Heterozygosity assessment of five STR loci located at 5q13 region for preimplantation genetic diagnosis of spinal muscular atrophy.

Preimplantation genetic diagnosis (PGD) has been considered as an alternative to prenatal diagnosis for prevention of genetic disorders while avoiding the subsequent termination of pregnancy. However, the limited amount of template DNA available in a single diploid cell used for PGD leads to number of problems including an increased incidence of detectable contamination; amplification failure and allele drop out. Due to their highly polymorphic and amplifiable characteristics, short tandem repeat (STR) analysis has been proposed as a mean to overcome these limitations. Heterozygosity of the applied STRs is of paramount importance in their informativity, and should therefore be studied in any certain population. Here, for the first time, we report on the heterozygosity analysis of five STR markers (D5S1408, D5S1417, D5S610, D5S629 and D5S637) flanking to SMA gene region, to examine their applicability in the PGD for SMA disease. We have also investigated other statistical features of these markers and found that all of the five studied STRs were informative and four meet the Hardy-Weinberg equilibrium for the studied population. Furthermore, our results propose that similar approaches can be used for the PGD of other single gene disorders.

Novel nucleotide changes in mutational analysis of mitochondrial 12SrRNA gene in patients with nonsyndromic and aminoglycoside-induced hearing loss.

Mitochondria have essential role in cellular energy metabolism and defects in their function lead to many metabolic diseases. Mitochondrial DNA (mtDNA) mutations have been associated with number diseases such as nonsyndromic and aminoglycoside-induced hearing loss. Mutational screening of entire 12SrRNA and tRNA (ser (UCN)) genes in 107 unrelated Iranian patients with amino glycoside-induced and nonsyndromic bilateral hearing loss by direct sequencing analysis method were performed. Twenty different homoplasmic sequence variants were identified; including fifteen common polymorphisms, two putatively pathogenic variants: m.921T>C and m.1005T>C, one 12SrRNA sequence variant m.739C>T and two nucleotides substitution; m.1245T>C and m.1545T>C. Deafness-associated mutation, m.1555A>G, was not found. In our patients we found the mutation 1005 was associated with R haplogroup. These finding show that m.1555A>G mutation is not important in our population. Nucleotide change, m.739C>T, previously reported with very low frequency. We suggested the variation of two nucleotides 1245 and 1545 that localized at conserved site of 12SrRNA may be new candidate for amino glycoside-induced and nonsyndromic hearing impairment associated mutations. However, aminoglycoside exposure is a risk factor for clinical phenotype appearance of these mutations.

High frequency of mutations in the PIK3CA gene helical and kinase coding regions in a group of Iranian patients with high-grade glioblastomas: five novel mutations.

Glioblastoma multiform (GBM; World Health Organization (WHO) grade IV) and anaplastic astrocytomas (AA; WHO grade III) are highly aggressive and lethal astrocytic brain tumors. To detect cancer-specific somatic mutations in two hot-spot regions of PIK3CA gene, the helical and kinase domains (encoded by exons 9 and 20, respectively) in GBM and AA, the authors examined the respective sequences 31 paraffin-embedded samples (23 GBM and 8 AA). The samples were obtained from a group of Iranian patients affected with high-grade glioblastoma (HGG). The overall prevalence of PIK3CA mutations was 23% (7/31) for both tumor types (22% in GBM, and 25% in AA). Five mutations were detected in exon 20, p.Arg992Gln (c.2976G→A), p.Met1005Val (c.3014A→G), p.Ile1019→Val (c.3056A→G), p.Ser1008Cys (c.3024C→G), and p.Asn1044Asp (c.3130A→G), and one mutation in exon 9, p.Glu545Ala (c.1634A→C). Additionally exons 4-8 of P53 gene in four unrelated young patients, who showed no mutations in PIK3CA exons 9 and 20, were analyzed. Three mutations were identified: p.Pro72Ala (c.214C→G), g.11608G→T (homozygote splice mutation), and p.Thr170Thr (c.510G→A silent mutation). In conclusion, mutation detection in PIK3CA in patients with a high degree of malignancy and early age at diagnosis should be included in molecular diagnostic protocols, also with regard to possible upcoming therapies.

Intraspecific variation within Phlebotomus sergenti Parrot (1917) (Diptera: Psychodidae) based on mtDNA sequences in Islamic Republic of Iran.

