Novel splicing variant and gonadal mosaicism in DYRK1A gene identified by whole-genome sequencing in multiplex autism spectrum disorder families.

Autism spectrum disorder (ASD) is a complex neurodevelopmental condition with considerable genetic heterogeneity. The disorder is clinically diagnosed based on DSM-5 criteria, featuring deficits in social communication and interaction, along with restricted and repetitive behaviours. Here, we performed whole-genome sequencing (WGS) on four individuals with ASD from two multiplex families (MPX), where more than one individual is affected, to identify potential single nucleotide variants (SNVs) and structural variants (SVs) in coding and non-coding regions. A rigorous bioinformatics pipeline was employed for variant detection, followed by segregation analysis. Our investigation revealed an unreported splicing variant in the DYRK1A gene (c.-77 + 2T > C; IVS1 + 2T > C; NM_001396.5), in heterozygote form in two affected children in one of the families (family B), which was absent in the healthy parents and siblings. This finding suggests the presence of gonadal mosaicism in one of the parents, representing the first documented instance of such inheritance for a variant in the DYRK1A gene associated with ASD. Furthermore, we identified a 50 bp deletion in intron 9 of the DLG2 gene in two affected patients from the same family, confirmed by PCR and Sanger sequencing. In Family A, we identified potential candidate variants associated with ASD shared by the two patients. These findings enhance our understanding of the genetic landscape of ASD, particularly in MPX families, and highlight the utility of WGS in uncovering novel genetic contributions to neurodevelopmental disorders.

Expression of ERBB gene family in females with breast cancer and its correlation with clinicopathological characteristics of the disease.

BACKGROUND: Breast cancer (BC) is the most prevalent and fatal cancer in women. Given that there are very few studies investigating the overexpression of four members of ERBB genes, we decided to investigate the correlation between these four genes with clinicopathological characteristics in breast cancer cases. METHODS: Tumoural tissue of 50 patients with sporadic invasive ductal BC was recruited. Also, control samples were provided from adjacent non-cancerous tissues (ANCTs) of the same patients. The expression of four ERBB genes was evaluated by real-time PCR and its correlation with clinicopathological characteristics was assessed. RESULTS: Only ERBB2 (HER2) was overexpressed in tumoural tissue compared with ANCTs. Our data showed a significant relationship between ERBB1 overexpression with triple-negative tumors, ER, and PR negativity (P < 0.05). Also, ERBB2 overexpression indicated a significant correlation with several pathological characteristics such as age < 50, tumor size larger than 2 cm, early and advanced stages, negative involved lymph nodes, luminal B, triple-negative, ERBB2-enrich, estrogen receptor (ER) and progesterone receptor (PR) negative tumors, Ki-67 mutation more than 15%, and finally HER2/neu immunohistochemistry (IHC) positive and intermediate (P < 0.05). Moreover, this study demonstrated that ERBB4 overexpression had a significant correlation with tumor size smaller than 2 cm, grade I and II tumors (early-stage tumors), luminal A, ER and PR positive tumors, HER-2/neu IHC intermediate, and tumors that had a Ki-67 mutation lower than 15% (P < 0.05). Besides, our analysis showed a significant correlation between the expression of ERBB1 with ERBB2 and ERBB3 with ERBB4 (P < 0.05). CONCLUSIONS: Our findings showed a significant relationship between unfavorable clinicopathological characteristics with ERBB1 and ERBB2 overexpression, but overexpression of ERBB4 was correlated with favorable outcomes.

BOD1 Is Required for Cognitive Function in Humans and Drosophila.

