In silico Derivation of HLA-Specific Alloreactivity Potential from Whole Exome Sequencing of Stem-Cell Transplant Donors and Recipients: Understanding the Quantitative Immunobiology of Allogeneic Transplantation

AbstractObjectiveThe objective of this study is to identify the genetic cause of disease in a congenital form of congenital spinal muscular atrophy and arthrogryposis (CSMAA).MethodsA 2-year-old boy was diagnosed with arthrogryposis multiplex congenita, severe skeletal abnormalities, torticollis, vocal cord paralysis and diminished lower limb movement. Whole exome sequencing was performed on the proband and family members. In silico modeling of protein structure and heterologous protein expression and cytotoxicity assays were performed to validate pathogenicity of the identified variant.ResultsWhole exome sequencing revealed a homozygous mutation in the TRPV4 gene (c.281C>T; p.S94L). The identification of a recessive mutation in TRPV4 extends the spectrum of mutations in recessive forms of the TRPV4-associated disease. p.S94L and other previously identified TRPV4 variants in different protein domains were compared in structural modeling and functional studies. In silico structural modeling suggests that the p.S94L mutation is in the disordered N-terminal region proximal to important regulatory binding sites for phosphoinositides and for PACSIN3, which could lead to alterations in trafficking and/or channel sensitivity. Functional studies by western blot and immunohistochemical analysis show that p.S94L reduces TRPV4 protein stability because of increased cytotoxicity and therefore involves a gain-of-function mechanism.ConclusionThis study identifies a novel homozygous mutation in TRPV4 as a cause of the recessive form of congenital spinal muscular atrophy and arthrogryposis.

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Hemolytic anemia with null PKLR mutations identified using whole exome sequencing and cured by hematopoietic stem cell transplantation combined with splenectomy

Bone Marrow Transplantation ◽

10.1038/bmt.2016.218 ◽

2016 ◽

Vol 51 (12) ◽

pp. 1605-1608 ◽

Cited By ~ 2

Author(s):

M Kim ◽

J Park ◽

J Lee ◽

W Jang ◽

H Chae ◽

...

Keyword(s):

Stem Cell ◽

Hematopoietic Stem Cell Transplantation ◽

Stem Cell Transplantation ◽

Exome Sequencing ◽

Cell Transplantation ◽

Whole Exome Sequencing ◽

Hematopoietic Stem Cell ◽

Hemolytic Anemia ◽

Hematopoietic Stem ◽

Whole Exome

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Whole exome sequencing identifies rare coding variants in novel human-mouse ortholog genes in African individuals diagnosed with non-syndromic hearing impairment

Experimental Biology and Medicine ◽

10.1177/1535370220960388 ◽

2020 ◽

pp. 153537022096038

Author(s):

Oluwafemi G Oluwole ◽

Kevin K Esoh ◽

Edmond Wonkam-Tingang ◽

Noluthando Manyisa ◽

Jean Jacques Noubiap ◽

...

Keyword(s):

Exome Sequencing ◽

Hearing Impairment ◽

Whole Exome Sequencing ◽

In Silico ◽

Homozygous Variant ◽

Whole Exome ◽

African Populations ◽

Mouse Orthologs ◽

Syndromic Hearing Impairment ◽

Functional Analyses

Physiologically, the human and murine hearing systems are very similar, justifying the extensive use of mice in experimental models for hearing impairment (HI). About 340 murine HI genes have been reported; however, whether variants in all human-mouse ortholog genes contribute to HI has been rarely investigated. In humans, nearly 120 HI genes have been identified to date, with GJB2 and GJB6 variants accounting for half of congenital HI cases, of genetic origin, in populations of European and Asian ancestries, but not in most African populations. The contribution of variants in other known genes of HI among the populations of African ancestry is poorly studied and displays the lowest pick-up rate. We used whole exome sequencing (WES) to investigate pathogenic and likely pathogenic (PLP) variants in 34 novel human-mouse orthologs HI genes, in 40 individuals from Cameroon and South Africa diagnosed with non-syndromic hearing impairment (NSHI), and compared the data to WES data of 129 ethnically matched controls. In addition, protein modeling for selected PLP gene variants, gene enrichment, and network analyses were performed. A total of 4/38 murine genes, d6wsu163e, zfp719, grp152 and minar2, had no human orthologs. WES identified three rare PLP variants in 3/34 human-mouse orthologs genes in three unrelated Cameroonian patients, namely: OCM2, c.227G>C p.(Arg76Thr) and LRGI1, c.1657G>A p.(Gly533Arg) in a heterozygous state, and a PLP variant MCPH1, c.2311C>G p.(Pro771Ala) in a homozygous state. In silico functional analyses suggest that these human-mouse ortholog genes functionally co-expressed interactions with well-established HI genes: GJB2 and GJB6. The study found one homozygous variant in MCPH1, likely to explain HI in one patient, and suggests that human-mouse ortholog variants could contribute to the understanding of the physiology of hearing in humans. Impact statement Despite, human and murine hearing system being very similar, the contribution of variants in relevant mouse-ortholog genes to hearing impairment (HI) has not been fully investigated. The contribution of variants in the known non-syndromic hearing impairment (NSHI) genes among Africans is poorly studied, suggesting that the novel gene(s) and mutations are yet to be discovered in NSHI in the African populations. Using whole exome sequencing (WES), this study identified rare candidate pathogenic and likely pathogenic (PLP) variants in 3/34 novel human-mouse ortholog genes in 3/40 individuals, with one homozygous variant, MCPH1, c.2311C>G p.(Pro771Ala), likely to explain HI in one patient. In silico functional analyses suggest that these human-mouse ortholog genes could contribute to the understanding of the physiology of hearing in humans and thus the variants identified in those genes deserve additional investigations.

