Preimplantation High-Resolution HLA Sequencing Using Next Generation Sequencing.

Hematopoietic stem cell transplantation (SCT) is the only therapeutic option in a number of heritable hematologic disorders and hematologic cancers. Many parents and families fail to find an HLA-identical donor for their affected family member. In such cases, conceiving for a "savior baby" remains the only option, especially in countries without access to national registries. By means of next generation sequencing (NGS) techniques, in a single experiment on single-cell products of in vitro fertilization, a healthy HLA-identical embryo can be implanted in the uterus of a concerned mother. The patient can therefore benefit from cord blood SCT along with confirming that the fetuses are not suffering from the heritable disorder. This study is an attempt to study the feasibility of preimplantation HLA sequencing on single blastomeres using NGS. Two couples who had previously undergone preimplantation genetic diagnosis of ß-thalassemia and their overall 10 embryos were studied and their 5 HLA loci were typed in high resolution through multiple displacement amplification and NGS of single cells. For 88.9% of the 90 HLA alleles, conclusive HLA typing in 4 digit sets was made. HLA alleles were typed; 1 ambiguity in the allelic group and 4 ambiguities in the protein level were observed that were then unraveled by haplotype analysis. Amplification efficiency was 93.3% with an allele drop-out (ADO) rate of 22.2% (6 alleles dropped from a maximum of 27 possible ADOs). In this study the feasibility of a new method of preimplantation HLA sequencing via combining the state-of-the-art techniques used in single-cell whole genome amplification, preimplantation genetic diagnosis, and high-resolution HLA typing by NGS has been shown. This method can make preimplantation HLA sequencing a practicable technique in families desperate for an HLA-matched donor.

A novel ACVR1 mutation detected by whole exome sequencing in a family with an unusual skeletal dysplasia.

"Disorganized Development of Skeletal Component" (DDSC) is a group of genetic skeletal dysplasia, caused by mutations in 9 genes including ACVR1. The most known ACVR1-related disorder is fibrodysplasia ossificans progressiva (FOP). FOP variants are frequently encountered with diagnostic challenges due to overlapping clinical manifestations and variable severity. Application of high throughput sequencing methods can overcome these limitations by simultaneous investigation of the entire ACVR1 gene together with other genes involved in disorders with similar manifestations. A 33-year-old man with an unusual skeletal dysplasia and no previous clinical diagnosis is presented in this study. Whole exome sequencing detected a novel c.737T>A (p.Phe246Tyr) mutation in ACVR1 gene. Detailed targeted variant analysis in 226 known genes associated with genetic skeletal disorders together with more specific targeted analysis in 9 genes associated with DDSC ruled out the involvement of other investigated genes. Proband's phenotypically normal father and brother had the same mutation in whom subsequent investigations showed subclinical radiographic findings. The clinical manifestations, the disease course, and the molecular findings of involvement of ACVR1 gene in this family are suggestive of "FOP variant" or an unusual ACVR1-related skeletal dysplasia. Moreover, this report has demonstrated the critical role of the next generation sequencing technique in characterizing such a rare disorder with variable and even no clinical manifestations, providing the opportunity for effective preventive measures such as preimplantation genetic diagnosis.

Prenatal Diagnosis of Tyrosinemia Type 1 Using Next Generation Sequencing.

Prenatal diagnosis using conventional molecular genetic techniques may be encountered with some limitations when the disease causing mutation is unknown. Here, we report on prenatal diagnosis of tyrosinemia in a family with consanguineous marriage and two affected children in whom no disease causing mutation had been identified before pregnancy. Mutation analyses of three genes associated with tyrosinemia including FAH, TAT and HPD were carried out in the fetal DNA sample using Next Generation Sequencing. A heterozygous nonsense mutation (p.Arg237Ter) in FAH gene was detected in the fetus. Further investigations suggested that the fetus was carrier of tyrosinemia type 1. This study demonstrates the successful application of Next Generation Sequencing in prenatal diagnosis, when the time is a limiting factor, more than one (especially large) responsible genes are involved, a "founder" or a "previously detected" mutation is not present and hence the conventional molecular genetic investigations cannot be employed.

Her2 amplification status in Iranian breast cancer patients: comparison of immunohistochemistry (IHC) and fluorescence in situ hybridisation (FISH).

INTRODUCTION: Her2/neu is a biomarker which is amplified and/or overexpressed in a subset of breast cancer patients who are eligible to receive trastuzumab. Her-2 gene amplification analysed by fluorescence in situ hybridisation (FISH) and/or protein over-expression detected by immunohistochemistry (IHC) are the two main methods used to detect Her-2 status in clinical practice. The concordance rate between the two techniques is controversial. METHODS: FISH analysis were performed on 104 tumoural samples from breast cancer patients with known IHC results to determine the Her2 gene status. The FISH/IHC analyses results were then compared and the concordance rate was determined. RESULTS: Her2 gene amplification was detected in 0 of IHC score 1+, 24/86 (27.91%) 2+, and 8/13 (61.54%) 3+. The IHC and FISH results concordance rates were 100%, 27.9%, and 61.5% for IHC scores of 1+, 2+, and 3+ respectively. CONCLUSION: The results of this study suggest that IHC 1+ should be considered as negative while IHC 2+ results need further confirmative analysis by FISH. Further quality control and standardization of IHC technique are required to improve the concordance rate between the two methods.

Human papillomavirus in saliva of patients with oral squamous cell carcinoma.

