Homozygous Null TBX4 Mutations Lead to Posterior Amelia with Pelvic and Pulmonary Hypoplasia.

The development of hindlimbs in tetrapod species relies specifically on the transcription factor TBX4. In humans, heterozygous loss-of-function TBX4 mutations cause dominant small patella syndrome (SPS) due to haploinsufficiency. Here, we characterize a striking clinical entity in four fetuses with complete posterior amelia with pelvis and pulmonary hypoplasia (PAPPA). Through exome sequencing, we find that PAPPA syndrome is caused by homozygous TBX4 inactivating mutations during embryogenesis in humans. In two consanguineous couples, we uncover distinct germline TBX4 coding mutations, p.Tyr113∗ and p.Tyr127Asn, that segregated with SPS in heterozygous parents and with posterior amelia with pelvis and pulmonary hypoplasia syndrome (PAPPAS) in one available homozygous fetus. A complete absence of TBX4 transcripts in this proband with biallelic p.Tyr113∗ stop-gain mutations revealed nonsense-mediated decay of the endogenous mRNA. CRISPR/Cas9-mediated TBX4 deletion in Xenopus embryos confirmed its restricted role during leg development. We conclude that SPS and PAPPAS are allelic diseases of TBX4 deficiency and that TBX4 is an essential transcription factor for organogenesis of the lungs, pelvis, and hindlimbs in humans.

Distinct genetic variation and heterogeneity of the Iranian population.

Iran, despite its size, geographic location and past cultural influence, has largely been a blind spot for human population genetic studies. With only sparse genetic information on the Iranian population available, we pursued its genome-wide and geographic characterization based on 1021 samples from eleven ethnic groups. We show that Iranians, while close to neighboring populations, present distinct genetic variation consistent with long-standing genetic continuity, harbor high heterogeneity and different levels of consanguinity, fall apart into a cluster of similar groups and several admixed ones and have experienced numerous language adoption events in the past. Our findings render Iran an important source for human genetic variation in Western and Central Asia, will guide adequate study sampling and assist the interpretation of putative disease-implicated genetic variation. Given Iran's internal genetic heterogeneity, future studies will have to consider ethnic affiliations and possible admixture.

Biallelic variants in TMEM222 cause a new autosomal recessive neurodevelopmental disorder.

PURPOSE: To elucidate the novel molecular cause in families with a new autosomal recessive neurodevelopmental disorder. METHODS: A combination of exome sequencing and gene matching tools was used to identify pathogenic variants in 17 individuals. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) and subcellular localization studies were used to characterize gene expression profile and localization. RESULTS: Biallelic variants in the TMEM222 gene were identified in 17 individuals from nine unrelated families, presenting with intellectual disability and variable other features, such as aggressive behavior, shy character, body tremors, decreased muscle mass in the lower extremities, and mild hypotonia. We found relatively high TMEM222 expression levels in the human brain, especially in the parietal and occipital cortex. Additionally, subcellular localization analysis in human neurons derived from induced pluripotent stem cells (iPSCs) revealed that TMEM222 localizes to early endosomes in the synapses of mature iPSC-derived neurons. CONCLUSION: Our findings support a role for TMEM222 in brain development and function and adds variants in the gene TMEM222 as a novel underlying cause of an autosomal recessive neurodevelopmental disorder.

Pre-implantation genetic diagnosis in an Iranian family with a novel mutation in MUT gene.

BACKGROUND: Methylmalonic acidemia (MMA), which is an autosomal recessive metabolic disorder, is caused by mutations in methylmalonyl-CoA mutase (MUT) gene. As a result, the conversion of methylmalonyl-CoA to succinyl-CoA is impaired in this disorder, leading to a wide range of clinical manifestations varying from no signs or symptoms to severe lethargy and metabolic crisis in newborn infants. Since identification of novel mutations in MUT gene can help discover the exact pathogenesis of MMA and also use these disease-causing mutations in prenatal diagnosis, this study was conducted to uncover the possible mutations in an Iranian couple with a deceased offspring clinically diagnosed as having organic acidemia. Moreover, to prevent the occurrence of the mutation in the next pregnancy, we took the advantage of pre-implantation genetic diagnosis (PGD), which resulted in a successful pregnancy. CASE PRESENTATION: The affected individual was a 15-month-old boy who passed away due to aspiration pneumonia. The child presented at the age of 3 months with lethargy, protracted vomiting, hypotonia, and decreased level of consciousness. To find the mutated gene, Next Generation Sequencing (NGS) was performed as carrier testing for the parents and the results revealed a novel (private) heterozygous missense mutation in MUT gene (c.1055A > G, p.Q352R). After performing PGD on three blastomeres, one was identified as being homozygous wild-type that was followed by successful pregnancy. CONCLUSIONS: Our study identified a novel, deleterious, heterozygous missense mutation in MUT gene in a couple and helps to consider the genetic counselling and prenatal diagnosis more seriously for this family with clinical phenotypes of organic acidemia.