Here we report a stop-mutation in the BOD1 (Biorientation Defective 1) gene, which co-segregates with intellectual disability in a large consanguineous family, where individuals that are homozygous for the mutation have no detectable BOD1 mRNA or protein. The BOD1 protein is required for proper chromosome segregation, regulating phosphorylation of PLK1 substrates by modulating Protein Phosphatase 2A (PP2A) activity during mitosis. We report that fibroblast cell lines derived from homozygous BOD1 mutation carriers show aberrant localisation of the cell cycle kinase PLK1 and its phosphatase PP2A at mitotic kinetochores. However, in contrast to the mitotic arrest observed in BOD1-siRNA treated HeLa cells, patient-derived cells progressed through mitosis with no apparent segregation defects but at an accelerated rate compared to controls. The relatively normal cell cycle progression observed in cultured cells is in line with the absence of gross structural brain abnormalities in the affected individuals. Moreover, we found that in normal adult brain tissues BOD1 expression is maintained at considerable levels, in contrast to PLK1 expression, and provide evidence for synaptic localization of Bod1 in murine neurons. These observations suggest that BOD1 plays a cell cycle-independent role in the nervous system. To address this possibility, we established two Drosophila models, where neuron-specific knockdown of BOD1 caused pronounced learning deficits and significant abnormalities in synapse morphology. Together our results reveal novel postmitotic functions of BOD1 as well as pathogenic mechanisms that strongly support a causative role of BOD1 deficiency in the aetiology of intellectual disability. Moreover, by demonstrating its requirement for cognitive function in humans and Drosophila we provide evidence for a conserved role of BOD1 in the development and maintenance of cognitive features.

Deep sequencing reveals 50 novel genes for recessive cognitive disorders.

Common diseases are often complex because they are genetically heterogeneous, with many different genetic defects giving rise to clinically indistinguishable phenotypes. This has been amply documented for early-onset cognitive impairment, or intellectual disability, one of the most complex disorders known and a very important health care problem worldwide. More than 90 different gene defects have been identified for X-chromosome-linked intellectual disability alone, but research into the more frequent autosomal forms of intellectual disability is still in its infancy. To expedite the molecular elucidation of autosomal-recessive intellectual disability, we have now performed homozygosity mapping, exon enrichment and next-generation sequencing in 136 consanguineous families with autosomal-recessive intellectual disability from Iran and elsewhere. This study, the largest published so far, has revealed additional mutations in 23 genes previously implicated in intellectual disability or related neurological disorders, as well as single, probably disease-causing variants in 50 novel candidate genes. Proteins encoded by several of these genes interact directly with products of known intellectual disability genes, and many are involved in fundamental cellular processes such as transcription and translation, cell-cycle control, energy metabolism and fatty-acid synthesis, which seem to be pivotal for normal brain development and function.

Mutation of the conserved polyadenosine RNA binding protein, ZC3H14/dNab2, impairs neural function in Drosophila and humans.

Here we report a human intellectual disability disease locus on chromosome 14q31.3 corresponding to mutation of the ZC3H14 gene that encodes a conserved polyadenosine RNA binding protein. We identify ZC3H14 mRNA transcripts in the human central nervous system, and we find that rodent ZC3H14 protein is expressed in hippocampal neurons and colocalizes with poly(A) RNA in neuronal cell bodies. A Drosophila melanogaster model of this disease created by mutation of the gene encoding the ZC3H14 ortholog dNab2, which also binds polyadenosine RNA, reveals that dNab2 is essential for development and required in neurons for normal locomotion and flight. Biochemical and genetic data indicate that dNab2 restricts bulk poly(A) tail length in vivo, suggesting that this function may underlie its role in development and disease. These studies reveal a conserved requirement for ZC3H14/dNab2 in the metazoan nervous system and identify a poly(A) RNA binding protein associated with a human brain disorder.

Phenotypical characterization of 13q deletion syndrome: Report of two cases.

Patients with 13q deletion syndrome are characterized with different phenotypical features depending on the size and location of the deleted region on chromosome 13. These patients fall into three groups: In Group 1, deleted region is in the proximal and does not extend into q32; in Group 2, deleted region involves proximal to the q32 and in Group 3 q33-q34 is deleted. We present two cases with 13q syndrome with two different deleted region and different severity on clinical features: One case with interstitial deletion belongs to the Group 1 with mild mental retardation and minor malformations and the other case with terminal deletion belongs to Group 3 with moderate to severe mental retardation and major malformations.

Genomic characterization of some Iranian children with idiopathic mental retardation using array comparative genomic hybridization.

