De Novo Mutations And Mosaicism PDF

Complications of mendelian inheritance patterns De novo mutations and mosaicism Child affected by autosomal dominant disease born to unaffected parents. Why? Non paternity De novo mutation...

Complications of mendelian inheritance patterns De novo mutations and mosaicism Child affected by autosomal dominant disease born to unaffected parents. Why? Non paternity De novo mutation Incomplete penetrance De novo mutation (DNM) Genetic alteration that is present for the first time in one family member Most arise during formation of gametes in one of the parents (germline variant) Rarely, arise post-zygotically during early embryogenesis (germline and/or somatic variants), can give rise to mosaicism NGS trio testing for the identification of de novo variants DNMs identified by WGS/WES in healthy trios (mother, father, child) (Acuna-Hidalgo et al., 2016): 40-80 SNVs per genome, 1-2 in coding sequences 3-9 indels and 0.015 CNVs per genome Inherited variant: De novo variant: mother and child are child is heterozygous for de both heterozygous novo variant, not detected in blood of either parent Figure modified from Acuna-Hidalgo et al, Genome Biology 17, 241 (2016) Acuna-Hidalgo et al, Genome Biology 17, 241 (2016) Box Fig. 1 Technical improvements to the detection of de novo mutations (DNMs). a Trio-based sequencing allows the identification of de novo mutations in an individual. b Increased sequencing coverage benefits the detection of de novo mutations (in blue). Low coverage (upper) reduces the probability that a de novo mutation will be sequenced and called, compared with high sequencing coverage (lower). c Using random tags or unique molecular identifiers (UMIs) decreases the number of false positives (in red) by making consensus calls from all reads with the same UMI. Furthermore, UMIs can be used to remove PCR-derived duplicate reads to determine accurately the allelic ratio. d Long sequencing reads improve mappability, even across difficult genomic regions such as those containing repeats (gray boxes). Additionally, long reads can be used to phase mutations (shown in blue and in green) and generate haplotypes, to help identify the parent of origin of a mutation. IV inherited variant. Parental origin of germline DNMs >80% of DNMs are of paternal origin arising during spermatogenesis (i.e., DNMs occur 3-4x more often in paternal germ cells than in maternal germ cells). DNMs increase in number with parental age – more pronounced with paternal ageing (paternal age effect). Goriely Nat Genet. 2016 Jul 27;48(8):823-4. Figure 1 Gametogenesis differs in females and males. The sperm produced by a 20-year-old male has gone through ∼190 cell divisions (mitoses), and this number increases to ∼650 by the age of 40 years. In contrast, eggs do not replicate after birth. These sex-specific differences in germline biology are likely to explain the 3:1 excess of paternally derived DNMs observed in the progeny. germline cells undergoing mitosis differentiating gametes undergoing meiosis Paternal age effect Goldmann et al Trends Genet. 2019 Nov;35(11):828-839. Figure 2. Estimated Genome Replications in the Male Germline versus Number of De Novo Mutations (DNMs) in Offspring. Green line and green axis indicate the estimated number of genome replications in the male germline. Black dots indicate observations of DNM numbers in parent–offspring trios; the black line indicates a linear fit to the data. Dark-gray area, prenatal period; light-gray area, childhood. DNMs detected in patients with genetic disorders Monogenic disorders frequent in serious dominant or X-linked recessive conditions, e.g., achondroplasia (80% of cases), neurofibromatosis (50%), osteogenesis imperfecta (35-60%), Duchenne muscular dystrophy (30%) rare in autosomal recessive conditions (2 mutational events required!) recurrence rate: empiric risk of 1-2% for a couple that already have a child with a genetic disease caused by a DNM; higher than risk of same disease in general population Chromosomal abnormalities aneuploidies (e.g. trisomy 21), macrodeletions (e.g. cri-du-chat syndrome, Prader-Willi syndrome …) Rare sporadic genetic disease, especially developmental disorders congenital malformations (e.g., congenital cardiac defects) neurodevelopmental disorders (e.g., intellectual disability, autism, epilepsy) neuropsychiatric disorders (e.g., schizophrenia) Neurofibromatosis type 1 (NF1) Neurocutaneous disorder: neurofibromas (nonmalignant peripheral nerve tumors); cafè-au-lait spots, freckling, learning disabilities in 50% of children Causative gene: NF1 Protein: neurofibromin-1, negative regulator of RAS signaling, highly expressed in neurons and myelinating cells Inheritance: autosomal dominant; 50% de novo mostly germline of paternal origin, rarely postzygotic Fig 19.7 Emery's Element of Medical Genetics and Genomics. Ed 16, Elsevier DNMs are a major cause of developmental disorders Deciphering Developmental Disorders Study (UK) DDD Study. Nature. 