Gene Panel Testing in Hereditary Breast Cancer.

BACKGROUND: Breast cancer (BC) is a highly complex, heterogeneous and multifactorial disease and is the most commonly diagnosed cancer and the leading cause of cancer-related mortality in women worldwide. Family history and genetic mutations are important risk factors for BC. While studies in twins have estimated that about 10%-30% of BC are due to hereditary factors, only 4%-5% of them are due to mutations in BRCA1 or BRCA2 genes. Our aim was to investigate the role of other BC genes in familial BC among the Iranian population. METHODS: We selected 61 BC patients who were wild-type for BRCA1 and BRCA2 mutations but who met the criteria for hereditary BC based on the American College of Medical Genetics and Genomics (ACMG) and the National Comprehensive Cancer Network (NCCN) guidelines. We performed targeted sequencing covering the exons of 130 known cancer susceptibility genes based on the Cancer Gene Census list. RESULTS: We found seven mutations in seven known BC susceptibility genes (RAD50, PTEN, TP53, POLH, DKC1, WRN and CHEK2) in seven patients including two pathogenic frameshift variants in RAD50 and WRN genes, four pathogenic missense variants in TP53, PTEN, POLH, and DKC1 genes and a pathogenic splice donor variant in the CHEK2 gene. The presence of all these variants was confirmed by Sanger sequencing and Gap reverse transcription-polymerase chain reaction (RT-PCR) for the splice variant. In silico analysis of all of these variants predicted them to be pathogenic. CONCLUSION: Panel testing of BC patients who met the established criteria for hereditary BC but who were negative for BRCA1/2 mutations provided additional relevant clinical information for approximately 11.5% of the families. Our findings indicate that next generation sequencing (NGS) is a powerful tool to investigative putative mutagenic variants among patients who meet the criteria for hereditary BC, but with negative results on BRCA1/2 testing.

First Report of a Dominantly Inherited ß-Thalassemia Caused by a Novel Elongated ß-Globin Chain.

A distinct set of mutations on the ß-globin gene leads to dominantly inherited ß-thalassemia (ß-thal) that is associated with a disease phenotype in a single mutant copy. We described molecular and hematological characteristics of a novel elongated ß-globin chain in combination with a known hemoglobin (Hb) variant (N-Baltimore or HBB: c.286A>G) in cis. The highly unstable Hb variant caused typical features of ß-thal major (ß-TM) or ß-thal intermedia (ß-TI) in two members of a family depending on their α-globin genotypes. The ß mutant allele of the mother was transmitted in an autosomal dominant fashion to her daughter. They resemble severe forms of ß-thal due to ineffective erythropoiesis. Taken together with previously published data, this result indicates that a dominant form of ß-thal should be regarded as a phenotypic term of hemoglobinopathies caused by ß chain variants that are highly unstable.

Characterization of Homozygous Hb Setif (HBA2: c.283G>T) in the Iranian Population.

Hemoglobin (Hb) variants are abnormalities resulting from point mutations in either of the two α-globin genes (HBA2 or HBA1) or the ß-globin gene (HBB). Various reports of Hb variants have been described in Iran and other countries around the world. Hb Setif (or HBA2: c.283G>T) is one of these variants with a mutation at codon 94 of of the α2-globin gene that is characterized in clinically normal heterozygous individuals. We here report clinical and hematological findings in two homozygous cases of Iranian origin for this unstable Hb variant.

Identification of Mutations Causing Aberrant Termination and Deficient Splice Donor Site on the HBA1 Gene.

α-Thalassemia (α-thal) is a common genetic disorder in Iran and many parts of the world. Genetic defects on the α-globin gene cluster can result in α-thal that may develop a clinical phenotype varying from almost asymptomatic to a lethal hemolytic anemia. In the present study, four Iranian individuals with hypochromic microcytic anemia, who revealed none of the known mutations responsible for α-thal, were subjected for further investigations. The thalassemic phenotype of these patients resulted from abnormal RNA splicing sites owing to a missense at the splice donor site, a truncated protein or hemoglobin (Hb) variants as a result of two different substitutions on the α1-globin gene. The clinical presentation of mild anemia in these individuals showed the contribution of these novel mutations in α-thal in spite of the dominantly expressed α2-globin gene. This study describes hematological manifestations of subjects carrying some novel mutations comparable to the reported phenotype of α(+)-thal trait.

Point mutations which should not be overlooked in Hb H disease.