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Whole exome sequencing to estimate alloreactivity potential between donors and recipients in stem cell transplantation

British Journal of Haematology ◽

10.1111/bjh.12898 ◽

2014 ◽

Vol 166 (4) ◽

pp. 566-570 ◽

Cited By ~ 23

Author(s):

Juliana K. Sampson ◽

Nihar U. Sheth ◽

Vishal N. Koparde ◽

Allison F. Scalora ◽

Myrna G. Serrano ◽

...

Keyword(s):

Stem Cell ◽

Stem Cell Transplantation ◽

Exome Sequencing ◽

Cell Transplantation ◽

Whole Exome Sequencing ◽

Whole Exome

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Whole Exome Sequencing Identifies Novel Lyst-Missense Mutations In Incomplete Childhood Chediak-Higashi-Syndrome Presenting As Hemphagocytic Lymphohistiocytosis (HLH)

Blood ◽

10.1182/blood.v122.21.3479.3479 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3479-3479

Author(s):

Joachim Kunz ◽

Tobias Rausch ◽

Obul R Bandapalli ◽

Adrian M Stuetz ◽

Johann Greil ◽

...

Keyword(s):

Stem Cell ◽

Genetic Counseling ◽

Stem Cell Transplantation ◽

Exome Sequencing ◽

Whole Exome Sequencing ◽

Missense Mutations ◽

Heterozygous Genotype ◽

Whole Exome ◽

Genomic Analyses ◽

Chediak Higashi Syndrome

Abstract Chediak Higashi Syndrome (CHS) is caused by defective membrane targeting of components of the lysosome, which results from inactivation of the lysosomal trafficking regulator LYST. Clinically, CHS is typically characterized by partial albinism, susceptibility to infection, lymphoproliferation with acceleration to HLH. The immunodeficiency can be cured by allogeneic stem cell transplantation (HSCT), but transplanted patients can develop picture resembling spinocerebellar degeneration in early adult life. Depending on the type of mutation, CHS can vary from a most severe childhood form with null-mutations to milder adult onset forms with hypomorphic mutations. We report on a previously healthy boy, who presented at the age of 3 years with life threatening features of HLH but no clinical features of CHS. The patient was treated with HSCT from an unrelated HLA-identical stem cell donor 4 years ago and is developing normally since. Analysis of HLH candidate genes did not result in the identification of the genetic cause at that time. At the time of the next pregnancy whole exome sequencing of DNA that had been obtained before HSCT was performed to enable specific genetic counseling. The Agilent SureSelect Target Enrichment Kit was used and the captured fragments were sequenced as 100 bp paired reads using an Illumina HiSeq2000 sequencing instrument. All sequenced DNA reads were preprocessed using Trimmomatic (Lohse et al. 2012) to clip adapter contaminations and to trim reads for low quality bases. The remaining reads greater than 36bp were mapped to build hg19 of the human reference genome with Stampy (Lunter & Goodson, 2011), using default parameters. Following such preprocessing, the number of mapped reads was >95% for all samples. Single-nucleotide variants (SNVs) were called using SAMtools mpileup (Li et al. 2009). The number of exonic SNVs varied between 23,741 and 31,418 per sample. To facilitate a fast classification and identification of candidate driver mutations, all identified coding SNVs were comprehensively annotated using the ANNOVAR framework (Wang et al., Nat. Rev. Genet., 2010). To identify possible pathogenic mutations, candidate SNVs were filtered for nonsynonymous, stopgain or stoploss SNVs, requiring an SNV quality greater or equal to 100, and requiring absence of segmental duplications. Only SNVs that were not contained in dbSNP were considered for further analysis. No homozygous and 122 heterozygous SNVs meeting those requirements were identified. Only one gene, LYST, was affected by two different SNVs and was selected for further analysis because of its known relationship to HLH. Sanger sequencing confirmed the compound heterozygous genotype for the two novel LYST missense mutations Q3057K and R3785H in the patient and the heterozygous genotype for one of these mutations in the parents. We then specifically searched for typical features of CHS in the pre-HSCT diagnostic material. The typical large lysosomal granules in blood cells could not be identified. By contrast, light