Whole exome sequencing identifies novel pathogenic variants in TGM1 and ALOX12B in patients with hereditary ichthyosis.

BACKGROUND: Hereditary ichthyosis is a clinically and genetically heterogeneous disorder associated with more than 50 genes with TGM1, ALOX12B, and ALOXE3 being the most prevalent. Establishing an accurate diagnosis is important for effective genetic counseling and optimal patient management. OBJECTIVE: We studied the diagnostic value of whole exome sequencing (WES) in a small case series with hereditary ichthyosis. METHODS: During a 1-year period, index cases of 5 unrelated families clinically diagnosed with hereditary ichthyosis went through WES, followed by extensive segregation analysis. Prenatal diagnosis (PND) was conducted where indicated. RESULTS: We identified 4 homozygous variants-2 in TGM1 (c.655A > G and c.797A > G) and 2 in ALOX12B (c.527 + 2 T > G and c.1654G > T)-alongside a heterozygous variant in TGM1 (c.428G > A) in 5 families. The variants were all pathogenic/likely pathogenic according to the ACMG classification and segregation analysis, except for c.797A > G in TGM1 which remained a variant of unknown clinical significance. Four variants were novel. All families were referred either during pregnancy or before reproductive planning; 4 benefited from WES as it identified the mutation in the probands and enabled carrier detection in at-risk relatives; PND was conducted in 2 families. CONCLUSION: Our findings further support WES is a powerful tool for the comprehensive, accurate, and rapid molecular diagnosis of hereditary ichthyosis and can offer opportunities for reproductive planning, carrier screening and prenatal diagnosis to at-risk families.

Molecular characterization of a large cohort of mucopolysaccharidosis patients: Iran Mucopolysaccharidosis RE-diagnosis study (IMPRESsion).

Mucopolysaccharidoses (MPSs) are rare, heterogeneous inborn errors of metabolism (IEM) diagnosed through a combination of clinical, biochemical, and genetic investigations. The aim of this study was molecular characterization of the largest cohort of Iranian MPS patients (302 patients from 289 unrelated families), along with tracking their ethnicity and geographical origins. 185/289 patients were studied using an IEM-targeted NGS panel followed by complementary Sanger sequencing, which led to the diagnosis of 154 MPS patients and 5 non-MPS IEMs (diagnostic yield: 85.9%). Furthermore, 106/289 patients who were referred with positive findings went through reanalysis and confirmatory tests which confirmed MPS diagnosis in 104. Among the total of 258 MPS patients, 225 were homozygous, 90 harbored novel variants, and 9 had copy number variations. MPS IV was the most common type (34.8%) followed by MPS I (22.7%) and MPS VI (22.5%). Geographical origin analysis unveiled a pattern of distribution for frequent variants in ARSB (c.430G>A, c.962T>C [p.Leu321Pro], c.281C>A [p.Ser94*]), GALNS (c.319G>A [p.Ala107Thr], c.860C>T [p.Ser287Leu], c.1042A>G [p.Thr348Ala]), and IDUA (c.1A>C [p.Met1Leu], c.1598C>G [p.Pro533Arg], c.1562_1563insC [p.Gly522Argfs*50]). Our extensive patient cohort reveals the genetic and geographic landscape of MPS in Iran, which provides insight into genetic epidemiology of MPS and can facilitate a more cost-effective, time-efficient diagnostic approach based on the region-specific variants.

Identification of a novel de novo variant in OTX2 in a patient with congenital microphthalmia using targeted next-generation sequencing followed by prenatal diagnosis.

BACKGROUND: Next-generation sequencing has been proven to be a reliable method for the detection of genetic causes in heterogeneous ocular disorders. In this report an NGS-based diagnostic approach was taken to uncover the genetic etiology in a patient with coloboma and microphthalmia, a highly heterogeneous disease with intrafamilial phenotypic variability. MATERIALS AND METHODS: Next generation sequencing using a targeted panel of 316 genes, was carried out in the proband. Prioritized variants were then identified and confirmed using Sanger sequencing. Prenatal diagnosis of the detected variant was then performed in the family. RESULTS: A novel de novo frameshift variant c.157_164delTTCACTCG (p.Phe53fs) in OTX2, leading to a truncated protein, was identified. Prenatal diagnosis identified the same variant in the fetus. CONCLUSIONS: This report demonstrates the importance of genetic counseling and underscores the efficiency and effectiveness of targeted NGS as a means of detecting variants in inherited eye disorders.

Detection of Aneuploidies in Products of Conception and Neonatal Deaths in Iranian Patients Using the Multiplex Ligation-Dependent Probe Amplification (MLPA).

BACKGROUND: Around 70% of all pregnancies (Including 15% of clinically-recognized ones) are lost due to various fetal or maternal disorders. Chromosomal aneuploidies are among the most common causes of pregnancy loss. Standard chromosome analysis using G-banding technique (Karyotype) is the technique of choice in studying such abnormalities; however, this technique is time-consuming and sensitive, and limited by vulnerabilities such as cell culture failure. The use of molecular cytogenetic techniques, including array-based techniques and Multiplex Ligation-Dependent Probe Amplification (MLPA), has been proposed to overcome the limitations of this method to study the products of conception. This study has been designed to investigate the feasibility of using MLPA technique as a standalone genetic testing, with histopathologic examinations and genetic counseling to detect aneuploidies in products of conception and neonatal deaths. METHODS: Forty-two verified fetal and neonatal samples were studies and genetic counseling was scheduled for all parents. Histopathologic examinations were carried out on the products of conception, and appropriate fetal tissues were separated for genetic studies. Following DNA extraction and purification, MLPA was carried out to investigate chromosomal aneuploidies. RESULTS: Nine samples (21.42%) were diagnosed to be affected with aneuploidy. Detected aneuploidies were trisomy 22 (n=3), trisomy 21(n=1), trisomy 18 (n=2), trisomy 16 (n=1), trisomy 13 (n=1), and monosomy of chromosome X (n=1). The MLPA analysis results were conclusive for all of the fetal samples (Success rate: 100%). CONCLUSION: These results suggest that MLPA, as a standalone genetic testing, is an accurate, rapid, and reliable method in overcoming the limitations of standard cytogenetic techniques in genetic investigation of products of conception.

