A Stop-Gain Mutation within MLPH Is Responsible for the Lilac Dilution Observed in Jacob Sheep

A coat color dilution, called lilac, was observed within the Jacob sheep breed. This dilution results in sheep appearing gray, where black would normally occur. Pedigree analysis suggested an autosomal recessive inheritance. Whole-genome sequencing of a dilute case, a known carrier, and sixteen non-dilute sheep was used to identify the molecular variant responsible for the coat color change. Through investigation of the genes MLPH, MYO5A, and RAB27A, we discovered a nonsynonymous mutation within MLPH, which appeared to match the reported autosomal recessive nature of the lilac dilution. This mutation (NC_019458.2:g.3451931C>A) results in a premature stop codon being introduced early in the protein (NP_001139743.1:p.Glu14*), likely losing its function. Validation testing of additional lilac Jacob sheep and known carriers, unrelated to the original case, showed a complete concordance between the mutation and the dilution. This stop-gain mutation is likely the causative mutation for dilution within Jacob sheep.

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Mutation in the RPE65 gene causing hereditary retinal dystrophy in the Briard dogs: application of a new detection method

Czech Journal of Animal Science ◽

10.17221/372-cjas ◽

2008 ◽

Vol 53 (No. 4) ◽

pp. 176-179

Author(s):

R. Bechyňová ◽

J. Dostál ◽

A. Stratil ◽

F. Jílek ◽

P. Horák

Keyword(s):

Autosomal Recessive ◽

Clinical Symptoms ◽

Stop Codon ◽

Retinal Dystrophy ◽

Premature Stop Codon ◽

Causative Mutation ◽

Functional Protein ◽

Hereditary Retinal Dystrophy ◽

Briard Dogs ◽

Autosomal Recessive Manner

Inherited eye diseases are widespread in most of the pure dog breeds and they show a severe impact on canine health, welfare and working ability. Congenital stationary night blindness (CSNB) was originally described in Briards. CSNB is slow progressive retinal degeneration with very early onset of clinical symptoms and is inherited in an autosomal recessive manner. The causative mutation (Y16567.1:c.487_490delAAGA) for CSNB was identified in exon 5 of the RPE65 gene. This deletion results in a frameshift and leads to a premature stop codon and expression of a non-functional protein. To date, only expensive, laborious or unpractical methods have been used for detection of the mutation in the canine RPE65 gene. The main goals of this study were to develop a new method for routine genotyping of the causative mutation and to assess its occurrence in the Czech population of Briards. The method of electrophoresis in the gel Spraedex EL600 can be widely used for genotyping of the RPE65 gene as a basis of proper genetic counselling and an improvement of genetic health in the Briard populations. In the studied population, the following frequencies of alleles + (wild) and – (mutant) were observed – 0.939 and 0.061, respectively.

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An Autosomal-Recessive GFI1B Mutation Defines the Splice Isoform p37 As Essential for Biogenesis of Functional Human Platelets, but Dispensable for Erythropoiesis

Blood ◽

10.1182/blood.v128.22.2644.2644 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2644-2644

Author(s):

Harald Schulze ◽

Axel Schlagenhauf ◽

Georgi Manukjan ◽

Christine Beham-Schmid ◽

Oliver Andres ◽

...

Keyword(s):

