Molecular genetics of a cohort of 635 cases of phenylketonuria in a consanguineous population.

Phenylketonuria (PKU) is an inborn error of amino acid metabolism caused by mutations in the phenylalanine hydroxylase (PAH) gene, characterized by intellectual deficit and neuropsychiatric complications in untreated patients with estimated frequency of about one in 10,000 to 15,000 live births. PAH deficiency can be detected by neonatal screening in nearly all cases with hyperphenylalaninemia on a heel prick blood spot. Molecular testing of the PAH gene can then be performed in affected family members. Herein, we report molecular study of 635 patients genetically diagnosed with PKU from all ethnicities in Iran. The disease-causing mutations were found in 611 (96.22%) of cases. To the best of our knowledge, this is the most comprehensive molecular genetics study of PKU in Iran, identifying 100 distinct mutations in the PAH gene, including 15 previously unreported mutations. Interestingly, we found unique cases of PKU with uniparental disomy, germline mosaicism, and coinheritance with another Mendelian single-gene disorder that provides new insights for improving the genetic counseling, prenatal diagnosis (PND), and/or pre-implantation genetic diagnosis (PGD) for the inborn error of metabolism group of disorders.

Human papillomavirus (HPV) prevalence and types among women attending regular gynecological visit in Tehran, Iran.

BACKGROUND: Persistent infection by HPV is now recognized as the main cause of cervical cancer. HPV prevalence data is not yet available in Iran. This study is organized to evaluate type-specific HPV prevalence and to compare it with Pap smear results among Iranian women attending regular gynecological visits. METHODS: A total of 851 women aged 18 - 65 years, attending regular gynecological visits, were retrospectively evaluated. HPV detection and genotyping was performed by use of Polymerase Chain Reaction (PCR) and Restriction Fragment Length Polymorphism (RFLP). Cytological evaluation was done by Papanicolaou method and the association between cytological results and HPV status was analyzed. RESULTS: 19 different HPV types were detected in 265 of the 851 specimens (31.1%). Overall HPV infection as well as infection with High Risk (HR) HPV types was highest in women aged 18 - 25 years and decreased with age. Type-specific prevalence of HPV-16 and 18 was 7.3% and 2.8%, respectively, and a large number of women (20.2%) were infected by HR HPV types other than HPV-16 and/or HPV-18. There was also an upward trend in the prevalence of HR HPV infections as the abnormality in cytology increased. The prevalence of HPV co-infection was 29.1% of HPV positive patients and declined from LSIL (18.2%) to HSIL (3.9%). CONCLUSIONS: Our study indicated that the burden of HPV infection among Iranian females was higher in comparison with previous estimates reported from Iran. Furthermore, higher prevalence of premalignant changes in Iranian women infected with HR HPV types, other than vaccine types, should be considered in immunization programs and development of population-specific HPV vaccines. This remarkable difference in prevalence of HPV types among previous studies, confirms our need to further investigations on epidemiology of HPV infection in Iran.

A novel PCBD gene mutation in an Iranian patient with hyperphenylalaninemia.

BACKGROUND: Neonatal screening for PKU is carried out nationally and our center is one of the referral centers for molecular analysis of PKU in Iran. Hyperphenylalaninemias are common disorders of phenyalanine catabolism. Six genes, including PAH, PTPS, DHPR, GTPCH, SR, and PCBD, independently play a role in this disorder. METHODS: A 2-year-old boy was referred to our center for genetic diagnosis of PKU. PAH gene was sequenced but no mutation was found. Using the STR based linkage mapping approach, BH4-metabolizing genes were screened. RESULT: A pattern of autozygosity by descent (ABD) suggested that the PCBD gene may be involved in this family. The PCBD gene was sequenced and a homozygous T > C substitution (X105Q) was found in the termination codon. CONCLUSIONS: Although most of the reported mutations in PCBD gene are single substitutions or premature stop codons causing a benign or transient form of BH4 deficiency, this novel mutation was found in the stop codon.

Investigation of GJB6 large deletions in Iranian patients using quantitative real-time PCR.

