 11: 279-285Crossref PubMed Scopus (41) Google Scholar9KRT146c.1130T>CAD/SemidominantHeterozygous/Homozygousp.Ile377ThrMissenseEBS, localized in heterozygous state; EBS, generalized severe in homozygous state0.999 (Probably damaging)–4.646 (Deleterious)Rugg et al., 2007Rugg E.L. Horn H.M. Smith F.J. Wilson N.J. Hill A.J. Magee G.J. et al.Epidermolysis bullosa simplex in Scotland caused by a spectrum of keratin mutations.J Invest Dermatol. 2007; 127: 574-580Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar10KRT146c.1241del18ADHeterozygous—In-frame deletionGeneralized EBS with severe palmoplantar hyperkeratosisN/AN/AThis study11KRT147c.1253T>GADHeterozygousp.Leu418ArgMissenseEBS, localized1.000 (Probably damaging)–5.504 (Deleterious)This studyAbbreviations: AD, autosomal dominant; AR, autosomal recessive; N/A, not applicable; PROVEAN, Protein Variation Effect Analyzer.1 Epidermolysis bullosa simplex.2 http://genetics.bwh.harvard.edu/pph2/. The values range from 0–1.000, the latter being most damaging.3 http://provean.jcvi.org/index.php. The values below the cutoff score –2.5 indicate deleterious mutations. Open table in a new tab Abbreviations: AD, autosomal dominant; AR, autosomal recessive; N/A, not applicable; PROVEAN, Protein Variation Effect Analyzer. In addition to these putative autosomal dominant families with EBS, three families with a constellation suggestive of an autosomal recessive inheritance pattern were found to harbor homozygous KRT14 mutations, c.92delT, p.Trp305Ter, and c.200delG, the last mutation being previously unreported to our knowledge. Both parents of the probands were heterozygous carriers of the corresponding mutations (Figure 1). Of particular interest was a large multiplex family (family 9) with affected individuals in five generations (Figure 1h). Three of the family members had generalized spontaneous blistering (see Supplementary Figure S1b), and the rest of the affected individuals had a relatively mild blistering tendency limited to palms and soles when subjected to considerable trauma or heat (see Supplementary Figure S1). Initial attempts to identify candidate genes in this family were performed with whole genome-wide single nucleotide polymorphism-based homozygosity mapping using the Illumina Infinium Exome-24 Bead Chip (240,000 markers) (Illumina, San Diego, CA) which showed conserved homozygosity blocks in all three affected individuals on chromosome band 17q21, corresponding to ITGA3, JUP, and KRT14 as candidate genes (see Supplementary Figure S2a and c). The phenotypes in patients reported with mutations in ITGA3 and JUP are very different from those with KRT14 mutations, the latter patients not having renal and pulmonary involvement (Has et al., 2012Has C. Sparta G. Kiritsi D. Weibel L. Moeller A. Vega-Warner V. et al.Integrin alpha3 mutations with kidney, lung, and skin disease.N Engl J Med. 2012; 366: 1508-1514Crossref PubMed Scopus (170) Google Scholar) or cardiomyopathy and curly hair (Boente et al., 2016Boente M.D. Nanda A. Baselaga P.A. Kelsell D.P. McGrath J.A. South A.P. Cardiomyopathy diagnosed in the eldest child harboring p.S24X mutation in JUP.Br J Dermatol. 2016; ([e-pub ahead of print])http://dx.doi.org/10.1111/bjd.14617Google Scholar); accordingly, sequencing of JUP did not show any pathogenic sequence variants. Because the patients with ITGA3 mutation show early lethality due to renal and pulmonary complications (Has et al., 2012Has C. Sparta G. Kiritsi D. Weibel L. Moeller A. Vega-Warner V. et al.Integrin alpha3 mutations with kidney, lung, and skin disease.N Engl J Med. 2012; 366: 1508-1514Crossref PubMed Scopus (170) Google Scholar), this gene was not sequenced here. Sequencing of KRT14 showed the presence of a c.1130T→C transition that resulted in amino acid substitution p.Ile377Thr (Figure 1h). Segregation analysis of all 12 patients showed that individuals with generalized blistering and with consanguineous parents (first cousin marriages) were homozygous for the C allele (C/C), whereas the remaining nine patients with milder disease were heterozygous for T/C (family 9, Figure 1h). The wild-type sequence T/T was found in a clinically unaffected individual. Thus, this family shows mild localized blistering in heterozygous carriers of the mutation, whereas homozygous mutations result in generalized, more severe disease, consistent with semidominant inheritance. Such a semidominant mode of inheritance was also reported in an EBS family with DST-e mutation (Groves et al., 2010Groves R.W. Liu L. Dopping-Hepenstal P.J. Markus H.S. Lovell P.A. Ozoemena L. et al.A homozygous nonsense mutation within the dystonin gene coding for the coiled-coil domain of the epithelial isoform of BPAG1 underlies a new subtype of autosomal recessive epidermolysis bullosa simplex.J Invest Dermatol. 