BACKGROUND: Hb H disease is an alpha-thalassemia (α-thal) syndrome characterized by chronic hemolytic anemia that occurs when three of total four α-globin genes lost their function due to completely deletions or different kind of mutations. OBJECTIVE: We here described 66 patients who have been diagnosed for Hb H disease during the last five years in our center. The genotypes involving point mutations present more severe phenotype than deletional forms that make them of primary important to health management. STUDY DESIGN: Hb H subjects carry different α-globin genotypes including deletional and non-deletional mutations showing heterogenous clinical manifestations. RESULTS: The Hb H patients presenting a wide range of phenotype carried different deletional, non-deletional mutations or compound heterozygosity of them. CONCLUSION: We emphasize the importance of some point mutations responsible for more severe form of Hb H disease in Iranian population and the necessity for consideration of prenatal diagnosis (PND) in high-risk couples.

Copy number variations of six and seven α-globin genes in a family with intermedia and major thalassemia phenotypes.

BACKGROUND: Copy number variations in α-globin genes are results of unequal crossover between homologous segments in the α-globin gene cluster that misalign during the meiosis phase of the gametogenesis process. Reduction or augmentation of α-globin genes leads to imbalance of α/ß chains in hemoglobin tetramer and consequently attenuate or worsen the ß-thal clinical symptoms, respectively. OBJECTIVE: Multiplications in α-globin genes have been found in some populations, justifying unexpected severe phenotype of ß-thal carriers. STUDY DESIGN: Unexpected severe phenotype in the family members may result from coexistence of extra α-globin genes, which is an important factor in the causation of thalassemia intermedia and major in heterozygous ß-thalassemia. RESULTS: We described different multiplications in α-globin locus in an Iranian family with one, two or three extra α-globin genes (ααα/αα, αααα/αα and αααα/ααα). CONCLUSION: The excess α-globin gene/genes cause increment in ß/α chain imbalance and leads to worsening pathophysiology and clinical severity of ß-thalassemia carriers.

Mutations on the α2-Globin Gene That May Trigger α(+)-Thalassemia.

In the present study, a total of 11 individuals with hypochromic microcytic anemia who did not reveal the most common α-thalassemia (α-thal) deletions or mutations, were subjected to more investigations by DNA sequencing of the α-globin genes. Seven novel nondeletional α-thal mutations localized on the α2-globin gene in the heterozygous state were identified. These mutations either corrupted regulatory splice sites and consequently affected RNA processing or created unstable hemoglobin (Hb) variants. The mutations described here produced globin gene variants that lead to amino acid changes in critical regions of the globin chain. The clinical presentation of most patients was a persistent mild microcytic anemia similar to an α(+)-thal. In the last decade, numerous α-globin mutations have been observed leading to an α-thal phenotype and these studies have been considered to be important as discussed here.

Homozygosity for the AATAAA > AATA- - Polyadenylation Site Mutation on the α2-Globin Gene Causing Transfusion-Dependent Hb H Disease in an Iranian Patient: A Case Report.

We describe a case of Hb H disease associated with homozygosity for a two nucleotide deletion in the polyadenylation signal of the α2-globin gene (HBA2: c.*93_*94delAA). The patient, a 27-year-old son of a consanguineous couple, needs regular blood transfusions every 6 months.

Interaction of an α-Globin Gene Triplication with ß-Globin Gene Mutations in Iranian Patients with ß-Thalassemia Intermedia.

The 3.7 kb triplicated α-globin gene (ααα(anti 3.7)) mutation has been found in most populations. It results from an unequal crossover between misaligned homologous segments in the α-globin gene cluster during meiosis. The pathophysiology and clinical severity of ß-thalassemia (ß-thal) are associated with the degree of α chain imbalance. The excess of α-globin chains plays an important role in the pathophysiology of ß-thal. When heterozygous/homozygous ß-thal coexists with an α gene numerical alteration, the clinical and hematological phenotype of thalassemia could change to mild anemia in case of an α deletion (-α/αα) or severe anemia in the case of an α triplication (αα/ααα). The coexistence of an ααα(anti 3.7) triplication is considered an important factor in the severity of ß-thal, exacerbating the phenotypic severity of ß-thal by causing more globin chain imbalance. This study shows that the ααα(anti 3.7) triplication is an important factor in the causation of ß-thal intermedia (ß-TI) in heterozygous ß-thal. This type of phenotype modification has rarely been observed and reported in the Iranian population. Here we report the coinheritance of a triplicated α-globin gene arrangement and heterozygous/homozygous ß-thal in 23 cases, presenting with a ß-TI or ß-thal major (ß-TM) phenotype. Some of these patients were considered to have a mild ß-TI phenotype as they needed no blood transfusions; some occasionally received blood transfusions in their lifetime (for example on delivery) but some are dependent on regular blood transfusions (every 20 to 40 days). Our study was focused on the importance of detecting the α-globin gene triplication in genotype/phenotype prediction in Iranian thalassemia patients.