Genetics of intellectual disability in consanguineous families.

Autosomal recessive (AR) gene defects are the leading genetic cause of intellectual disability (ID) in countries with frequent parental consanguinity, which account for about 1/7th of the world population. Yet, compared to autosomal dominant de novo mutations, which are the predominant cause of ID in Western countries, the identification of AR-ID genes has lagged behind. Here, we report on whole exome and whole genome sequencing in 404 consanguineous predominantly Iranian families with two or more affected offspring. In 219 of these, we found likely causative variants, involving 77 known and 77 novel AR-ID (candidate) genes, 21 X-linked genes, as well as 9 genes previously implicated in diseases other than ID. This study, the largest of its kind published to date, illustrates that high-throughput DNA sequencing in consanguineous families is a superior strategy for elucidating the thousands of hitherto unknown gene defects underlying AR-ID, and it sheds light on their prevalence.

Diagnostic accuracy of sFlt1/PlGF ratio as a marker for preeclampsia.

BACKGROUND: Preeclampsia (PE) is a serious complication of pregnancy and one of the main causes of maternal and neonatal mortality and morbidity in the world. Finding a biomarker with high sensitivity and specificity could lead to prediction and early diagnosis of the disease and reduces its complications. In this study, we evaluated diagnostic accuracy of Soluble fms-like tyrosine kinase-1 (sFlt-1) to Placental growth factor (PlGF) ratio for diagnosis of PE. METHODS: The cases included 23 mild, 15 severe preeclamptic patients, and 20 normal term pregnant women as control referred to GYN ward of the Persian Gulf Hospital in Bandar Abbas from 2014 to 2016. Levels of sFlt-1 and PlGF were measured. Receiver Operating Characteristic (ROC) curve analysis was applied to calculate diagnostic accuracy of sFlt-1/PlGF ratio. RESULTS: The mean Level of sFlt-1/PlGF in PE patients (91.33 ng/ml) was significantly higher than control women (17.62) (P<0.001). ROC curve analysis showed sFlt-1/PlGF ratio diagnostic accuracy in preeclamptic patients with Area Under Curve (AUC) of 0.90, the best cut-off value of 24.96, sensitivity and specificity of 84.2 and 85.0%, respectively. CONCLUSIONS: Our data showed sFlt-1/PlGF ratio has higher accuracy for differentiating PE patients from non-PEs in comparison with its power for differentiating severe or early onset forms of the disease.

Iranome: A catalog of genomic variations in the Iranian population.

Considering the application of human genome variation databases in precision medicine, population-specific genome projects are continuously being developed. However, the Middle Eastern population is underrepresented in current databases. Accordingly, we established Iranome database (www.iranome.com) by performing whole exome sequencing on 800 individuals from eight major Iranian ethnic groups representing the second largest population of Middle East. We identified 1,575,702 variants of which 308,311 were novel (19.6%). Also, by presenting higher frequency for 37,384 novel or known rare variants, Iranome database can improve the power of molecular diagnosis. Moreover, attainable clinical information makes this database a good resource for classifying pathogenicity of rare variants. Principal components analysis indicated that, apart from Iranian-Baluchs, Iranian-Turkmen, and Iranian-Persian Gulf Islanders, who form their own clusters, rest of the population were genetically linked, forming a super-population. Furthermore, only 0.6% of novel variants showed counterparts in "Greater Middle East Variome Project", emphasizing the value of Iranome at national level by releasing a comprehensive catalog of Iranian genomic variations and also filling another gap in the catalog of human genome variations at international level. We introduce Iranome as a resource which may also be applicable in other countries located in neighboring regions historically called Greater Iran (Persia).

Effect of inbreeding on intellectual disability revisited by trio sequencing.