An intraspecific study on the morphological and molecular characteristics of Phlebotomus sergenti s.l., the main vector of Leishmania tropica, was performed on 28 Iranian populations from 11 provinces and a few samples from Greece, Morocco, Lebanon, Turkey, Pakistan, and Syria. Three morphotypes were identified as A, B and C, with some intermediate forms in the samples under investigation. Based on the number of setae and the width of basal lobe of coxite, differences between A and B morphotypes were highly significant. Excluding one unusual haplotype, sequence analysis of approximately 439 bp of mtDNA (a fragment of cytochrome B gene, tRNA for serine gene, and a fragment of NADH1 gene) revealed a 6-7% genetic distance within the Iranian populations and among the specimens of other countries. Neighbor-Joining (NJ) analysis confirmed the existence of three main groups within our samples. Although there was no consistency between morphotypes and genotypes, but an interrelationship was found between morphometry and morphotypes. Morphotype A, which was considered as P. sergenti sergenti, was the most prevalent in collection sites. Morphotype B, which was identified as Phlebotomus sergenti similis, is the first record of this subspecies in Iran, and was found to be sympatric with other morphotypes. Morphotype C had an elongated style in comparison with P. sergenti sergenti. Molecular database showed three main genetic structures. This is the first combined morphological and molecular studies on P. sergenti s.l. in Iran.

Transforming growth factor-beta1 codon 10 polymorphism is associated with acute GVHD after allogenic BMT in Iranian population.

BACKGROUND: Certain cytokine genotypes are associated with acute graft versus host disease (aGVHD) after bone marrow transplantation (BMT). The present study aimed to determine existing association between TGF-beta1 codon 10 polymorphism and aGVHD after HLA-identical sibling BMT in the Iranian population. MATERIAL/METHODS: In a retrospective case-control study, 168 subjects including 84 Iranian HLA-identical sibling BMT donor/recipient pairs were recruited. All of the patients were affected by hematological malignancies (AML=39, ALL=23 and CML=22). PCR-SSP method was performed to determine TGF-beta1 codon 10 T/C polymorphism genotypes. RESULTS: The frequency of TGF-beta1 codon 10 TT, TC and CC genotypes among all subjects were 26.8%, 33.3% and 39.9% respectively. Recipients with the T allele developed aGVHD significantly less than those without the T allele (odds ratio =0.334, P=0.026). CONCLUSIONS: Genetic background of TGF-beta1 may be involved as a protective factor in the development of aGVHD in HLA-matched sibling BMT in Iranian population. Moreover, this finding may indicate that the genetic markers in Iranians are, at least to some extent, linked to distinct genetic event from Japanese.

Molecular analysis of the SMN1 and NAIP genes in Iranian patients with spinal muscular atrophy.

INTRODUCTION: Childhood-onset proximal spinal muscular atrophies (SMAs) are an autosomal recessive, clinically heterogeneous group of neuropathies characterised by the selective degeneration of anterior horn cells. SMA has an estimated incidence of 1 in 10,000 live births. The causative genes are survival motor neuron (SMN) gene and neuronal apoptosis inhibitory protein (NAIP) gene. Deletions of the telomeric copy of SMN gene (SMN1) have been reported in 88.5% to 95% of SMA cases, whereas the deletion rate for NAIP gene (NAIP) is between 20% and 50% depending on the disease severity. The main objective of this study was to genetically characterise the childhood onset of SMA in Iran. MATERIALS AND METHODS: Molecular analysis was performed on a total of 75 patients with a clinical diagnosis of SMA. In addition to common PCR analysis for SMN1 exons 7 and 8, we analysed NAIP exons 4 and 5, along with exon 13, as a internal control, by bi-plex PCR. RESULTS: The homozygous-deletion frequency rate for the telomeric copy of SMN exons 7 and 8 in all types of SMA was 97%. Moreover, exons 5 and 6 of NAIP gene were deleted in approximately 83% of all SMA types. Three deletion haplotypes were constructed by using SMN and NAIP genotypes. Haplotype A, in which both genes are deleted, was seen in approximately 83% of SMA types I and II but not type III. It was also found predominantly in phenotypically severe group with an early age of onset (i.e., less than 6-month-old). We also report 34 of our prenatal diagnosis. CONCLUSIONS: To our knowledge, the present study is the first one giving detailed information on SMN and NAIP deletion rates in Iranian SMA patients. Our results show that the frequency of SMN1 homozygous deletions in Iran is in agreement with previous studies in other countries. The molecular analysis of SMA-related gene deletion/s will be a useful tool for pre- and postnatal diagnostic.