Prenatal Screening for Aneuploidies Using QF-PCR and Karyotyping: A Comprehensive Study in Iranian Population.

BACKGROUND: We have investigated the efficacy of QF-PCR for the prenatal recognition of common aneuploidy and compared our findings with cytogenetic results in our laboratories. METHODS: A total of 4058 prenatal samples (4031 amniotic fluid and 27 chorionic villous samples) were analyzed by QF-PCR using several selected STR markers together with amelogenin. Results were compared to those obtained by conventional cytogenetic analysis. RESULTS: We detected 139 (3.42%) numerical abnormalities in our subjects by QF-PCR. Concordant QF-PCR and karyotype results were obtained in 4001 (98.59%) of the samples. An abnormal karyotype associated with adverse clinical outcome undetected by QF-PCR was found in 16.66% (n = 28) of samples. Using QF-PCR alone, we were able to detect abnormalities in 98.59% of all referred families; however the karyotyping results improved the detection rate to 99.85% of the referred cases. Individuals with neonatal screening result with 1:10 risk ratio showed 11.29% abnormal karyotype while this number was 2.16% in mothers with risk ratio of 1:250 or less. CONCLUSION: In countries where large scale conventional cytogenetic is hampered by its high cost and lack of technical expertise, QF-PCR may be used as the first line of screening for detection of chromosomal abnormalities. We also recommend QF-PCR for all the families that are seeking prenatal diagnosis of single gene disorders aneuploidies screening to be added to their work up.

Hb Dartmouth (HBA2: c.200T>C): An α2-Globin Gene Associated with Hb H Disease in One Homozygous Patient.

Hb H (ß4) disease is caused by deletion or inactivation of three out of four α-globin genes. A high incidence of Hb H disease has been reported all over the world. There is a wide spectrum of phenotypic presentations, from clinically asymptomatic to having significant hepatosplenomegaly and requiring occasional or even regular blood transfusions, even more severe anemia, Hb Bart's (γ4) hydrops fetalis syndrome that can cause death in the affected fetuses late in gestation. We here present a case who was diagnosed with Hb H disease that represents a new genotype for this hereditary disorder. Hb Dartmouth is a variant caused by a missense mutation at codon 66 of the α2-globin gene (HBA2: c.200T>C), resulting in the substitution of leucine by proline. We here emphasize the importance of this point mutation involving Hb H disease and also the necessity for prenatal diagnosis (PND) for those who carry this point mutation in the heterozygous state.

A 21 Nucleotide Duplication on the α1- and α2-Globin Genes Involves a Variety of Hypochromic Microcytic Anemias, From Mild to Hb H Disease.

α-Thalassemia (α-thal) is a common genetic disorder in Iran and many parts of the world. Genetic defects in the α-globin gene cluster can result in α-thal that may develop into a clinical phenotype varying from almost asymptomatic to a lethal hemolytic anemia. Loss of one functional α gene, indicated as heterozygous α(+)-thal, shows minor hematological abnormalities. Homozygosity for α(+)- or heterozygosity for α(0)-thal have more severe hematological abnormalities due to a markedly reduced α chain output. At the molecular level, the absence of three α-globin genes resulting from the compound heterozygous state for α(0)- and α(+)-thal, lead to Hb H disease. Here we present a 21 nucleotide (nt) duplication consisting of six amino acids and 3 bp of intronic sequence at the exon-intron boundary, in both the α-globin genes, detected by direct DNA sequencing. This duplication was identified in three patients originating from two different Iranian ethnic groups and one Arab during more than 12 years. The clinical presentation of these individuals varies widely from a mild asymptomatic anemia (heterozygote in α1-globin gene) to a severely anemic state, diagnosed as an Hb H individual requiring blood transfusion (duplication on the α2-globin gene in combination with the - -(MED) double α-globin gene deletion). The third individual, who was homozygous for this nt duplication on the α1-globin gene, showed severe hypochromic microcytic anemia and splenomegaly. In the last decade, numerous α-globin mutations have demonstrated the necessity of prenatal diagnosis (PND) for α-thal, and this study has contributed another mutation as important enough that needs to be considered.

Novel mutations responsible for α-thalassemia in Iranian families.