2010; 130: 1551-1557Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Most previous reports on KRT14 mutations have been limited to families with few affected individuals, and it is therefore unclear if other KRT14 mutations are also semidominant. To our knowledge, our study expands the mutation database by reporting previously undescribed findings on dominant negative missense mutations in KRT5 and KRT14 and adding a previously unreported in-frame six-amino acid deletion in keratin 14. This study further adds one previously unreported frameshift, premature termination codon-causing mutation to the KRT14 database in autosomal recessive EBS. Demonstration of the dose effect of a mutation in one family, consistent with semidominant inheritance, further increases our understanding of the genotype/phenotype correlations in this group of disorders. Regarding the mode of inheritance of EBS, in general the autosomal dominant form is by far more common than the recessive variant, with only 16 distinct mutations of the latter type having been published in KRT14 so far (Garcia et al., 2011Garcia M. Santiago J.L. Terron A. Hernandez-Martin A. Vicente A. Fortuny C. et al.Two novel recessive mutations in KRT14 identified in a cohort of 21 Spanish families with epidermolysis bullosa simplex.Br J Dermatol. 2011; 165: 683-692Crossref PubMed Scopus (21) Google Scholar). In our cohort, four out of 11 families, with a total of seven patients with homozygous mutations, were noted, attesting to the effect of consanguinity on heritable diseases in the Iranian population. Another example of the preponderance of homozygous mutations in EBS in the Iranian cohort of consanguineous families is the report of Kindler syndrome with complete homozygosity of mutations in FERMT1 in all families (Youssefian et al., 2015Youssefian L. Vahidnezhad H. Barzegar M. Li Q. Sotoudeh S. Yazdanfar A. et al.The Kindler syndrome: a spectrum of FERMT1 mutations in Iranian families.J Invest Dermatol. 2015; 135: 1447-1450Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Knowledge of family-specific mutations provides information useful for genetic counseling regarding the risk of recurrence in future pregnancies in the same or subsequent generations (Uitto, 2009Uitto J. Progress in heritable skin diseases: translational implications of mutation analysis and prospects of molecular therapies.Acta Derm Venereol. 2009; 89: 228-235Crossref PubMed Scopus (30) Google Scholar). Specific mutations can also allow prognostication at the newborn stage of disease severity. An example of such prognostic utility of the knowledge of mutations in the keratin genes is the recent study indicating that a specific mutation, p.Glu477Lys in KRT5, is strongly associated with mortality in EBS (Sathishkumar et al., 2016Sathishkumar D. Orrin E. Terron-Kwiatkowski A. Browne F. Martinez A.E. Mellerio J.E. et al.The p.Glu477Lys mutation in keratin 5 is strongly associated with mortality in generalized severe epidermolysis bullosa simplex.J Invest Dermatol. 2016; 136: 719-721Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). Thus, the expanded mutation database forms the basis for application of precision medicine for management of these patients. The authors state no conflict of interest. The authors thank Mina Tabrizi, Mohsen Siavashi, and Maryam Abiri (Tehran University of Medical Sciences) for assistance in sample collection and processing and Hakon Hakonarson and Cecilia Kim (Children's Hospital of Philadelphia) for assistance in homozygosity mapping. Carol Kelly assisted in manuscript preparation. This work partially fulfills requirements for the PhD thesis of HV. Download .pdf (.51 MB) Help with pdf files Supplementary Figures S1 and S2