BACKGROUND: Hearing loss is a serious sensory defect in the world. Mutations in the GJB2 and GJB6 genes are the major causes of autosomal recessive nonsyndromic hearing loss (NSHL). Recently, three major large deletions in the GJB6 gene including del(GJB6-D13S1830), del(GJB6-D13S1854), and a > 920 kb deletion have been reported to form double heterozygosity with GJB2. This may suggest that deletions involving GJB6 may be responsible for some NSHL. METHODS: We designed a real time SYBR green-based PCR to quantify a common deleted region in GJB6 gene. The amplified region covers the area which has been seen to be deleted in all of the above reports. We selected nine families heterozygous for different mutations in GJB2 gene to investigate the presence of deletions in the GJB6 gene. The samples were run along with controls for normal hearing and heterozygous and homozygous for GJB2 mutations to optimize our method. As a reference gene or external standard, a segment of the CLCN7 gene was also quantified as well. RESULTS: We did not detect any deletion in the GJB6 gene. CONCLUSIONS: Using this method, any deletion involving GJB6 gene can be detected in a rapid and sensitive way.

Unexpected heterogeneity due to recessive and de novo dominant mutations of GJB2 in an Iranian family with nonsyndromic hearing loss: implication for genetic counseling.

Mutations in the GJB2 gene are the most common cause of nonsyndromic autosomal recessive sensorineural hearing loss (HL). A few mutations in GJB2 have also been reported to cause dominant nonsyndromic HL. Here we report a large inbred family including two individuals with nonsyndromic sensorineural hearing loss. A dominant GJB2 mutation, c.551G>A (p.R184Q), was detected in the proband, yet his parents were negative for the mutation. The second affected person had heterozygous c.35delG mutation, which was inherited from his father. Large deletions of the GJB6 gene were not detected in this family. This study highlights the importance of mutation analysis in all affected cases within a pedigree.

Development of a quantitative real-time PCR assay for detection of unknown alpha-globin gene deletions.

BACKGROUND: Alpha-Thalassemia is the most common inherited disorder of hemoglobin (Hb) synthesis in the world. Unlike beta-thalassemia, in which non-deletional mutations predominate, most of recognized alpha-thalassemia mutations include deletion of one or both alpha-globin genes. The importance of alpha-thalassemia detection is mainly due to its shared blood parameters with beta-thalassemia and its impact on discrimination between unknown alpha-thalassemia and normal HbA2 beta-thalassemia during thalassemia prevention program. MATERIALS AND METHODS: Cases with hematologic profile of low MCV, MCH, and normal HbA2 were enrolled in this study. Common alpha-globin deletional mutations including alpha(3.7)kb, alpha(4.2)kb, alpha(20.5)kb, and alpha(MED) and point mutation including 5 nt, Constant Spring (CS), and C19 were checked using either GAP-PCR or ARMS-PCR. Cases with unknown molecular defects were investigated further by direct gene sequencing. Finally, further study was done for probable unknown deletions by gene dosage analysis using real-time PCR. For this, five pairs of primers were used spanning from theta-globin gene up to the 3' upstream of alpha(2) gene. RESULTS: After validation of primers specificity and performing serial dilution analysis in order to calculate PCR efficiency, the assay was performed on normal samples and cases with known alpha-globin gene deletions as positive and negative controls, respectively. The assay was able to diagnose the control groups successfully. In 21 out of 29 unknown cases (72.4%), the assay showed various patterns of deletions in at 2 to 5 screened regions (theta gene up to the upstream of alpha2 gene). In 8 (27.6%) cases, deletions were seen in all regions. CONCLUSION: Gene dosage study by quantitative real-time PCR can be suggested as a rapid and reliable assay to screen probable carrier of alpha-thalassemia for unknown alpha-globin gene deletions.

Detection of unknown deletions in beta-globin gene cluster using relative quantitative PCR methods.

beta-Thalassemia is mainly caused by mutations involving single base substitution and small deletions. However, a considerable number of carriers are suspected to have large deletions in beta-globin gene cluster. Common strategy for identifying deletions with definite breakpoints is based on Gap PCR. There are, however, some cases with indefinite breakpoints which usually cannot be detected by this method. We developed and optimized a quantitative real-time PCR assay for copy number analysis of beta-globin gene cluster. The copy number of target fragments (i.e. beta, delta or (G)gamma-globin genes) was determined using comparative threshold cycle method. In addition, gene dosage was analyzed using multiplex ligation-dependent probe amplification (MLPA) method in all suspected carriers. Using these relative quantitative assays, normal or carrier statuses of all 26 unknown samples were successfully determined according to the ranges obtained from the ratios of normal and definite carrier samples. Interestingly, large deletions involving the entire beta-globin gene cluster were observed in six carrier individuals. This study showed that the MLPA as a preliminary screening test can be followed by SYBR Green real-time PCR for analysis of copy number variations in beta-globin gene cluster. Combination of these relative quantitative PCR methods could be an appropriate approach for accurate diagnosis of unknown beta-thalassemia deletions in routine diagnosis of beta-thalassemia mutations.