microscopy of the patient’s hair showed a silvery aspect and chunky dyspigmentation in the medulla. Little granular melanin was detected in the hair cortex. Electron microscopy revealed an uneven distribution of pigment and giant melanosomes in some keratinocytes, compatible with a partial albinism. We thus conclude that this patient suffers from an incomplete albeit immunologically most severe Chediak-Higashi syndrome, which led to an early accelerated phase resembling primary HLH. This report highlights the diagnostic power of whole exome sequencing, which enables an unbiased mutation analysis and the identification of unexpected causes of genetic diseases with atypical phenotypes. At the same time, this case also highlights some of the ethical challenges associated with diagnostic genomic analyses: While a specific and clinically validated diagnosis enabled specific genetic counseling, the family now has to face the unexpected uncertainty about the neurologic prognosis of incomplete Chediak-Higashi-syndrome, which may possibly progress into untreatable neurodegeneration during early adulthood despite successful allogeneic stem cell transplantation. Apart from adding to the knowledge of the genetic and phenotypic complexity of CHS, this patient also underlines the necessity of careful counseling before diagnostic genomic analyses are offered to patients and their families. Disclosures: No relevant conflicts of interest to declare.

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A de novo frameshift variant of ANKRD11 (c.1366_1367dup) in a Chinese patient with KBG syndrome

BMC Medical Genomics ◽

10.1186/s12920-021-00920-3 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Jing Chen ◽

Zhongmin Xia ◽

Yulin Zhou ◽

Xiaomin Ma ◽

Xudong Wang ◽

...

Keyword(s):

Short Stature ◽

Exome Sequencing ◽

Developmental Delay ◽

Whole Exome Sequencing ◽

Slow Wave ◽

In Silico ◽

De Novo ◽

Triangular Face ◽

Whole Exome ◽

Kbg Syndrome

Abstract Background KBG syndrome is a rare autosomal dominant genetic disease mainly caused by pathogenic variants of ankyrin repeat domain-containing protein 11 (ANKRD11) or deletions involving ANKRD11. Herein, we report a novel de novo heterozygous frameshift ANKRD11 variant via whole exome sequencing in a Chinese girl with KBG syndrome. Case presentation A 2-year-2-month-old girl presented with a short stature and developmental delay. Comprehensive physical examinations, endocrine laboratory tests and imaging examination were performed. Whole‐exome sequencing and Sanger sequencing were used to detect and confirm the variant associated with KBG in this patient, respectively. The pathogenicity of the variant was further predicted by several in silico prediction tools. The patient was diagnosed as KBG syndrome with a short stature and developmental delay, as well as characteristic craniofacial abnormalities, including a triangular face, long philtrum, wide eyebrows, a broad nasal bridge, prominent and protruding ears, macrodontia of the upper central incisors, dental crowding, and binocular refractive error. Her skeletal anomalies included brachydactyly, fifth finger clinodactyly, and left-skewed caudal vertebrae. Electroencephalographic results generally showed normal background activity with sporadic spikes and slow wave complexes, as well as multiple spikes and slow wave complexes in the bilateral parietal, occipital, and posterior temporal regions during non-rapid-eye-movement sleep. Brain MRI showed a distended change in the bilateral ventricles and third ventricle, as well as malformation of the sixth ventricle. Whole exome sequencing revealed a novel heterozygous frameshift variant in the patient, ANKRD11 c.1366_1367dup, which was predicted to be pathogenic through in silico analysis. The patient had received physical therapy since 4 months of age, and improvement of gross motor dysfunction was evident. Conclusions The results of this study expand the spectrum of ANKRD11 variants in KBG patients and provide clinical phenotypic data for KBG syndrome at an early age. Our study also demonstrates that whole exome sequencing is an effective method for the diagnosis of rare genetic disorders.

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