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.

Heterozygosity assessment of five STR loci located at 5q13 region for preimplantation genetic diagnosis of spinal muscular atrophy.

Preimplantation genetic diagnosis (PGD) has been considered as an alternative to prenatal diagnosis for prevention of genetic disorders while avoiding the subsequent termination of pregnancy. However, the limited amount of template DNA available in a single diploid cell used for PGD leads to number of problems including an increased incidence of detectable contamination; amplification failure and allele drop out. Due to their highly polymorphic and amplifiable characteristics, short tandem repeat (STR) analysis has been proposed as a mean to overcome these limitations. Heterozygosity of the applied STRs is of paramount importance in their informativity, and should therefore be studied in any certain population. Here, for the first time, we report on the heterozygosity analysis of five STR markers (D5S1408, D5S1417, D5S610, D5S629 and D5S637) flanking to SMA gene region, to examine their applicability in the PGD for SMA disease. We have also investigated other statistical features of these markers and found that all of the five studied STRs were informative and four meet the Hardy-Weinberg equilibrium for the studied population. Furthermore, our results propose that similar approaches can be used for the PGD of other single gene disorders.

"Familial" versus "Sporadic" intellectual disability: contribution of common microdeletion and microduplication syndromes.

BACKGROUND: Interstitial Microdeletion and Microduplication syndromes have been proposed as a significant cause of sporadic intellectual disability (ID) but the role of such aberrations in familial ID has not yet been investigated. As the balanced chromosomal abnormalities commonly lead to the recurrent ID or multiple congenital anomalies, this study was designed to evaluate whether it was justified to investigate such aberrations in familial ID patients. Three hundred and twenty eight patients from 101 unrelated Iranian families with more than two ID patients in the first-degree relatives, have been investigated. Assessment of a panel of 21 common Microdeletion and Microduplication syndromes (CMMS) was carried out using Multiplex Ligation-Dependent Probe Amplification (MLPA) technique. RESULTS: Among the families studied, 27.7% had 4-12, 35.6% had 3 and 36.6% had 2 affected individuals in the first-degree relatives. An autosomal dominant inheritance of Williams-Beuren syndrome (WBS) was detected in a family with no clinical suspicion of WBS. The prevalence of CMMS was therefore,0.99%. CONCLUSION: This is the first investigation of a panel of CMMS in a large sample set of "familial ID patients". The findings of this study showed the low prevalence of CMMSs in "familial ID" patients in spite of the significant contribution of such aberrations in "sporadic ID" which has a very useful practical impact by avoiding unnecessary diagnostic tests in "familial ID" patients.

"Familial" versus "sporadic" intellectual disability: contribution of subtelomeric rearrangements.

BACKGROUND: Cryptic subtelomeric rearrangements have been proposed as a significant cause of sporadic intellectual disability (ID) but the role of such aberrations in familial ID has not yet been studied. As positive family history of ID had been proposed as an important and significant predicting factor of subtelomeric rearrangements, it was assumed that the contribution of subtelomeric aberrations in familial ID would be much more than the sporadic ones. Three hundred and twenty two patients from 102 unrelated families with more than two ID patients in the first degree relatives have been investigated. Assessment of subtelomeric rearrangements were carried out using Multiplex Ligation-Dependent Probe Amplification (MLPA) technique. Detected aberrations were then confirmed by Fluorescence in Situ Hybridization (FISH) method. RESULTS: Among the families studied, 27.4% had 4-12, 36.3% had 3 and 36.3% had 2 affected individuals in the first degree relatives. One unbalanced translocation and 4 polymorphic changes were detected. The prevalence of clinically significant subtelomeric rearrangements was 0.98%. CONCLUSION: This is the first investigation of subtelomeric aberrations in a large sample set of familial ID patients. Our results show that the contribution of subtelomeric rearrangements to familial ID is not as much as what had been determined for sporadic ones in the literature. Moreover, this study shows that the positive family history by alone, cannot be the most important and determining indicator of subtelomeric aberrations while it would be a good predicting factor when associated with dysmorphism or congenital malformations. These findings propose that other cryptic chromosomal abnormalities or even single gene disorders may be the main cause of familial ID rather than subtelomeric aberrations.

Effect of vitamin C on salivary total antioxidant capacity in smokers.

This study was designed to elucidate the effect of ascorbic acid on salivary total antioxidant capacity in smokers. In this single blind crossover clinical trial, the whole unstimulated saliva of 30 smokers, who were randomly divided into two groups, was collected. In the first phase after the saliva collection, one group of patients took 500 mg of vitamin C powder, for 3 weeks. Then, saliva of all patients was collected. After a one-week wash-out period, vitamin C was given to the other group. The collection of saliva was done after 3 weeks. Total antioxidant capacity was measured. Statistic evaluation was performed by Repeated Measured ANOVA, Independent sample t-test and Covariate test. The mean of total antioxidant capacity with and without using vitamin C was 0.511 ± 0.155 (U/mL) and 0.555 ± 0.171 (U/mL), respectively. This variability was not significant (p = 0.605). Oxidative stress from cigarette smoke was not decreased significantly with using vitamin C.