In outbred Western populations, most individuals with intellectual disability (ID) are sporadic cases, dominant de novo mutations (DNM) are frequent, and autosomal recessive ID (ARID) is very rare. Because of the high rate of parental consanguinity, which raises the risk for ARID and other recessive disorders, the prevalence of ID is significantly higher in near- and middle-east countries. Indeed, homozygosity mapping and sequencing in consanguineous families have already identified a plethora of ARID genes, but because of the design of these studies, DNMs could not be systematically assessed, and the proportion of cases that are potentially preventable by avoiding consanguineous marriages or through carrier testing is hitherto unknown. This prompted us to perform whole-exome sequencing in 100 sporadic ID patients from Iran and their healthy consanguineous parents. In 61 patients, we identified apparently causative changes in known ID genes. Of these, 44 were homozygous recessive and 17 dominant DNMs. Assuming that the DNM rate is stable, these results suggest that parental consanguinity raises the ID risk about 3.6-fold, and about 4.1 to 4.25-fold for children of first-cousin unions. These results do not rhyme with recent opinions that consanguinity-related health risks are generally small and have been "overstated" in the past.

Clinical application of next generation sequencing for Mendelian disease diagnosis in the Iranian population.

Next-generation sequencing (NGS) has been proven to be one of the most powerful diagnostic tools for rare Mendelian disorders. Several studies on the clinical application of NGS in unselected cohorts of Middle Eastern patients have reported a high diagnostic yield of up to 48%, correlated with a high level of consanguinity in these populations. We evaluated the diagnostic utility of NGS-based testing across different clinical indications in 1436 patients from Iran, representing the first study of its kind in this highly consanguineous population. A total of 1075 exome sequencing and 361 targeted gene panel sequencing were performed over 8 years at a single clinical genetics laboratory, with the majority of cases tested as proband-only (91.6%). The overall diagnostic rate was 46.7%, ranging from 24% in patients with an abnormality of prenatal development to over 67% in patients with an abnormality of the skin. We identified 660 pathogenic or likely pathogenic variants, including 241 novel variants, associated with over 342 known genetic conditions. The highly consanguineous nature of this cohort led to the diagnosis of autosomal recessive disorders in the majority of patients (79.1%) and allowed us to determine the shared carrier status of couples for suspected recessive phenotypes in their deceased child(ren) when direct testing was not possible. We also highlight the observations of recessive inheritance of genes previously associated only with dominant disorders and provide an expanded genotype-phenotype spectrum for multiple less-characterized genes. We present the largest mutational spectrum of known Mendelian disease, including possible founder variants, throughout the Iranian population, which can serve as a unique resource for clinical genomic studies locally and beyond.

Prenatal diagnosis of Sex determining region Y -box transcription factor 2 anophthalmia syndrome caused by germline mosaicism using next-generation sequencing: A case report.

Background: Sex determining region Y box transcription factor 2 (SOX2) mutations lead to bilateral anophthalmia with autosomal dominant human inheritance. SOX2 mutations could result in severe ocular phenotypes usually associated with variable systemic defects. Most patients described with SOX2 anophthalmia syndrome possessed de novo mutations in this gene. Case Presentation: In this case report, we describe 2 brothers with mental retardation and bilateral anophthalmia caused due to SOX2 germline mosaicism in unaffected parents. Next-generation DNA sequencing was carried out to determine the family's possible cause of genetic mutation. Sanger sequencing was performed on the patients and their parents. Prenatal diagnosis was done in both pregnancies of the older brother's wife via chorionic villus sampling. A novel heterozygous pathogenic frameshift deletion variant (exon1:c.58_80del:p.G20fs) was identified in the SOX2 gene, which was confirmed by Sanger sequencing in both affected brothers and did not exist in healthy parents, indicating germline mosaicism. Conclusion: Most SOX2 mutations known look to arise de novo in probands and are diagnosed through anophthalmia or microphthalmia. Prenatal diagnosis should be offered to healthy parents with a child with SOX2 mutation every pregnancy.

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 Pediatric Parathyroid Carcinoma: An Unusual Clinical Presentation and Mini-review.

INTRODUCTION: Primary hyperparathyroidism (PHPT) is a rare condition in the pediatric population. Parathyroid carcinoma (PC) is a very uncommon cause of PHPT, accounting for < 1% of pediatric PHPT cases. It is challenging to distinguish between parathyroid adenoma (PA), the most common cause of PHPT, and PC. In this report, we described a young female who presented with a history of progressive limping and was finally diagnosed with PC. CASE PRESENTATION: A 15-year-old girl presented with progressive limping and bone pain for 8 years. She was referred by an orthopedic surgeon because of elevated intact parathyroid hormone (iPTH) for further evaluation. Physical examination revealed a large, firm, and non-tender neck mass, left hip tenderness, and limited range of motion. The initial biochemistry tests showed a borderline high calcium level of 10.8 mg/dl, an elevated iPTH level of 2876 pg/mL, and a decreased phosphorus level of 2.4 mg/dL. The 99mTechnetium (Tc) sestamibi scan displayed early intense activity in the right thyroid lobe persisting in the three-hour repeat scan, compatible with a parathyroid lesion. The patient underwent right-sided neck exploration and parathyroidectomy. Intraoperative and pathology findings confirmed the diagnosis of PC. Immunohistochemistry (IHC) staining revealed creatine kinase (CK) and CD31 in endothelial cells of the tumor. Ki67 staining was also positive in 2% - 3% of tumor cells. The whole exome sequencing (WES) study was negative for cell division cycle 73 (CDC73) and multiple endocrine neoplasia 1 (MEN1) genes. CONCLUSIONS: PC should be considered as a differential diagnosis of PHPT in the pediatric population, even in the presence of mild hypercalcemia.

