Targeted Double-Stranded cDNA Sequencing-Based Phase Analysis to Identify Compound Heterozygous Mutations and Differential Allelic Expression

There are two combinations of heterozygous mutation, i.e., in trans, which carries mutations on different alleles, and in cis, which carries mutations on the same allele. Because only in trans compound heterozygous mutations have been implicated in autosomal recessive diseases, it is important to distinguish them for clinical diagnosis. However, conventional phase analysis is limited because of the large target size of genomic DNA. Here, we performed a genetic analysis on a patient with Wilson disease, and we detected two heterozygous mutations chr13:51958362;G>GG (NM_000053.4:c.2304dup r.2304dup p.Met769HisfsTer26) and chr13:51964900;C>T (NM_000053.4:c.1841G>A r.1841g>a p.Gly614Asp) in the causative gene ATP7B. The distance between the two mutations was 6.5 kb in genomic DNA but 464 bp in mRNA. Targeted double-stranded cDNA sequencing-based phase analysis was performed using direct adapter ligation library preparation and paired-end sequencing, and we elucidated they are in trans compound heterozygous mutations. Trio analysis showed that the mutation (chr13:51964900;C>T) derived from the father and the other mutation from the mother, validating that the mutations are in trans composition. Furthermore, targeted double-stranded cDNA sequencing-based phase analysis detected the differential allelic expression, suggesting that the mutation (chr13:51958362;G>GG) caused downregulation of expression by nonsense-mediated mRNA decay. Our results indicate that targeted double-stranded cDNA sequencing-based phase analysis is useful for determining compound heterozygous mutations and confers information on allelic expression.

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Three Cases of Primary Ciliary Dyskinesia Combined With Reduced Exhaled Nitric Oxide

Annals of Otology Rhinology & Laryngology ◽

10.1177/0003489419861119 ◽

2019 ◽

Vol 128 (11) ◽

pp. 1081-1085

Author(s):

Sung Min Han ◽

Chi Sang Hwang ◽

Hyun Jong Jeon ◽

Ho Young Lee ◽

Hyung-Ju Cho ◽

...

Keyword(s):

Nitric Oxide ◽

Primary Ciliary Dyskinesia ◽

Exhaled Nitric Oxide ◽

Diagnostic Tools ◽

Compound Heterozygous Mutation ◽

Heterozygous Mutation ◽

Compound Heterozygous ◽

Causative Gene ◽

Tem Analysis ◽

Ciliary Dyskinesia

Objectives: The diagnosis of primary ciliary dyskinesia (PCD) is often delayed in part related to the limitations of the available diagnostic tests. We present 3 cases of PCD diagnosed using an exhaled nitric oxide (eNO) measurement. Methods: Three cases with a clinical phenotype consistent with PCD were evaluated using an eNO assay with additional transmission electron microscopy (TEM) and/or genetic panel testing. Results: One male and 2 female patients presented with common symptoms included recurrent respiratory infection from early childhood and a history of neonatal respiratory distress as term newborn. Two of them had situs inversus totalis. Fractional eNO measurement revealed extremely low NO levels, and subsequently, TEM analysis confirmed ciliary ultrastructural defects in all patients. One patient had compound heterozygous mutation of the PCD-causative gene ( DNAH5) identified using next generation sequencing. Conclusion: Our report stresses the reliability of eNO measurement in the diagnosis of PCD, accompanied by clinical phenotypes and additional diagnostic tools, such as TEM analysis and genetic testing.

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The Novel Compound Heterozygous Mutations of ECEL1 Identified in a Family with Distal Arthrogryposis Type 5D

BioMed Research International ◽

10.1155/2020/2149342 ◽

2020 ◽

Vol 2020 ◽

pp. 1-6

Author(s):

Jie-Yuan Jin ◽

Dan-Yu Liu ◽

Zi-Jun Jiao ◽

Yi Dong ◽

Jie Li ◽

...

Keyword(s):