BACKGROUND: Mental retardation (MR) has a prevalence of 1-3% and genetic causes are present in more than 50% of patients. Chromosomal abnormalities are one of the most common genetic causes of MR and are responsible for 4-28% of mental retardation. However, the smallest loss or gain of material visible by standard cytogenetic is about 4 Mb and for smaller abnormalities, molecular cytogenetic techniques such as array comparative genomic hybridization (array CGH) should be used. It has been shown that 15-25% of idiopathic MR (IMR) has submicroscopic rearrangements detectable by array CGH. In this project, the genomic abnormalities were investigated in 32 MR patients using this technique. MATERIALS AND METHODS: Patients with IMR with dysmorphism were investigated in this study. Karyotype analysis, fragile X and metabolic tests were first carried out on the patients. The copy number variation was then assessed in a total of 32 patients with normal results for the mentioned tests using whole genome oligo array CGH. Multiple ligation probe amplification was carried out as a confirmation test. RESULTS: In total, 19% of the patients showed genomic abnormalities. This is reduced to 12.5% once the two patients with abnormal karyotypes (upon re-evaluation) are removed. CONCLUSION: The array CGH technique increased the detection rate of genomic imbalances in our patients by 12.5%. It is an accurate and reliable method for the determination of genomic imbalances in patients with IMR and dysmorphism.

A novel mutation in MCPH1 gene in an Iranian family with primary microcephaly.

Primary microcephaly (MCPH) is a genetic disorder in which affected individuals present with a head circumference 3 standard deviations (SDs) below the age- and sex-related mean and is accompanied by mental retardation without further associated malformations. Here we report a patient with sporadic MCPH from Northwest of Iran who was investigated for MCPH1 locus. Clinical examination and karyotype analyses were performed and microsatellite based mapping was done by using flanking and intragenic short tandem repeat (STR) markers for MCPH1 locus. For these markers the affected individual was homozygote and the parents were heterozygote. According to this pattern of allele sharing and also the cytogenetic findings, mutation screening of Microcephalin gene was performed and subsequent sequencing revealed a novel mutation in Microcephalin gene.

Exploring the Genetic Causality of Discordant Phenotypes in Familial Apparently Balanced Translocation Cases Using Whole Exome Sequencing.

Familial apparently balanced translocations (ABTs) are usually not associated with a phenotype; however, rarely, ABTs segregate with discordant phenotypes in family members carrying identical rearrangements. The current study was a follow-up investigation of four familial ABTs, where whole exome sequencing (WES) was implemented as a diagnostic tool to identify the underlying genetic aetiology of the patients' phenotypes. Data were analysed using an in-house bioinformatics pipeline alongside VarSome Clinical. WES findings were validated with Sanger sequencing, while the impact of splicing and missense variants was assessed by reverse-transcription PCR and in silico tools, respectively. Novel candidate variants were identified in three families. In family 1, it was shown that the de novo pathogenic STXBP1 variant (NM_003165.6:c.1110+2T>G) affected splicing and segregated with the patient's phenotype. In family 2, a likely pathogenic TUBA1A variant (NM_006009.4:c.875C>T, NP_006000.2:p.(Thr292Ile)) could explain the patient's symptoms. In family 3, an SCN1A variant of uncertain significance (NM_006920.6:c.5060A>G, NP_008851.3:p.(Glu1687Gly)) required additional evidence to sufficiently support causality. This first report of WES application in familial ABT carriers with discordant phenotypes supported our previous findings describing such rearrangements as coincidental. Thus, WES can be recommended as a complementary test to find the monogenic cause of aberrant phenotypes in familial ABT carriers.

Autosomal recessive mental retardation: homozygosity mapping identifies 27 single linkage intervals, at least 14 novel loci and several mutation hotspots.