Prenatal diagnosis for beta-thalassemia major in the Iranian Province of Hormozgan.

beta-Thalassemias are a group of heterogenous recessive disorders common in many parts of the world. Despite the great advances in the treatment of thalassemia, there is so far no cure, but perhaps bone marrow transplantation (BMT) is a possibility. Prevention, using prenatal diagnosis and selective abortion in the cases where the fetus is found to be affected, should be considered as a sensible alternative. During the past 5 years, 112 couples have been referred to our Center for detection of their beta-thalassemia (beta-thal) carrier status. In this group, common and rare mutations were detected. Of these, 106 couples (94.6%) came for counseling during pregnancy and six (5.4%) came before becoming pregnant. Prenatal diagnosis was performed for the 106 couples at risk. Fetal DNA was obtained from both chorionic villus sampling (CVS) (99) and amniotic fluid (7). Using reverse hybridization, 64 (60.4%) were found to be heterozygous for a beta-thal mutation and 24 (22.6%) were normal. Eighteen (17.0%) were found to carry an affected fetus and these pregnancies were terminated.

Accuracy of Soluble Endoglin for Diagnosis of Preeclampsia and its Severity

Background: The use of biomarkers for diagnosis of Preeclampsia (PE), a life-threatening pregnancy disorder, could reduce serious complications of this disease. In this study, we investigated dysregulation of endoglin (Eng) expression and diagnostic accuracy of soluble endoglin (sEng) in PE patients. Methods: For this case-control study, 26 mild and 15 severe preeclamptic women along with 20 normotensive controls were recruited. The expression level of Eng (the co-receptor of TGF-ß1) was evaluated using qRT-PCR. Also, the serum concentration of soluble Eng and expression of membranous Eng were determined by ELISA and immunohistochemistry. Results: A significant up-regulation in Eng mRNA and sEng levels was observed in PE patients versus normal controls. Immunohistochemistry (IHC) showed up-regulation of membranous Eng staining in syncytiotrophoblast and cytotrophoblast cells of PE patients. The serum levels of sEng were significantly increased in all patients (mild, sever, early- and late-onset) as compared to healthy pregnant women (P˂0.001). Receiver-operating characteristic (ROC) curve analysis revealed that sEng had the highest accuracy in distinguishing PE from normal pregnancies with cut-off value of 20.4, sensitivity of 92.1%, specificity of 90%, and area under the curve (AUC) of 0.94 (95% CI: 0.88-1.00). Conclusion: Our data showed that the up-regulation of Eng mRNA along with its membranous and soluble form in PE patients leads to defect in angiogenesis pathway. Also, the results of this study revealed sEng potential as a marker for diagnosis of PE and its severity.

Expression of placental growth factor mRNA in preeclampsia.

BACKGROUND: Preeclampsia (PE) is a serious complication of pregnancy with hallmarks of incomplete placentation, placental ischemia and endothelial dysfunction. Imbalance between vascular endothelial growth factor (VEGF), placenta growth factor (PlGF) and their receptors play important role in pathophysiology of PE. OBJECTIVE: This study was aimed to asses PlGF mRNA expression in placenta of women affected with PE. MATERIAL AND METHODS: In this cross-sectional study, expression of PlGF mRNA was evaluated in 26 mild PE cases, 15 severe preeclamptic women and 20 normotensive controls. Patients were sub-classified as early onset PE (9) and late onset (32). After RNA extraction, PlGF expression was quantified with qRT-PCR. RESULTS: The results of PlGF mRNA expression between mild-severe, and early-late onset PE patients showed no statistically significant difference compared with the control group (p=0.661, p=0.205 respectively). CONCLUSION: Despite we found no distinct differential expression of PlGF mRNA in placental tissue of PE patients compared with control women, but according to decreased level of this angiogenic factor in PE even before clinical onset of the disease, determining molecular mechanisms related to reduced secretion of PlGF into the maternal circulation may be useful for future therapeutics.