ITS2-rDNA Sequence Variation of Phlebotomus sergenti s.l. (Dip: Psychodidae) Populations in Iran.

BACKGROUND: Phlebotomus sergenti s.l. is considered the most likely vector of Leishmania tropica in Iran. Although two morphotypes- P. sergenti sergenti (A) and P. sergenti similis (B)-have been formally described, further morphological and a molecular analysis of mitochondrial cytochrome oxidase I (mtDNA-COI) gene revealed inconsistencies and suggests that the variation between the morphotypes is intraspecific and the morphotypes might be identical species. METHODS: We examined the sequence of the ITS2-rDNA of Iranian specimens of P. sergenti s.l., comprising P. cf sergenti, P. cf similis, and intermediate morphotypes, together with available data in Genbank. RESULTS: Sequence analysis showed 5.2% variation among P. sergenti s.l. morphotypes. Almost half of the variation was due to the number of an AT microsatellite repeats in the center of the spacer. Nine haplotypes were found in the species constructing three main lineages corresponding to the origin of the colonies located in southwest (SW), northeast (NE), and northwest-center-southeast (NCS). Lineages NCS and NE included both typical P. cf sergenti and P. cf similis and intermediate morphotypes. CONCLUSION: Phylogenetic sequence analysis revealed that, except for one Iranian sample, which was close to the European samples, other Iranian haplotypes were associated with the northeastern Mediterranean populations including Turkey, Cyprus, Syria, and Pakistan. Similar to the sequences of mtDNA COI gene, ITS2 sequences could not resolve P. sergenti from P. similis and did not support the possible existence of sibling species or subspecies within P. sergenti s.l..

Mutation analysis of androgen receptor gene: multiple uses for a single test.

Androgen receptor gene mutations are one of the leading causes of disorders of sex development (DSD) exhibited by sexual ambiguity or sex reversal. In this study, 2 families with patients whom diagnosed clinically as androgen insensitivity syndrome (AIS) were physically and genetically examined. This evaluation carried out by cytogenetic and molecular analysis including karyotype and sequencing of SRY and AR genes. In family 1, two brothers and their mother were hemizygous and heterozygous respectively for c.2522G>A variant, while one of their healthy brother was a completely normal hemizygote. Family 2 assessment demonstrated the c.639G>A (rs6152) mutation in two siblings who were reared as girls. The SRY gene was intact in all of the study's participants. Our findings in family 1 could be a further proof for the pathogenicity of the c.2522G>A variant. Given the importance of AR mutations in development of problems such as sex assignment in AIS patients, definitive diagnosis and phenotype-genotype correlation could be achieved by molecular genetic tests that in turn could have promising impacts in clinical management and also in prenatal diagnosis of prospect offspring. In this regard, phenotype-genotype correlation could be helpful and achieved by molecular genetic tests. This could influence the clinical management of the patients as well as prenatal diagnosis for the prospective offspring.

Novel human mitochondrial tRNA phe mutation in a patient with hearing impairment: a case study.

We present a patient with non-syndromic and sensorineural hearing impairment with a novel mitochondrial DNA transition. A 7-year-old boy showed progressive deafness. He gradually lost his hearing ability and his hearing function did not improve with hearing aids. Laboratory data revealed normal blood lactate and pyruvate levels. Genetic analyses for mitochondrial DNA and GJB2 and GJB6 genes were performed. Mitochondrial genes analysis revealed a novel heteroplasmic nucleotide substitution, m.628C>T, in the phenylalanine transfer RNA gene. This case study reveals m.628C>T transition as a novel mitochondrial nucleotide change which may be important in mitochondrial deafness.

Partial distal 10q trisomy due to de novo amplification: A new case without furrows or ridges in fingers and palms.

BACKGROUND: Here we describe a new case of partial distal 10q trisomy in a 6-year-old Iranian girl from healthy parents with mental, growth, and psychomotor retardations. METHODS: Additional clinical features include dysmorphic craniofacial features, microcephaly, bilateral hydronephrosis without heart problems, small and rotated low-set ears, bow-shaped mouth, abnormal teeth, short neck, and as a first case reported, fingers with camptodactly (i.e., without any furrows or ridges in the palms and fingers). RESULTS: Cytogenetic analysis (GTG-banding) revealed an unbalanced female karyotype with additional bands at the end of the long arm of chromosome 10, karyotype: 46,XX,dup(10)(q25q26). CONCLUSION: According to the banding pattern it is most likely that a duplication of the distal part of the long arm of chromosome 10 occurred.