α-Thalassemia (α-thal) is usually caused by deletions on the α-globin gene cluster and the role of point mutations is less well investigated. In the present study, a total of 1048 individuals with hypochromic microcytic anemia, who did not present the most common α-thal deletions, were referred for α-globin gene DNA sequencing. The nucleotide changes were studied and a total of five new mutations was identified, of which three were located on the α2 gene [codon7 (Lys→Stop), codon 34 (Leu→Pro) and codon 83 (Leu→Arg)] and two on the α1 gene [IVS-I-116 (A>G) and codon 44 (+C)]. These novel mutations not only explain new findings by molecular analysis of the α-globin gene but also have clinical importance due to their changes in α-globin production in means of decreased hemoglobin (Hb) related values. Moreover, considerations of its role in combination with other mutations, and the possibility of causing Hb H (ß4) are yet to be studied.

The spectrum of α-thalassemia mutations in the Kurdish population of Northeastern Iraq.

In an attempt to determine the spectrum of α-thalassemia (α-thal) mutations in the Kurdish population of Northeastern (NE) Iraq, a total of 101 unrelated adults with unexplained hypochromia and/or microcytosis were enrolled. α-Thalasssemia mutations were characterized by gap polymerase chain reaction (gap-PCR), multiplex PCR (m-PCR) and reverse hybridization and sequencing for both α genes. A total of nine α-thal mutations were characterized including four deletional ones: -α(3.7) (rightward), - -(MED-I), -(α)(20.5), -α(4.2) (leftward) and five nondeletional ones: α(polyA1)α, αα(Adana), α(-5 nt)α, α(CS)α and α(polyA2)α. These determinants were arranged in 12 different genotypes, the most frequent of which were: -α(3.7)/αα, - -(MED-I)/αα, -α(3.7)/-α(3.7), α(polyA1)α/αα, αα(Adana)/αα and -(α)(20.5)/αα. This pattern is similar to that reported in Turkey, western (W) Iran, Cyprus and Greece, and to some extent, different from the pattern observed in the Arabian Peninsula.

Genotype-phenotype correlation in Iranian patients with Hb H disease.

Thalassemia is the most common genetic disorder in Iran. Some α-globin genotypes leading to Hb H disease may cause severe anemia and dependence on regular blood transfusions. In this study, 40 patients were analyzed for the molecular basis and the genotype-phenotype correlation of Hb H disease in Iran. α-Globin molecular analysis was performed by polymerase chain reaction (PCR) followed by agarose gel electrophoresis, reverse hybridization test strips or DNA sequencing. The most frequently observed α-globin genotypes were -α(3.7)/- -(MED) in 10 patients (25%), - -(20.5)/α(-5nt)α in six patients (15%) and - -(20.5)/-α(3.7) in four patients (10%). A subset of the identified Hb H genotypes, including - -(MED)/α(CS)α, - -(MED)/α(PolyA2)α and α(CS)α/α(CS)α, was associated with a need for regular or irregular blood transfusions. Our findings provide a basis for predicting phenotype severity by identifying the Hb H genotype and making more selective decisions for prenatal diagnosis.

Screening of Iranian thalassemic families for the most common deletions of the beta-globin gene cluster.

Deltabeta-thalassemia (thal) is a disorder, characterized by increased levels of fetal hemoglobin (Hb F) in adult life. A considerable number of deletions of variable size and position in the beta-globin gene cluster are associated with the clinical manifestation of deltabeta-thal. In this study we have determined the presence of the eight most common deletions in Iranian patients. Thirty-two patients from 19 families were referred to the Kariminejad-Najmabadi Pathology and Genetics Center, Tehran, Iran (a private genetics center), within the past 3 years with elevated levels of Hb F and low mean corpuscular volume (MCV). After obtaining their informed consent, DNA was extracted from whole blood by the salting-out method. Detection of eight deletions was performed using polymerase chain reaction (PCR). These deletions included the hereditary persistence of fetal Hb (HPFH) 1 (Black) and 3 (Indian), Spanish (-114 kb), Sicilian (-13,377 bp), Chinese (G)gamma((A)gammadeltabeta)(0)-thal (-100 kb), Asian-Indian inversion-deletion (G)gamma((A)gammadeltabeta)(0)-thal, and the Turkish form of inversion-deletion (deltabeta)(0)-thal, as well as the Hbs Lepore, which are characterized by unequal crossovers between the delta- and beta-globin genes. We found the Sicilian (-13,377 bp) and Hb Lepore deletions as well as the Asian-Indian (G)gamma((A)gammadeltabeta)(0)-thal in 11 (57.89%), three (15.78%) and five (26.31%) families, respectively. None of the aforementioned deletions were found in one of the patients. This is the first study of the deletions involved in deltabeta-thal in Iranian patients. Our study highlights the importance of detecting these mutations for prenatal diagnosis carrier detection and genotype/phenotype prediction.