Mutational Analysis ◽

Clinical Symptoms ◽

Prenatal Development ◽

Compound Heterozygous ◽

Arthrogryposis Multiplex Congenita ◽

Causative Gene ◽

Distal Arthrogryposis ◽

Axonal Arborization ◽

Motor Nerves ◽

Compound Heterozygous Mutations

Introduction. Distal arthrogryposis type 5D (DA5D) is an autosomal recessive disease. The clinical symptoms include contractures of the joints of limbs, especially camptodactyly of the hands and/or feet, unilateral ptosis, a round-shaped face, arched eyebrows, and micrognathia, without ophthalmoplegia. ECEL1 is a DA5D causative gene that encodes a membrane-bound metalloprotease. ECEL1 plays important roles in the final axonal arborization of motor nerves in limb skeletal muscles and neuromuscular junction formation during prenatal development. Methods. A DA5D family with webbing of the elbows and fingers was recruited. We performed whole-exome sequencing (WES) and filtered mutations by disease-causing genes of arthrogryposis multiplex congenita (AMC). Mutational analysis and cosegregation confirmation were then performed. Results. We identified novel compound heterozygous mutations of ECEL1 (NM_004826: c.69C>A, p.C23∗ and c.1810G>A, p.G604R) in the proband. Conclusions. We detected causative mutations in a DA5D family, expanding the spectrum of known ECEL1 mutations and contributing to the clinical diagnosis of DA5D.

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Unique three-site compound heterozygous mutation in the WFS1 gene in Wolfram syndrome

BMC Endocrine Disorders ◽

10.1186/s12902-021-00823-5 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Ziyu Ren ◽

Jixiu Yi ◽

Min Zhong ◽

Yunting Wang ◽

Qicong Liu ◽

...

Keyword(s):

Genetic Disease ◽

Target Genes ◽

Novel Mutation ◽

Clinical Manifestations ◽

Wolfram Syndrome ◽

Compound Heterozygous Mutation ◽

Heterozygous Mutation ◽

Compound Heterozygous ◽

Compound Heterozygous Mutations ◽

Wfs1 Gene

Abstract Background Wolfram syndrome (WFS) is a rare autosomal recessive genetic disease whose main cause is mutations in the WFS1 and CISD2 genes. Its characteristic clinical manifestations are diabetes insipidus, diabetes mellitus, optic atrophy and deafness. Methods In this study, two patients from this particular family underwent complete routine biochemical and ophthalmic tests. Blood, urine, routine stool test, visual acuity (VA) examination, visual field assessment, funduscope, optical coherence tomography and periorbital magnetic resonance imaging (MRI) scans were performed for each patient to evaluate whether the nerve fiber layer around the optic nerve head was atrophied and next-generation sequencing of target genes was performed in two patients. Results When the patients were diagnosed with Wolfram syndrome, their genetic analyses suggested unique three-site compound heterozygous mutations (c.2314C > T + c.2194C > T + c.2171C > T) in exon 8 of both patients’ chromosome 4. One mutation (c.2314C > T) was a novel mutation in the known reports of Wolfram syndrome. As a degenerative genetic disease, the types of gene mutations in the Chinese population are generally homozygous mutations at the unit point or compound heterozygous mutations at two nucleotide change sites. However, the two patients reported in this study are the first known cases of compound heterozygous mutations with three mutation sites coexisting on the WFS1 gene in China or even globally. Conclusions This study expands the phenotypic spectrum of Wolfram syndrome and may reveal a novel mutation pattern of pathogenesis of Wolfram syndrome. The implications of this discovery are valuable in the clinical diagnosis, prognosis, and treatment of patients with WFS1.

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New Compound Heterozygous Mutations of GPIIb in Patient with Glanzmann Thrombasthenia.

Blood ◽

10.1182/blood.v110.11.3921.3921 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3921-3921

Author(s):

Ziqiang Yu ◽

Jian Su ◽

Xia Bai ◽

Zhaoyue Wang ◽

Changgeng Ruan

Keyword(s):

Flow Cytometry ◽

Autosomal Recessive ◽

Protein Translation ◽

Compound Heterozygous Mutation ◽

Heterozygous Mutation ◽

Compound Heterozygous ◽

Sequencing Analysis ◽

Glanzmann Thrombasthenia ◽

Compound Heterozygous Mutations ◽

Average Fluorescence

Abstract Glanzmann thrombasthenia (GT) is a homozygous or compound heterozygous autosomal recessive bleeding disorder caused by the qualitative or quantitative deficiency of integrin GPIIb-IIIa, which acts as the receptor of platelet fibrinogen. Here we report a case of GT with a compound heterozygous mutation in GPIIb according to the results of flow cytometry and genetic investigation.The flow cytometry was used to measure the average amounts of integrin GPIIb-IIIa on the patient’s platelets, and all 30 exons of GPIIb were amplified and sequenced with the corresponding primers.The average fluorescence intensity of integrin GPIIb-IIIa were 3.07 and 12.5, respectively, compared with 23.7 and 254, respectively, in the normal healthy individuals. And sequencing analysis of all exons of GPIIb demonstrated that there existed following compound heterozygous mutations in GPIIb gene: one heterozygote mutation (68 C→A) in the 1st exon, which resulted in Pro 23 His substitution in signal peptide domain; one nonsense heterozygous mutation (1750 C→T) in the 17th exon, which result in premature termination; one heterozygote mutation (2159 T→C) in the 21stexon, which resulted in Leu 720 Pro substitution. According to Glanzmann thrombasthenia database of ISTH (http://sinaicentral.mssm.edu/intranet/research/glanzmann/listmutations?mut=GPIIb), 68 C→A mutation and 2159 T→C mutation are novel mutations in the GPIIb heavy chain. These compound heterozygous mutations in GPIIb gene might be a novel pathogenetic mechanism of GT, which impaired the protein translation and co-expression with GPIIIa on the membrane of platelet.