A genetic assay of three patients in the same family with Holt-Oram syndrome; a case report.

Holt-Oram syndrome (HOS) is a developmental disorder inherited in an autosomal-dominant pattern. Affected organs are the heart and forelimbs with upper extremity skeletal defects and congenital heart malformation. In this study we present three cases of HOS in the same family. In one of these three individuals we detected a transition of C to T (CTG-GTT, V205V) in exon 7 of the TBX5 gene. This nucleotide change causes no amino acid change and potential pathologic effects remain unknown.

Partial trisomy 7q and monosomy 13q in a child with disorder of sex development: phenotypic and genotypic findings.

Terminal 7q duplication and terminal 13q deletion are two conditions with variable phenotypes including microcephaly, thumb a-/hypoplasia, cortical dysplasia, microphtalmia, intellectual disability and dysmorphic features. We describe a boy born to a mother with a reciprocal t (7;13) who combines both a terminal 7q33-qter duplication and terminal 13q33-qter deletion through the inheritance of a derivative chromosome 13 (der (13)). The patient presented with developmental delay, facial and non-facial dysmorphic features, hypertonia, genital abnormality and skeletal malformation but no thumb a-/hypoplasia or microphtalmia. Knowing the exact breakpoints of his chromosomal aberrations using high resolution array CGH (aCGH) and comparison of his phenotypes with those of 24 and 59 previously published cases of 7q duplication and 13q deletion, respectively, allow us to further narrow the size of the proposed critical regions for microcephaly, thumb a-/hypoplasia and hypo/hypertonia on chromosome 13.

Frequent homozygous deletion of p16/CDKN2A gene in malignant gliomas of Iranian patients.

Homozygous deletion is the main mechanism of CDKN2A gene inactivation in malignant gliomas. However different frequencies were reported for its deletion. In order to find the homozygous deletion frequency among Iranian patients, we have analyzed the status of CDKN2A gene in 40 malignant gliomas and examined their lalpha and 2 exons by comparative multiplex Polymerase Chain Reaction (PCR), using D9S171 chromosomal marker as an internal control. We found homozygous deletion in 6 out of 7 cases (85.7%) of anaplastic astrocytomas and 20 out of 33 cases (60.6%) of glioblastoma multiforme, in total 26 out of 40 cases (65%) of malignant gliomas. We also found that CDKN2A deleted patients were younger than CDKN2A non-deleted patients and that exon 2 was deleted more than exon 1alpha.

Distribution of beta-thalassemia mutations in the northern provinces of Iran.

Beta-Thalassemia (thal) is one of the most common autosomal recessive disorders in Iran. There are more than two million carriers of beta-thal and over 15,000 people affected with beta-thal major who live in Iran. Prevalent mutations were identified by examining genomic DNAs isolated from 392 blood samples of beta-thal carriers from three northern provinces of Iran. Furthermore, 172 pregnant women were analyzed from the 196 couples who requested pregnant diagnosis for beta-thal. Allele identification was carried out using routine reverse dot-blot, amplification refractory mutation system (ARMS), and genomic sequencing. The most common mutation, IVS-II-1 (GA), is followed, in order of frequency, by codon 30 (GC), frameshift codons (FSC) 8,9 (+G), FSC 22/23/24 (-AAGTTGG), IVS-I-110 (GA), IVS-I-5 (GC), IVS-II-745 (CG), IVS-I-2 (TC), FSC 8 (-AA), IVS-I,3'-end (-25 bp), IVS-I-1 (GA), FSC 36/37 (-T), IVS-I-6 (TC), FSC 5 (-CT), -28 (AC), codon 37 (GA), IVS-II-2,3 (+11/-2), -30 (TA), and -88 (CA). We have also revealed the existence of five new mutations from northern Iran, one of which (codon 37) is the first reported for Iran. Furthermore, the rate of unknown mutations is significantly reduced in our study (about 6%). These results could help with establishing a center for prenatal diagnosis, prevention, and control of thalassemia in the northern provinces of Iran.