Predictive value of miR-210 as a novel biomarker for pre-eclampsia: a systematic review protocol.

INTRODUCTION: Pre-eclampsia (PE) is a serious condition affecting 3-5% of all pregnancies worldwide. However, underlying molecular pathogenesis of this disease has largely remained unknown. Recently, several studies have indicated the possibility role of microRNAs, especially miR-210, in the aetiology of PE. The aim of this systematic review is to assess the possible role of miR-210 as a novel biomarker for the prediction of PE. METHODS AND ANALYSIS: Using a combination of mesh terms 'preeclampsia', 'microRNA' and their equivalents, an electronic search will be performed for all observational studies (cross sectional, case-control and cohort) in PubMed, Web of Science, Scopus, Embase, Cochrane, LILACS and OvidSP MEDLINE from January 2005 to December 2015. Furthermore, other sources are searched, including grey literature, reference lists of relevant primary studies as well as key journals. Study selection, data extraction and quality assessment of studies will be performed independently by 2 reviewers, and any disagreement will be resolved by consensus. If sufficient data are available, it will be combined by either fixed or random effects models. We will investigate the source)s(and degree of heterogeneity using 'Heterogeneity χ2' and I2. Heterogeneity would be investigated through either subgroup analysis or metaregression. Stata V.11.1 will be used for data analysis. ETHICS AND DISSEMINATION: The results of this study are disseminated in peer-reviewed journal articles and academic presentations. Formal ethical approval is not required, since the secondary data will be collected. TRIAL REGISTRATION NUMBER: CRD42015032345.

The imbalance in expression of angiogenic and anti-angiogenic factors as candidate predictive biomarker in preeclampsia.

Preeclampsia is an important pregnancy disorder with serious maternal and fetal complications which its etiology has not been completely understood yet. Early diagnosis and management of disease could reduce its potential side effects. The vascular endothelial growth factor (VEGF) family including VEGF-A is the most potent endothelial growth factor which induces angiogenesis and endothelial cell proliferation and has basic role in vasculogenesis. VEGF and its tyrosine kinase receptors (Flt1 and KDR) are major factors for fetal and placental angiogenic development. Finding mechanisms involved in expression of angiogenic factors may lead to new prognostic and therapeutic points in management of preeclampsia. Recent researches, has shown capability of some anti-angiogenic factors as potential candidate to be used as early predictors for preeclampsia. Soluble fms-like tyrosin kinase-1 (sFlt1) is a truncated splice variant of the membrane-bound VEGF receptor Flt1, that is produced by the placenta and it can bind to angiogenic growth factors and neutraliz, their effects. It is also observed that the ratio of sFlt1 to placental growth factor is valuable as prognostic marker. In this review, VEGF family member's role in angiogenesis is evaluated as biomarkers to be used for prediction of preeclampsia.

Effect of predictive value of progesterone level on the day of HCG injection for IVF success in women with infertility due to tubal factor or polycystic ovarian syndrome referred to the women hospital, Tehran, 2009.

BACKGROUND: Polycystic ovarian syndrome is one of the most common causes of endocrine disorders and main reason of infertility due to anovulation and recurrent abortions. Progesterone has been shown to have an important role in fertilization of oocyte and fetal implantation. OBJECTIVE: The purpose of this study was to compare the predictive value of progesterone level on IVF success in women with infertility due to tubal factor or PCOS. MATERIALS AND METHODS: In a stratified cohort study, we assigned 76 infertile women of 20-38 years old who referred to women hospital into two equal groups with fallopian tube factor infertility and PCOS. We measured the plasma levels of progesterone and estradiol on the day of HCG administration. The patients were divided into two groups based on progesterone level cut off point of 1.2ng/ml. Thereafter the incidence of pregnancy (chemical by ß-HCG measurement and clinical by ultrasonography up to the 6 weeks after fetal transfer) was compared in these groups. RESULTS: Total pregnancy rates were 15.8% in patients with tubal factor infertility and 26.3% in women with PCOS. In women with PCOS, the pregnancy rate was less in patients with progesterone level <1.2 ng/ml. However this difference was not statistically significant. Likewise, we did not observe any significant differences in pregnancy rate in patients with fallopian tube factor infertility. CONCLUSION: Serum progesterone level on the day of HCG administration is not well predictive of the IVF success in infertile women due to fallopian tube factor or PCOS. To obtain more uniform results, we recommend use of larger samples while the bias variable is taken into account and the ROC curve is used for determination of the unique serum progesterone level.