2017 Feb 23;542(7642):433-438. Strategy: WES of 4,293 families with members having severe undiagnosed developmental disorders; mostly, only 1 affected member. Meta-analysis of published data from 3,287 individuals with similar disorders. Results: >40% of patients with a severe developmental disorder have a pathogenic DNM in a protein-coding gene. DDD Study. Nature. 2017 Feb 23;542(7642):433- 94 genes enriched in damaging DNMs 438. developmental disorders caused by DNMs have an average prevalence of 1/448 to 1/213 births, increasing with parental age  Postzygotic DNMs can lead to mosaicism Presence in an individual of two or more genetically different cell lines, all derived from one original zygote 3 SNVs estimated to arise per cell division in early human embryogenesis (Ju et al., 2017 Nature 543, 714–718) Degree of mosaicism depends on timing of DNM: DNM in first few cell divisions after fertilization → high-level mosaicism present in many different tissues, including germline. DNMs arising later in development or postnatally → low-level mosaicism restricted to a small number of somatic cells or a single tissue Image: McNulty et al., Am J Hum Genet. 2019;105(4):734-746 Mosaicism Three types of mosaicism depending on timing and cell type (somatic vs. germline): : Somatic mosaicism mutation occurs in somatic cells at any stage of life (embryonic/fetal development, after birth, childhood, adulthood) mutation cannot be transmitted to offspring Biesecker & Spinner. Nature Reviews Genetics 14, 307–320 (2013) Mosaicism Three types of mosaicism depending on timing and cell type (somatic vs. germline): Germline/gonadal mosaicism: mutation occurs in germline during early embryonic development mutation can be transmitted to offspring Biesecker & Spinner. Nature Reviews Genetics 14, 307–320 (2013) Mosaicism Three types of mosaicism depending on timing and cell type (somatic vs. germline): Gonosomal mosaicism: mutation occurs during early embryogenesis involving both germ cell and somatic cell lineages mutation can be transmitted to offspring Biesecker & Spinner. Nature Reviews Genetics 14, 307–320 (2013) Freed et al. Genes (Basel). 2014 Dec 11;5(4):1064-94. Figure 1. Overview of categories of variation including inherited (panels A–C), de novo (panels D,E), and somatic variation (panels F,G).  Somatic mosaicism in segmental neurofibromatosis type 1 Rare cases of NF1 due to postzygotic somatic mutations. Manifestations are limited to a segment of the body - narrow strip, one quadrant or one half of body. Ma and Hu.N Engl J Med. 2015;372(10):963. Germline mosaicism in monogenic disorders Parental germline mosaicism suspected when 2 or more offspring present with an autosomal dominant condition when there is no family history of the condition. Rare cases of osteogenesis imperfecta type 2 and achondroplasia Rare example of pedigree with sibling recurrence of AD disorder (father is a germline mosaic) https://www.ncbi.nlm.nih.gov/books/NBK5191/ box/further_illus-33/?report=objectonly Detection of mosaicism Difficult to detect, especially low-level mosaicism May be tissue-specific, may or may not be detectable in blood Ideally requires: (i) analysis of various tissues, even single cells, (ii) comparison of affected vs. nonaffected tissues BUT relevant tissues may not be accessible Paternal germline mosaicism quantified in sperm Methods include NGS and droplet digital PCR Expressed as variant allele frequency (VAF), i.e., frequency at which the variant is detected in a specimen; → provides an estimate of risk of recurrence Fig 5.19 Strachan and Read. HMG5 Garland Detection of mosaicism by NGS Figure 1. Next-generation sequencing reads for three mosaic variants. The reads are sorted by base such that positive reads are grouped together. All the positive reads for the three mosaic variants are displayed. Mosaic variants are located within the parallel vertical lines and are indicated by the letter of the substituted nucleotide. Brewer et al. J Mol Diagn. (2020) May;22(5):670-678. Detection of mosaicism by droplet digital PCR (ddPCR) ddPCR technology uses a combination of microfluidics and surfactant