Alternative Splicing ◽

Growth Factor ◽

Autosomal Recessive ◽

Stop Codon ◽

Zinc Fingers ◽

Premature Stop Codon ◽

Index Patient ◽

Homozygous Mutation ◽

Recessive Trait ◽

Erythroid Progenitor

Abstract Growth factor independent 1 (GFI1) and Growth Factor Independent 1B (GFI1B) are zinc finger transcriptional repressors that share about 90% homology on amino acid sequence and are expressed during hematopoiesis. While GFI1 is most important for granulocyte-monocyte lineage commitment, GFI1B is an essential master regulator of erythroid and megakaryocytic lineages. Mice lacking Gfi1b are embryonic lethal due to anemia and thrombocytopenia. In humans, alternative splicing leads to a shorter p32 isoform that lacks the first 2 of 6 zinc fingers. GFI1B germline mutations have been reported to cause autosomal-dominant macrothrombocytopenia with a grey-platelet syndrome phenotype, implying that the mutant protein acts in a dominant-negative manner. We report on a Chechen family from eastern Georgia whose affected family members all present with severe, life-threatening bleeding diathesis. The female index patient had recurrent hematomata and multiple petechiae since childhood. Both of her children (age 9 and 7) present with very low platelet counts (below 45/nL) and a similar cutaneous bleeding pattern like her mother. The brother also had thrombocytopenia and died at age 33 in reponse to a spontaneous cerebral hemorrhage. In contrast, the index patient's husband, her parents and the children of the deceased brother were clinically unaffected. Blood smears of affected patients showed macrothrombocytopenia with reduced May-Grünwald-Giema staining and decreased staining for alpha-granule markers von Willebrand factor (vWF) and P-selectin (CD62P). Platelet function testing revealed reduced responses to ADP, collagen, TRAP-6 and arachidonic acid. White and red blood cell parameters were overall normal in the index patient and the two affected children. We analyzed DNA from the index patient by targeted next generation sequencing for 59 genes relevant for platelet formation or function. We found a novel homozygous single nucleotide insertion in GFI1B (NM_004188.5; c.551insG), which was confirmed by Sanger sequencing and is expected to cause a premature stop-codon. The homozygous mutation co-segregated with the phenotype. The unaffected mother, the husband and two unaffected nephews were heterozygous, suggesting a local founder variant and an unexpected autosomal-recessive trait. Bone marrow analysis showed unaffected myeloid and erythroid cells, but dysplastic micromegakaryocytes with increased CD34 staining. Peripheral blood platelets were also positive for CD34. We performed quantitative real-time PCR of platelet RNA and found residual homozygous c.551_G insertion in the p37 transcript and an unexpected expression of the p32 variant. The p37 transcript was markedly reduced in context with an increased p32/p37 ratio compared to controls. Our findings indicate that the mutated transcript was not completely degraded by nonsense-mediated decay, but mostly subjected to alternative splicing skipping the mutated exon 9. Our findings imply that the first two zinc fingers of GFI1B are dispensable for human erythropoiesis, but essential for normal megakaryopoiesis and the production of functional platelets. While previous mutations affect both isoforms, the insertion variant presented here, results in a premature stop-codon and affects only the p37 isoform due to alternative splicing. This splice variant defines an important node at the megakaryocytic-erythroid progenitor stage and we conclude that the transcriptional regulation of erythropoiesis is uncoupled from that of megakaryopoiesis through alternative splicing of GFI1B. Disclosures No relevant conflicts of interest to declare.

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Premature Stop Codon in MMP20 Causing Amelogenesis Imperfecta

Journal of Dental Research ◽

10.1177/154405910808700109 ◽

2008 ◽

Vol 87 (1) ◽

pp. 56-59 ◽

Cited By ~ 51

Author(s):

P. Papagerakis ◽

H.-K. Lin ◽

K.Y. Lee ◽

Y. Hu ◽

J.P. Simmer ◽

...

Keyword(s):

Autosomal Recessive ◽

Proteolytic Enzymes ◽

Stop Codon ◽

Translation Termination ◽

Premature Stop Codon ◽

Dental Enamel ◽

Amino Acid Position ◽

Amelogenesis Imperfecta ◽

Exon 1 ◽

Kallikrein 4

Proteolytic enzymes are necessary for the mineralization of dental enamel during development, and mutations in the kallikrein 4 ( KLK4) and enamelysin ( MMP20) genes cause autosomal-recessive amelogenesis imperfecta (ARAI). So far, only one KLK4 and two MMP20 mutations have been reported. We have identified an ARAI-causing point mutation (c.102G>A, g.102G>A, and p.W34X) in exon 1 of MMP20 in a proband with autosomal-recessive hypoplastic-hypomaturation amelogenesis imperfecta. The G to A transition changes the tryptophan (W) codon (TGG) at amino acid position 34 into a translation termination (X) codon (TGA). No disease-causing sequence variations were detected in KLK4. The affected enamel is thin, with mild spacing in the anterior dentition. The enamel layer is hypomineralized, does not contrast with dentin on radiographs, and tends to chip away from the underlying dentin. An intrinsic yellowish pigmentation is evident, even during eruption. The phenotype supports current ideas concerning the function of enamelysin.