Mental retardation (MR) has a worldwide prevalence of around 2% and is a frequent cause of severe disability. Significant excess of MR in the progeny of consanguineous matings as well as functional considerations suggest that autosomal recessive forms of MR (ARMR) must be relatively common. To shed more light on the causes of autosomal recessive MR (ARMR), we have set out in 2003 to perform systematic clinical studies and autozygosity mapping in large consanguineous Iranian families with non-syndromic ARMR (NS-ARMR). As previously reported (Najmabadi et al. in Hum Genet 121:43-48, 2007), this led us to the identification of 12 novel ARMR loci, 8 of which had a significant LOD score (OMIM: MRT5-12). In the meantime, we and others have found causative gene defects in two of these intervals. Moreover, as reported here, tripling the size of our cohort has enabled us to identify 27 additional unrelated families with NS-ARMR and single-linkage intervals; 14 of these define novel loci for non-syndromic ARMR. Altogether, 13 out of 39 single linkage intervals observed in our cohort were found to cluster at 6 different loci on chromosomes, i.e., 1p34, 4q27, 5p15, 9q34, 11p11-q13 and 19q13, respectively. Five of these clusters consist of two significantly overlapping linkage intervals, and on chr 1p34, three single linkage intervals coincide, including the previously described MRT12 locus. The probability for this distribution to be due to chance is only 1.14 × 10(-5), as shown by Monte Carlo simulation. Thus, in contrast to our previous conclusions, these novel data indicate that common molecular causes of NS-ARMR do exist, and in the Iranian population, the most frequent ones may well account for several percent of the patients. These findings will be instrumental in the identification of the underlying genes.

A defect in the TUSC3 gene is associated with autosomal recessive mental retardation.

Recent studies have shown that autosomal recessive mental retardation (ARMR) is extremely heterogeneous, and there is reason to believe that the number of underlying gene defects goes into the thousands. To date, however, only four genes have been implicated in nonsyndromic ARMR (NS-ARMR): PRSS12 (neurotrypsin), CRBN (cereblon), CC2D1A, and GRIK2. As part of an ongoing systematic study aiming to identify ARMR genes, we investigated a large consanguineous family comprising seven patients with nonsyndromic ARMR in four sibships. Genome-wide SNP typing enabled us to map the relevant genetic defect to a 4.6 Mbp interval on chromosome 8. Haplotype analyses and copy-number studies led to the identification of a homozygous deletion partly removing TUSC3 (N33) in all patients. All obligate carriers of this family were heterozygous, but none of 192 unrelated healthy individuals from the same population carried this deletion. We excluded other disease-causing mutations in the coding regions of all genes within the linkage interval by sequencing; moreover, we verified the complete absence of a functional TUSC3 transcript in all patients through RT-PCR. TUSC3 is thought to encode a subunit of the endoplasmic reticulum-bound oligosaccharyltransferase complex that catalyzes a pivotal step in the protein N-glycosylation process. Our data suggest that in contrast to other genetic defects of glycosylation, inactivation of TUSC3 causes nonsyndromic MR, a conclusion that is supported by a separate report in this issue of AJHG. TUSC3 is only the fifth gene implicated in NS-ARMR and the first for which mutations have been reported in more than one family.

Prenatal diagnosis in a mentally retarded woman with mosaic ring chromosome 18.

We present a pregnant woman with mental retardation and mosaic for ring 18 referred for prenatal diagnosis. Major clinical features included short stature with clinodactyly in feet, foot deformity and club feet, hypotonia, kyphosis, and absence of breast development, low set ears, high arched palate, dental decay and speech disorder. Prenatal diagnosis was carried. Using amniocentesis. The fetus had a normal karyotype described as 46,XX. The fetus was evaluated for clinical features after delivery; she was healthy with no abnormal clinical characterizations.

The Relationship between KIT Copy Number Variation, Protein Expression, and Angiogenesis in Sporadic Breast Cancer.