chemistries to divide samples into thousands of water-in-oil droplets to run multiple PCR amplifications in parallel. Modification of the Taqman qPCR assay using allele-specific fluorescently-labelled probes Fig 5.19 Strachan and Read. HMG5 Garland Taqman assay Real time quantitative PCR assay TaqMan probes consist of sequence-specific oligonucleotides with a reporter (R) fluorophore covalently attached to the 5’-end and a quencher (Q) at the 3’-end. The intact probe is not fluorescent because the quencher absorbs energy released by the reporter fluorophore via FRET (fluorescence resonance energy transfer). During PCR amplification, the exonuclease activity of Taq polymerase cleaves the annealed probe, releasing the fluorophore which will no longer be subject to quenching → increase in fluorescence signal proportional to quantity of template DNA For genotyping, two parallel assays are run with allele- specific probes, one for the normal allele, the other for the variant allele, each labelled with a different reporter fluorophore (e.g., FAM and VIC) https://commons.wikimedia.org/wiki/File:TaqMan_GX_cartoon.jpg  ddPCR workflow A mixture of PCR reagents including fluorescent probes, primers and genomic DNA is partitioned into picoliter to nanoliter droplets where theoretically less than one DNA molecule is distributed in one droplet. Each droplet represents a microreactor for PCR amplification using the Taqman assay The fluorescence intensity of each individual droplet is detected and analysed. Postel et al. Expert Rev Mol Diagn. 2018 Jan;18(1):7-17. Deng et al. (2022). Detection of Very Low-Level Somatic Mosaic COL4A5 Splicing Variant in Asymptomatic Female Using Droplet Digital PCR. Front Med 9:847056 Alport syndrome: hereditary glomerulopathy featured by haematuria, proteinuria, and progressive renal failure, most commonly, X-linked due to COL4A5 variants Figure 1. Pedigree (A) and Sanger sequencing results (B) A. The black arrow indicates the proband. The filled black squares indicate the individuals presenting renal phenotypes, and the filled gray circle indicates the suspected mosaic. B. The red rectangle indicates a c.2245-1G>A mutation in COL4A5 detected in blood of affected individuals (III1 and III2, top 2 chromatograms), but NOT in multiple tissues (blood, saliva urine sediments and hair) of the mother. Deng et al. (2022). Detection of Very Low-Level Somatic Mosaic COL4A5 Splicing Variant in Asymptomatic Female Using Droplet Digital PCR. Front Med 9:847056. Alport syndrome: hereditary glomerulopathy featured by haematuria, proteinuria, and progressive renal failure, most commonly, X-linked due to COL4A5 variants Fuorescence intensity scatter plots Figure 2. COL4A5 wild-type and mutant-type (c.2245- 1G>A) alleles were identified in multiple samples (blood, saliva, and urine sediments) from proband's mother by ddPCR. Green dots - droplets containing wild-type alleles (WT, VIC probe) Blue dots - droplets containing mutant (MT) alleles (MT, FAM probe) Orange dots - droplets containing both alleles (VIC and FAM) Grey dots – droplets with no template DNA (NA, no amplification) Counts of mutant and wildtype droplets are used to calculate the variant allele frequency (MAF = minor allele frequency). Risk of recurrence in siblings of affected child ? Empiric risk of 1% Risk depends on the degree of parental germline mosaicism (i.e., proportion of gametes bearing variant, expressed in terms of VAF)  Paternal origin Maternal origin Offspring Bernkopf et al. (2023). Personalized recurrence risk assessment following the birth of a child with a pathogenic de novo mutation. Nature Comm., 2023; 14:853 Targeted ultra-deep NGS of multiple tissues in trios, allows the stratification of most couples into one of seven discrete categories associated with substantially different risks to future offspring. * * * VAF quantifiable in sperm Note: mosaicism is different from chimerism Mosaic has two or more genetically different cell lines derived from a single zygote. Chimera is derived from two zygotes which are genetically distinct (50% relatedness): extremely rare, includes intersex XX/XY individuals Fig 5.16 Strachan and Read. HMG5 Garland Reading Strachan and Read. Human Molecular Genetics, 5th edition, 2018 Chapter 5 Patterns of inheritance Thompson and Thompson. Genetics in Medicinen 8th edition, 2016 Chapter 7 Patterns of single-gene inheritance Review article (extra reading if interested): Acuna-Hidalgo et al. New insights into the generation and role of de novo mutations in health and disease. Genome Biology (2016) 17:241.