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Novel Mutations Segregating with Complete Androgen Insensitivity Syndrome and their Molecular Characteristics

International Journal of Molecular Sciences ◽

10.3390/ijms20215418 ◽

2019 ◽

Vol 20 (21) ◽

pp. 5418

Author(s):

Malcher ◽

Jedrzejczak ◽

Stokowy ◽

Monem ◽

Nowicka-Bauer ◽

...

Keyword(s):

Stop Codon ◽

Premature Stop Codon ◽

Androgen Insensitivity Syndrome ◽

Molecular Characteristics ◽

Heterozygous Mutation ◽

Transactivation Domain ◽

Rna Seq ◽

Nonsynonymous Mutation ◽

Complete Androgen Insensitivity Syndrome ◽

Androgen Insensitivity

We analyzed three cases of Complete Androgen Insensitivity Syndrome (CAIS) and report three hitherto undisclosed causes of the disease. RNA-Seq, Real-timePCR, Western immunoblotting, and immunohistochemistry were performed with the aim of characterizing the disease-causing variants. In case No.1, we have identified a novel androgen receptor (AR) mutation (c.840delT) within the first exon in the N-terminal transactivation domain. This thymine deletion resulted in a frameshift and thus introduced a premature stop codon at amino acid 282. In case No.2, we observed a nonsynonymous mutation in the ligand-binding domain (c.2491C>T). Case No.3 did not reveal AR mutation; however, we have found a heterozygous mutation in CYP11A1 gene, which has a role in steroid hormone biosynthesis. Comparative RNA-Seq analysis of CAIS and control revealed 4293 significantly deregulated genes. In patients with CAIS, we observed a significant increase in the expression levels of PLCXD3, TM4SF18, CFI, GPX8, and SFRP4, and a significant decrease in the expression of SPATA16, TSACC, TCP10L, and DPY19L2 genes (more than 10-fold, p < 0.05). Our findings will be helpful in molecular diagnostics of patients with CAIS, as well as the identified genes could be also potential biomarkers for the germ cells differentiation process.

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Autosomal Recessive Oculodentodigital Dysplasia: A Case Report and Review of the Literature

Cytogenetic and Genome Research ◽

10.1159/000489000 ◽

2018 ◽

Vol 154 (4) ◽

pp. 181-186 ◽

Cited By ~ 2

Author(s):

Elifcan Taşdelen ◽

Ceren D. Durmaz ◽

Halil G. Karabulut

Keyword(s):

Autosomal Recessive ◽

Autosomal Dominant ◽

Stop Codon ◽

Premature Stop Codon ◽

Rare Condition ◽

Clinical Findings ◽

Homozygous Mutation ◽

Nasal Bridge ◽

Oculodentodigital Dysplasia ◽

Autosomal Recessive Manner

Oculodentodigital dysplasia (ODDD) is a rare condition characterized by a typical facial appearance and variable findings of the eyes, teeth, and fingers. ODDD is caused by mutations in the GJA1 gene in chromosome 6q22 and inherited in an autosomal dominant manner in the majority of the patients. However, in recent clinical reports, autosomal recessive ODDD cases due to by GJA1 mutations were also described. Here, we report on a 14-year-old boy with microphthalmia, microcornea, narrow nasal bridge, hypoplastic alae nasi, prominent columnella, hypodontia, dental caries, and partial syndactyly of the 2nd and 3rd toes. These clinical findings were concordant with the diagnosis of ODDD, and a novel homozygous mutation (c.442C>T, p.Arg148Ter) was determined in the GJA1 gene leading to a premature stop codon. His phenotypically normal parents were found to be carriers of the same mutation. This is the third family in the literature in which ODDD segregates in an autosomal recessive manner.

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Patient-specific iPSC-derived photoreceptor precursor cells as a means to investigate retinitis pigmentosa

eLife ◽

10.7554/elife.00824 ◽

2013 ◽

Vol 2 ◽

Cited By ~ 114

Author(s):

Budd A Tucker ◽

Robert F Mullins ◽

Luan M Streb ◽

Kristin Anfinson ◽

Mari E Eyestone ◽

...