BACKGROUND: KIT is a protooncogene that encodes for the KIT oncoprotein, which is a transmembrane tyrosine kinase growth factor receptor that holds a critical role in a variety of normal physiological and pathological processes including angiogenesis. KIT has been shown to be involved in tumorigenesis, contributing to the development of gastrointestinal carcinoma and leukemia. A link between KIT overexpression and breast cancer development has previously been reported. In the current study, we explored KIT gene expression and exonic copy number variants (CNV) and the relationship with angiogenesis (CD34) and the clinicopathological features of breast cancer. METHODS: MLPA technique was used to determine the CNV in 64 breast cancer tumor samples from patients diagnosed with primary sporadic breast cancer. Results were confirmed by quantitative PCR. Expression of KIT and CD34 was determined using immunohistochemistry (IHC). RESULTS: Our results show that 28.1% of the tumor samples from patients with primary sporadic breast cancer had CNV in the KIT gene. Among the breast tumor samples, 54.7% showed positive KIT expression. The expression of the CD34 angiogenesis marker was reported in 43.8% of the tumor samples as low, 42.2% as moderate and 14.1% as high. A significant correlation between increased CNV of KIT exons, a high level of angiogenesis (CD34) and increased tumor grade was observed (p< 0.05). CONCLUSION: A significant correlation between the KIT CNV and the angiogenesis marker was found. Examining KIT expression and CNV has the potential to function as a biomarker for tyrosine kinase inhibitor drugs in breast cancer.

Investigation of Aneusomy of Chromosome 21 in the Micronuclei of 13 Patients with Early Onset Alzheimer's Disease Using Fluorescence in Situ Hybridization: A Pilot Study.

BACKGROUND: Alzheimer's disease is one of the most common neurodegenerative and dementia disorders in people between the ages of 30 and 65. When symptoms appear in this age group, the disease is referred to as early-onset Alzheimer's disease (EOAD). Unfortunately, the symptoms are progressive and no current treatments are effective. METHODS: In this research, 13 patients, aged 37 to 65 years with symptoms of early-onset Alzheimer's disease, were studied. First, patient lymphocytes were isolated and cultured in RPMI 1640 medium using a special micronucleus (MN) culture method. Next, the lymphocytes were harvested and prepared on slides. The slides were then examined by fluorescent microscopy using a unique FISH protocol specific for MNs. The patients were divided into groups aged 30-39, 40-49, and 50-65. RESULTS: We found that 19.76% of the MNs from our EOAD patients originated in chromosome 21. Micronuclei originated in chromosome 21 in 21.20 and 16.52% of patients without and with family histories of Alzheimer's, respectively. This difference was not significant. Also, the percentage of micronuclei originating in chromosome 21 was not dependent on the patient age at the time of the study, or symptom onset age or duration. CONCLUSION: This study shows that the rate of micronuclei with the origin of chromosome 21 is high in these patients. However, the micronucleus increased has no significant relationship with age and duration of disease or family history of it.

Whole-Exome Sequencing Identifies Three Candidate Homozygous Variants in a Consanguineous Iranian Family with Autism Spectrum Disorder and Skeletal Problems.

Autism spectrum disorder (ASD) is characterized by 3 core symptoms with impaired social communication, repetitive behavior, and/or restricted interests in early childhood. As a complex neurodevelopmental disorder (NDD), the phenotype and severity of autism are extremely heterogeneous. Genetic factors have a key role in the etiology of autism. In this study, we investigated an Azeri Turkish family with 2 ASD-affected individuals to identify probable ASD-causing variants. First, the affected individuals were karyotyped in order to exclude chromosomal abnormalities. Then, whole-exome sequencing was carried out in one affected sibling followed by cosegregation analysis for the candidate variants in the family. In addition, SNP genotyping was carried out in the patients to identify possible homozygosity regions. Both proband and sibling had a normal karyotype. We detected 3 possible causative variants in this family: c.5443G>A; p.Gly1815Ser, c.1027C>T; p.Arg343Trp, and c.382A>G; p.Lys128Glu, which are in the FBN1, TF, and PLOD2 genes, respectively. All of the variants cosegregated in the family, and SNP genotyping revealed that these 3 variants are located in the homozygosity regions. This family serves as an example of a multimodal polygenic risk for a complex developmental disorder. Of these 3 genes, confluence of the variants in FBN1 and PLOD2 may contribute to the autistic features of the patient in addition to skeletal problems. Our study highlights the genetic complexity and heterogeneity of NDDs such as autism. In other words, in some patients with ASD, multiple rare variants in different loci rather than a monogenic state may contribute to the development of phenotypes.