1 Pediatrics Center of Excellence. Children's Medical Center, Tehran University of Medical Sciences. Tehran (Iran) 2 Research Center for Immunodeficiencies. Children's Medical Center, Tehran University of Medical Sciences. Tehran (Iran) 3 Department of Pediatrics. Faculty of Medicine, Hacettepe University. Ankara (Turkey) 4 Institute of Human Genetics, University Hospital of Magdeburg. Magdeburg (Germany) 5 Department of Immunology. Molecular Immunology Research Center, Research Center for Immunodeficiencies, Children's Medical Center, Tehran University of Medical Sciences. Tehran (Iran)

Ataxia telangiectasia (AT) is an autosomal recessive multisystem disorder characterized by variable immunodeficiency, progressive neurodegeneration, occulocutaneous telangiectasia, and an increased susceptibility to malignancies. This study was designed to study the role of proapoptotic BAK, BAX, and NBK/BIK genes in a group of patients with AT to elucidate the possible role of these genes in progression of malignancies in this disease.

Background. Recurrent and chronic infections of ear, nose, and throat (ENT) such as sinusitis (rhinosinusitis) and otitis media are one of the most common health care problems worldwide and significantly impact quality of life in both children and adults. Antibody deficiencies are the most common type of primary immunodeficiency and also the most likely to present with recurrent ENT infections.Methods: A study was carried out to search for underlying immunodeficiencies in 103 patients with recurrent or chronic ear, nose and throat infections. Serum total IgG, IgA, and IgM levels were measured by kinetic nephelometry, and IgG subclasses by enzymelinked immunosorbent assay (ELISA). All patients were immunized intramuscularly with polyvalent pneumococcal vaccine (PENEUMO 23). Blood samples were drawn immediately before and 21 days after vaccination and antibodies to pneumococcal antigens were measured using a modified ELISA technique.Finding: Of 103 patients twenty one (20%) were found to have an immunodeficiency. One had a common variable immunodeficiency (CVID), 5 had selective IgA deficiency (one of them was associated with IgG2 deficiency and one with specific antibody deficiency). Eight patients had IgG-subclass deficiency including seven with an IgG2 deficiency and one patient with IgG3 deficiency. In 75 patients antibody titers of whole pneumococcal antigens were determined before and 21 days after immunization. Ten patients were found to have abnormally low antibody titers.Conclusion: The results of this study suggest that in a subpopulation of patients with a long standing history of ENT infections, a low serum immunoglobulin concentration or hypo responsiveness to pneurnococcal antigen would be associated with susceptibility to recurrent infections.

Ataxia telangiectasia (AT) is an autosomal recessive disorder with an estimated prevalence of 1/40,000 to 1/100,000 in reported populations. There is a 25% possibility for having an affected child when parents are carriers for the ATM gene mutation. There is no cure available for this disease and prenatal testing is strongly recommended for prevention of this disease. Although the preferred method is the direct mutation analysis of the ATM gene, the large size of the ATM gene with 63 exons and the large number of possible mutations in patients considerably limit efficiency of mutation analysis as a diagnostic choice. Indirect method is a better tool when parents are not carriers of founder mutation and pass different mutations to their children. Indirect molecular diagnosis using ATM-related molecular markers facilitates prenatal diagnosis of AT children. In this study, four molecular markers: D11S2179, D11S1787, D11S535, D11S1343 are genotyped in 19 unrelated families from different regions of Iran. Those markers are amplified using extracted sequence primers from the Gene Bank with their described PCR conditions. Amplified products were separated using denaturing PAGE gels, and data were analyzed to detect their pattern of inheritance in each family. In all families, segregation of alleles was according to Mendelian inheritance, and affected chromosomes were distinguishable from unaffected ones. All carriers and affected patients were diagnosed accurately. Thus, this method is effectively useful in prenatal diagnosis of AT.