Keyword(s):

Pluripotent Stem Cells ◽

Protein Misfolding ◽

Autosomal Recessive ◽

Stop Codon ◽

Premature Stop Codon ◽

Photoreceptor Cells ◽

Precursor Cells ◽

Patient Specific ◽

Basal Bodies ◽

Induced Pluripotent

Next-generation and Sanger sequencing were combined to identify disease-causing USH2A mutations in an adult patient with autosomal recessive RP. Induced pluripotent stem cells (iPSCs), generated from the patient’s keratinocytes, were differentiated into multi-layer eyecup-like structures with features of human retinal precursor cells. The inner layer of the eyecups contained photoreceptor precursor cells that expressed photoreceptor markers and exhibited axonemes and basal bodies characteristic of outer segments. Analysis of the USH2A transcripts of these cells revealed that one of the patient’s mutations causes exonification of intron 40, a translation frameshift and a premature stop codon. Western blotting revealed upregulation of GRP78 and GRP94, suggesting that the patient’s other USH2A variant (Arg4192His) causes disease through protein misfolding and ER stress. Transplantation into 4-day-old immunodeficient Crb1−/− mice resulted in the formation of morphologically and immunohistochemically recognizable photoreceptor cells, suggesting that the mutations in this patient act via post-developmental photoreceptor degeneration.

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A Third MLPH Variant Causing Coat Color Dilution in Dogs

Genes ◽

10.3390/genes11060639 ◽

2020 ◽

Vol 11 (6) ◽

pp. 639

Author(s):

Samantha L. Van Buren ◽

Katie M. Minor ◽

Robert A. Grahn ◽

James R. Mickelson ◽

Jennifer C. Grahn ◽

...

Keyword(s):

Genetic Testing ◽

Coat Color ◽

Stop Codon ◽

Premature Stop Codon ◽

Low Frequency ◽

Allelic Heterogeneity ◽

Recessive Inheritance ◽

Compound Heterozygotes

Altered melanosome transport in melanocytes, resulting from variants in the melanophilin (MLPH) gene, are associated with inherited forms of coat color dilution in many species. In dogs, the MLPH gene corresponds to the D locus and two variants, c.−22G > A (d1) and c.705G > C (d2), leading to the dilution of coat color, as described. Here, we describe the independent investigations of dogs whose coat color dilution could not be explained by known variants, and who report a third MLPH variant, (c.667_668insC) (d3), which leads to a frameshift and premature stop codon (p.His223Profs*41). The d3 allele is found at low frequency in multiple dog breeds, as well as in wolves, wolf-dog hybrids, and indigenous dogs. Canids in which the d3 allele contributed to the grey (dilute) phenotype were d1/d3 compound heterozygotes or d3 homozygotes, and all non-dilute related dogs had one or two D alleles, consistent with a recessive inheritance. Similar to other loci responsible for coat colors in dogs, this, alongside likely additional allelic heterogeneity at the D locus, or other loci, must be considered when performing and interpreting genetic testing.

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A Frameshift Mutation in MC1R and a High Frequency of Somatic Reversions Cause Black Spotting in Pigs

Genetics ◽

10.1093/genetics/158.2.779 ◽

2001 ◽

Vol 158 (2) ◽

pp. 779-785 ◽

Cited By ~ 2

Author(s):

J M H Kijas ◽

M Moller ◽

G Plastow ◽

L Andersson

Keyword(s):

Frameshift Mutation ◽

Coat Color ◽

Stop Codon ◽

Phenotypic Expression ◽

Premature Stop Codon ◽

Black Spot ◽

White Background ◽

Coding Region ◽

Black Spots ◽

Color Sequence

Abstract Black spotting on a red or white background in pigs is determined by the EP allele at the MC1R/Extension locus. A previous comparison of partial MC1R sequences revealed that EP shares a missense mutation (D121N) with the ED2 allele for dominant black color. Sequence analysis of the entire coding region now reveals a second mutation in the form of a 2-bp insertion at codon 23 (nt67insCC). This mutation expands a tract of six C nucleotides to eight and introduces a premature stop codon at position 56. This frameshift mutation is expected to cause a recessive red color, which was in fact observed in some breeds with the EP allele present (Tamworth and Hereford). RT-PCR analyses were conducted using skin samples taken from both spotted and background areas of spotted pigs. The background red area had transcript only from the mutant nt67insCC MC1R allele, whereas the black spot also contained a transcript without the 2-bp insertion. This indicates that black spots are due to somatic reversion events that restore the frame and MC1R function. The phenotypic expression of the EP allele is highly variable and the associated coat color ranges from red, red with black spots, white with black spots, to almost completely solid black. In several breeds of pigs the phenotypic manifestation of this allele has been modified by selection for or against black spots.