OBJECTIVE: The aim of this study was to evaluate the presence of human papillomavirus (HPV) in saliva rinses of patients with oral squamous cell carcinoma and to analyze the possibility of using saliva as a diagnostic method for screening high-risk patients. STUDY DESIGN: The saliva sample of 22 patients with oral squamous cell carcinoma and 20 age-sex matched healthy controls were obtained. The presence of HPV 6, 11, 16, 18, 31, and 33 was evaluated by polymerase chain reaction (PCR). RESULTS: In 40.9% of the patients and in 25% of the controls, the saliva was shown to be positive for HPV. In 27.3% of the patients and in 20% of the controls, the saliva was shown to be positive for HPV16; and none of the controls, except one patient was shown to be positive for HPV 18. Neither patients nor controls were positive for HPV 31 and 33. These differences were not statistically significant. CONCLUSIONS: The results of this study were unable to support the detection of HPV in saliva rinses as a diagnostic method for OSCC.

Chromosomal radiosensitivity in patients with common variable immunodeficiency.

Common variable immunodeficiency (CVID) is a heterogeneous group of primary immunodeficiency disorders. In addition to recurrent infections and autoimmunity, cancers are more prevalent in these patients than the normal population. Increased radiosensitivity may be a reason for the increased malignancies. To analyze chromosomal radiosensitivity of CVID patients, lymphocytes were cultured from 20 CVID patients. After irradiation (50, 100 cGy), metaphases were evaluated for chromosomal aberrations. Results were compared in patients, healthy individuals, and ataxia telangiectasia as positive controls. Before irradiation there was no difference between groups of patients, but after radiation, the incidence of all kinds of aberrations was higher in the CVID patients and this was statistically significant at 100 cGy (P<0.05). CVID patients appear to be susceptible to in vitro irradiation. These patients should be protected from unnecessary radiographic diagnostic and therapeutic procedures. Also, radiosensitivity may help classifying CVID patients.

Ataxia-telangiectasia in Iran: clinical and laboratory features of 104 patients.

Ataxia-telangiectasia is a multisystem disorder characterized by progressive neurologic impairment, variable immunodeficiency, impaired organ maturation, x-ray hypersensitivity, oculocutaneous telangiectasia, and a predisposition to malignancy. To evaluate clinical and immunologic features of Iranian patients with ataxia-telangiectasia, the records of 104 patients with ataxia-telangiectasia (54 male, 50 female) with the age range of 1.6-23.5 years were reviewed. The Iranian Primary Immunodeficiency Registry was used as the data source. Progressive ataxia was seen in all the patients. Other symptoms were eye movement disorders (n = 84), slurred speech (n = 70), mental retardation (n = 10), and ocular (n = 87) and cutaneous (n = 73) telangiectasia. Three patients developed leukemia and lymphoma, and 17 patients had family history of malignancy. Positive correlation was seen between clinical immunologic symptoms and immunoglobulin deficiencies (P = 0.004). The predominant infections were sinopulmonary and acute and recurrent infections (78 cases). Infections included pneumonia (56 patients), otitis media (34 patients), and sinusitis (50 patients). Average serum alpha-fetoprotein level was 149 +/- 137 ng/dL. The incidence of ataxia-telangiectasia in Iran is high, possibly due to familial marriages. Treatment should be focused on supportive management to prolong survival.

Prevalence of human papillomavirus genotypes in women with normal and abnormal cervical cytology in Iran.

INTRODUCTION: HPV infection has a prime etiologic role in development and progression of cervical cancer, one of the most frequent forms of cancer among women in developing countries. This study was designed to determine the most prevalent HPV genotypes in women with normal and abnormal cervical cytology in Iran. MATERIALS AND METHODS: Samples from134 patients, including 127 who attended gynecology clinics and 7 with solid cervical tumors were used. All 127 patients underwent routine Pap tests for cytological evaluation and at the same visit a sample of cervical epithelial cells was obtained by scraping the cervix osteum. In each case HPV infection was primarily evaluated by PCR using GP 5/6 primers and then subtyping was performed in proved infected samples with specific primers for HPV 16, 18, 31, 33, 11 and 6. After cytological evaluation, 50 patients with abnormal Pap tests were categorized as the abnormal group and the remaining 77 patients as the normal group. RESULTS: In the normal group, HPV infection was established in 10 cases (13% infection rate), while 30 HPV positive cases were discovered in the abnormal group (60% infected). The most prevalent genotypes among the infected samples were HPV 16 (76%), HPV18 (12.7%) and HPV11/6 (8.5%). Moreover, all 7 tumor samples were positive for HPV general primers of which, 5 samples were infected with HPV 16, two were co-infected with HPV16,18 and HPV16,31 genotypes and one was infected with HPV 18. CONCLUSIONS: Infection with HPV 16 was found to be significantly higher in abnormal group in comparison with normal group (42% vs. 11.6%, P value <0.005), likewise HPV18 genotypes were proved to be more prevalent in abnormal group (8% vs. 0%, P value <0.05). No significant relation between other HPV genotypes and pathologic cervical changes was obtained. According to our study high rates of infection with HPV genotypes in sexually active Iranian women makes molecular investigation for HPV16 and 18 very essential in clinical approaches to patients with proven dysplasia in their screening tests and also for those patients with borderline (i.e. ASCUS) or incongruous pathology reports. Larger studies are required to determine the most appropriate vaccine with highest protection in Iranian women.