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Aldosterone signaling defect in young infants: single-center report and review

BMC Endocrine Disorders ◽

10.1186/s12902-021-00811-9 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Melati Wijaya ◽

Huamei Ma ◽

Jun Zhang ◽

Minlian Du ◽

Yanhong Li ◽

...

Keyword(s):

Clinical Picture ◽

Heterozygous Mutation ◽

Compound Heterozygous ◽

Homozygous Mutation ◽

Hydroxylase Deficiency ◽

Salt Wasting ◽

Young Infants ◽

Adrenal Imaging ◽

Compound Heterozygous Mutations ◽

21 Hydroxylase Deficiency

Abstract Background Aldosterone (Ald) is a crucial factor in maintaining electrolyte and water homeostasis. Defect in either its synthesis or function causes salt wasting (SW) manifestation. This disease group is rare, while most reported cases are sporadic. This study aimed to obtain an overview of the etiology and clinical picture of patients with the above condition and report our rare cases. Methods A combination of retrospective review and case studies was conducted at the Pediatric Endocrine unit of The First Affiliated Hospital Sun Yat Sen University from September 1989 to June 2020. Results A total of 187 patients with SW were enrolled, of which 90.4% (n = 169) were diagnosed with congenital adrenal hyperplasia (CAH). SW type 21-hydroxylase deficiency accounted for 98.8% (n = 167) of CAH diagnosis, while 1.2% (n = 2) was of lipoid CAH. Non-CAH comprised 9.6% (n = 18) of the total patients whose etiologies included SF-1 gene mutation (n = 1), X-linked adrenal hypoplasia congenita (n = 9), aldosterone synthase deficiency (ASD, n = 4), and pseudo-hypoaldosteronism type 1 (PHA1, n = 1). Etiologies were not identified in three patients. All of patients with ASD and PHA1 exhibited SW syndrome in their early neonatal period. DNA sequencing showed mutations of CYP11B2 for P1-P4 and NR3C2 for P5. P1 and P2 were sibling brothers affected by compound heterozygous mutations of c.1121G > A (p.R374Q) and c.1486delC p.(L496fs); likewise, P4 was identified with compound heterozygous mutations of c.1200 + 1G > A and c.240–1 G > T; meanwhile P3 demonstrated c.1303G > A p.(G435S) homozygous mutation in CYP11B2 gene. Lastly, P5 showed c.1768 C > T p.(R590*) heterozygous mutation in the NR3C2 gene. Conclusion Etiology of infant with aldosterone defect was mostly congenital. Renal and adrenal imaging are recommended to exclude renal causes. If clinical picture is suggestive, normal plasma Ald in early infancy cannot rule out aldosterone insufficiency.

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Differential Allelic Expression among Long Non-Coding RNAs

Non-Coding RNA ◽

10.3390/ncrna7040066 ◽

2021 ◽

Vol 7 (4) ◽

pp. 66

Author(s):

Michael B. Heskett ◽

Paul T. Spellman ◽

Mathew J. Thayer

Keyword(s):

Rna Polymerase Ii ◽

Allelic Expression ◽

Protein Coding ◽

Differential Allelic Expression ◽

Cellular Processes ◽

Disease States ◽

Transcriptional Pattern ◽

In Trans ◽

Non Coding Rnas ◽

In Cis

Long non-coding RNAs (lncRNA) comprise a diverse group of non-protein-coding RNAs >200 bp in length that are involved in various normal cellular processes and disease states, and can affect coding gene expression through mechanisms in cis or in trans. Since the discovery of the first functional lncRNAs transcribed by RNA Polymerase II, H19 and Xist, many others have been identified and noted for their unusual transcriptional pattern, whereby expression from one chromosome homolog is strongly favored over the other, also known as mono-allelic or differential allelic expression. lncRNAs with differential allelic expression have been observed to play critical roles in developmental gene regulation, chromosome structure, and disease. Here, we will focus on known examples of differential allelic expression of lncRNAs and highlight recent research describing functional lncRNAs expressed from both imprinted and random mono-allelic expression domains.

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