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AKNA Frameshift Variant in Three Dogs with Recurrent Inflammatory Pulmonary Disease

Genes ◽

10.3390/genes10080567 ◽

2019 ◽

Vol 10 (8) ◽

pp. 567 ◽

Cited By ~ 1

Author(s):

Hug ◽

Anderegg ◽

Kehl ◽

Jagannathan ◽

Leeb

Keyword(s):

Pulmonary Disease ◽

Clinical Symptoms ◽

Stop Codon ◽

Family Members ◽

Premature Stop Codon ◽

Homozygosity Mapping ◽

Pedigree Analysis ◽

Gene Encoding ◽

Reading Frame ◽

Inflammatory Processes

We investigated three related Rough Collies with recurrent inflammatory pulmonary disease. The clinical symptoms were similar to primary ciliary dyskinesia (PCD). However, the affected dogs did not carry any known pathogenic PCD variants. Pedigree analysis suggested a recessive mode of inheritance. Combined linkage and homozygosity mapping in three cases and seven non-affected family members delineated 19 critical intervals on 10 chromosomes comprising a total of 99 Mb. The genome of one affected dog was sequenced and compared to 601 control genomes. We detected only a single private homozygous protein-changing variant in the critical intervals. The detected variant was a 4 bp deletion, c.2717_2720delACAG, in the AKNA gene encoding the AT-hook transcription factor. It causes a frame-shift introducing a premature stop codon and truncates 37% of the open reading frame, p.(Asp906Alafs*173). We genotyped 88 Rough Collies consisting of family members and unrelated individuals. All three available cases were homozygous for the mutant allele and all 85 non-affected dogs were either homozygous wildtype (n = 67) or heterozygous (n = 18). AKNA modulates inflammatory immune responses. Akna−/− knockout mice die shortly after birth due to systemic autoimmune inflammatory processes including lung inflammation that is accompanied by enhanced leukocyte infiltration and alveolar destruction. The perfect genotype-phenotype association and the comparative functional data strongly suggest that the detected AKNA:c.2717_2720delACAG variant caused the observed severe airway inflammation in the investigated dogs. Our findings enable genetic testing, which can be used to avoid the unintentional breeding of affected puppies.

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A Premature Stop Codon in RAF1 Is the Priority Candidate Causative Mutation of the Inherited Chicken Wingless-2 Developmental Syndrome

Genes ◽

10.3390/genes10050353 ◽

2019 ◽

Vol 10 (5) ◽

pp. 353

Author(s):

Ingrid Youngworth ◽

Mary E. Delany

Keyword(s):

Signaling Pathway ◽

Limb Development ◽

Cellular Proliferation ◽

Stop Codon ◽

Point Mutations ◽

Premature Stop Codon ◽

Mapk Signaling ◽

Candidate Region ◽

Causative Mutation ◽

Causative Gene

The chicken wingless-2 (wg-2) mutation is inherited in an autosomal recessive fashion, and the resulting phenotype in mutant (wg-2/wg-2) individuals is a developmental syndrome characterized by absent wings, truncated legs, craniofacial as well as skin and feather defects, and kidney malformations. Mapping and genotyping established that the mutation resides within 227 kilobases (kb) of chromosome 12 in a wg-2 congenic inbred line. A capture array was designed to target and sequence the candidate region along with flanking DNA in 24 birds from the line. Many point mutations and insertions or deletions were identified, and analysis of the linked variants indicated a point mutation predicted to cause a premature stop codon in the RAF1 gene. Expression studies were conducted inclusive of all genes in the candidate region. Interestingly, RAF1 transcription was elevated, yet the protein was absent in the mutants relative to normal individuals. RAF1 encodes a protein integral to the Ras/Raf/MAPK signaling pathway controlling cellular proliferation, and notably, human RASopathies are developmental syndromes caused by germline mutations in genes of this pathway. Our work indicates RAF1 as the priority candidate causative gene for wg-2 and provides a new animal model to study an important signaling pathway implicated in limb development, as well as RASopathies.

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