Bi-allelic Mutations in ALDH5A1 is associated with succinic semialdehyde dehydrogenase deficiency and severe intellectual disability.

Homozygous mutations of ALDH5A1 have been reportedly associated with Succinic semialdehyde dehydrogenase deficiency (SSADHD) that affects gamma-aminobutyric acid (GABA) catabolism and evinces a wide range of clinical phenotype from mild intellectual disability to severe neurodegenerative disorders. We report clinical and molecular data of a Lor family with 2 affected members presenting with severe intellectual disability, developmental delay, and generalized tonic-clonic seizures. A comprehensive genetic study that included whole-exome sequencing identified a homozygous missense substitution (NM_001080:c.G1321A:p.G441R) in ALDH5A1 (Aldehyde Dehydrogenase 5 Family Member A1) gene, consistent with clinical phenotype in the patients and co-segregating with the disease in the family. The non-synonymous mutation, p.G441R, affects a highly conserved amino acid residue, which is expected to cause a severe destabilization of the enzyme. Protein modeling demonstrated an impairment of the succinic semialdehyde (SSA) binding tunnel accessibility, and the anticipation of the protein folding stability and dynamics was a decrease in the free energy by 4.02 kcal/mol. Consistent with these in silico findings, excessive γ -hydroxybutyrate (GHB) could be detected in patients' urine as the byproduct of the GABA pathway. SSADHD, Succinic semialdehyde dehydrogenase deficiency; GABA, gamma-aminobutyric acid; ALDH5A1, Aldehyde Dehydrogenase 5 Family Member A1; GHB, γ -hydroxybutyrate; SSA, succinic semi aldehyde; WISC, Wechsler Intelligence Scale for Children; CNS, central nervous system ; EEG, electroencephalography; EEEF, empirical effective energy functions; ASD, autism spectrum disorder; ADHD, attention deficit hyperactivity disorder; IQ, intelligence quotient; EMG, electromyography; NCV, nerve conduction velocity; CP, cerebral palsy.

Combination of Genetics and Nanotechnology for Down Syndrome Modification: A Potential Hypothesis and Review of the Literature.

Down syndrome (DS) is one of the most prevalent genetic disorders in humans. The use of new approaches in genetic engineering and nanotechnology methods in combination with natural cellular phenomenon can modify the disease in affected people. We consider two CRISPR/Cas9 systems to cut a specific region from short arm of the chromosome 21 (Chr21) and replace it with a novel designed DNA construct, containing the essential genes in chromatin remodeling for inactivating of an extra Chr21. This requires mimicking of the natural cellular pattern for inactivation of the extra X chromosome in females. By means of controlled dosage of an appropriate Nano-carrier (a surface engineered Poly D, L-lactide-co-glycolide (PLGA) for integrating the relevant construct in Trisomy21 brain cell culture media and then in DS mouse model, we would be able to evaluate the modification and the reduction of the active extra Chr21 and in turn reduce substantial adverse effects of the disease, like intellectual disabilities. The hypothesis and study seek new insights in Down syndrome modification.

A Pathogenic Homozygous Mutation in The Pleckstrin Homology Domain of RASA1 Is Responsible for Familial Tricuspid Atresia in An Iranian Consanguineous Family.

OBJECTIVE: Tricuspid atresia (TA) is a rare life-threatening form of congenital heart defect (CHD). The genetic mechanisms underlying TA are not clearly understood. According to previous studies, the endocardial cushioning event, as the primary sign of cardiac valvulogenesis, is governed by several overlapping signaling pathways including Ras/ ERK pathway. RASA1, a regulator of cardiovascular development, is involved in this pathway and its haploinsufficiency (due to heterozygous mutations) has been identified as the underlying etiology of the autosomal dominant capillary malformation/arteriovenous malformation (CM/AVM). MATERIALS AND METHODS: In this prospective study, we used whole exome sequencing (WES) followed by serial bioinformatics filtering steps for two siblings with TA and early onset CM. Their parents were consanguineous which had a history of recurrent abortions. Patients were carefully assessed to exclude extra-cardiac anomalies. RESULTS: We identified a homozygous RASA1 germline mutation, c.1583A>G (p.Tyr528Cys) in the family. This mutation lies in the pleckstrin homology (PH) domain of the gene. The parents who were heterozygous for this variant displayed CM. CONCLUSION: This is the first study reporting an adverse phenotypic outcome of a RASA1 homozygous mutation. Here, we propose that the phenotypic consequence of the homozygous RASA1 p.Tyr528Cys mutation is more serious than the heterozygous type. This could be responsible for the TA pathogenesis in our patients. We strongly suggest that parents with CM/AVM should be investigated for RASA1 heterozygous mutations. Prenatal diagnosis and fetal echocardiography should also be carried out in the event of pregnancy in heterozygous parents.