Primary immunodeficiency disorders are a heterogeneous group of genetic disorders, with different modes of inheritance, consisting of more than 100 different types. We constructed the DNA banking of primary immunodeficiency disorders for the first time in Iran. The DNA of 31 immunodeficient patients and their families (total of 92 samples) were collected, as the first step for construction of DNA banking. DNA was isolated from whole blood by salting out method. Among our patients, Common variable immunodeficiency was the most common disorder, followed by X-linked agammaglobulinemia, Ataxia-telangiectasia, Chronic granulomatous disease, Severe combined immunodeficiency, Hyper IgM syndromes, and Leukocyte adhesion defects. DNA banking is a useful method for further detection of mutation in immunodeficient patients and prenatal diagnosis for presence or absence of the disorder in the fetus which can be confirmed by molecular genetics testing.

Ataxia-Telangiectasia (AT) is a rare human neurodegenerative autosomal recessive multisystem disease that is characterized by a wide range of features including, progressive cerebellar ataxia with onset during infancy, occulocutaneous telangiectasia, susceptibility to neoplasia, occulomotor disturbances, chromosomal instability and growth and developmental abnormalities. Mitochondrial DNA (mtDNA) has the only non-coding regions at the displacement loop (D-loop) region that contains two hypervariable segments (HVS-I and HVS-II) with high polymorphism. We investigated mt-DNA deletions and haplogroups in AT patients. In this study, 24 Iranian patients suffering from AT and 100 normal controls were examined. mt-DNA was extracted from whole bloodand examined by 6 primers for existence of mitochondrial deletions. We also amplified and sequenced the mtDNA HVS-I by standard sequencing techniques. mtDNA deletions were observed in 54.1% (13/24) of patients (8.9 kb deletion in all samples, 5.0 kb in one and 7.5 kb in two patients), representing mtDNA damage which may be due to oxidative stress in mitochondria. Our results showed that there is no association between mtDNA haplogroups and AT. This data may indicate involvement of mitochondrial damage in the pathogenesis of AT.

Ataxia-Telangiectasia (AT) is an autosomal recessive disorder involving cerebellar degeneration, immunodeficiency, radiation sensitivity and cancer predisposition. The ATM gene on human chromosome 11q22.3 has recently been identified as the gene responsible for ataxia-telangiectasia (AT). The gene mutated in AT, which has been designated as the ATM gene, encodes a large protein kinase with a PI-3 kinase-related domain. More than 100 mutations are broadly distributed throughout the ATM gene. The large size of the ATM gene (66 exons spanning ~150kb of genomic DNA) together with the diversity and broad distribution of mutations in AT patients, greatly limits the utility of direct mutation screening as a diagnostic tool. In this study, 20 families with at least one affected child clinically suspected to have ataxia-telangiectasia were examined and their DNA was extracted and amplified with standard methods. Sequencing methods were used to detect the new point mutation. Four exons which were hot spots for point mutations in ATM gene were detected by PCR-SSCP or PCR-RFLP.

MTT assay is designed for spectrophotometric quantification of cell growth and viability without using the radiactive isotopes. The aim of this study is comparison of LIT and MTT as sensitive poliferation assays in imrnunodeficient patients. 20 immunodeficient and 20 healthy subjects were selected in this study. All of them had normal lymphocytes count, normal CD3 and negative DIR reaction to PPD, DT and candida. The immunodeficient patients regularly suffered from herpes, candida and staph abscess. The lymphocytes of inimunodeficient control groups were treated with PHA as mitogen. The lymphocyte proliferation was evaluated by MTT and LTT. The results showed a strong correlation between LTT and MITT between immunodeficient patients and healthy controls. The sensitivity test for MTT was 90%, and the sensitivity test for LTT was 98%. MTT method can be considered as a non-radiactive evaluation of cell proliferation. Detecting cell proliferation with accurate, sensitive, fast, easy and safe method is more preferable than LTT.

Serum opsonic capacity againstB. pertussis was studied by using quantitative chemiluminescence eCL), a method known to have several advantages over conventional methods in evaluating opsonization and phagocytosis. Sera from unvaccinated infants was shown not to contain opsonins against B. pertussis and in unvaccinated infants suffering from whooping cough, no opsonins were detected. In adults fully vaccinated during childhood, antibody titers decreased with time. Therefore, antibody transfer to infants is negligible. The CL assay is simple, rapid, and reproducible, offering new possibilities to evaluate humoral immune mechanisms and phagocytosis in whooping cough. MJIRI, Vol. 10, No.1, 65-67, 1996.