Application of Multiplex Ligation-Dependent Probe Amplification in Determining the Copy Number Alterations of HER Gene Family Members in Invasive Ductal Breast Carcinoma.

BACKGROUND: The aim of this study was to assess the usability of multiplex ligation-dependent probe amplification (MLPA) for copy number determination of HER gene family members (ERBB1-4) in invasive breast carcinoma and to explore the association of ERBB1-4 gene copy numbers with clinicopathological characteristics of breast cancer (BC) patients. METHODS: Clinical and immunohistochemical characteristics were assessed in 104 BC patients and the molecular subtype was determined for each tumor sample. Furthermore, HER-2/neu status was assessed by immunohistochemistry (IHC) and equivocal results were confirmed by Fluorescent in situ hybridization (FISH). The copy numbers of ERBB1-4 genes were determined by MLPA. RESULTS: Twenty-five percent of all patients showed ERBB2 gene-amplification by MLPA, whereas 14.4% of cases showed ERBB-2/neu overproduction at the protein level (IHC). Moreover, only 2.9% and 1.9% of patients showed amplification in ERBB1 and ERBB4, respectively. No copy number changes were observed in ERBB3. Our results indicated a significant association between ERBB2 copy number gain and histological grade (p value= 0.01), stage (p value= 0.02), and tumor subtypes (p value= <0.001). In addition, we found MLPA more accurate in assessing HER2 status with 15.4% and 9.6% gene amplification detection in early stages (1, 2A and 2B) and advanced tumor stages (3A, 3B, and 4), respectively, compared to IHC (early stages= 13.5% and advanced stages= 4.7%). CONCLUSION: According to our findings, MLPA is a fast, precise and low-cost technique to detect ERBB2 amplification, especially in advanced tumor stages. However, due to infrequent amplification found in ERBB1 and ERBB4 as well as the lack of amplification in ERBB3, their importance in the prognostic evaluation of BC patients remains controversial.

A 57 kB Genomic Deletion Causing CTNS Loss of Function Contributes to the CTNS Mutational Spectrum in the Middle East.

Background: Nephropathic Cystinosis, the most common cause of renal Fanconi syndrome, is a lysosomal transport disorder with an autosomal recessive inheritance pattern. A large number of mutations in CTNS have been identified as causative to date. A 57 kb deletion encompassing parts of CTNS is most commonly identified in Caucasians but this allele has not been identified in individuals of Eastern Mediterranean, Middle Eastern, Persian, or Arab origin to date. Methods and Results: Implementing whole exome sequencing (WES) in a consanguineous Iranian family, we identified this large deletion affecting CTNS in a patient initially presenting with hypokalemic metabolic alkalosis symptoms and considerable proteinuria. Conclusion: We show WES is a cost and time efficient genetic diagnostics modality to identify the underlying molecular pathology in Cystinosis individuals and provide a summary of all previously reported CTNS alleles in the Middle east population. Our work also highlights the importance to consider the 57-kb deletion as underlying genetic cause in non-European populations, including the Middle East. Limited diagnostic modalities for Cystinosis in developing countries could account for the lack of previously reported cases in these populations carrying this allele. Further, our findings emphasize the utility of WES to define genetic causes in clinically poorly defined phenotypes and demonstrate the requirement of Copy number variation (CNV) analysis of WES data.