1. Mutations in TUBB8 and Human Oocyte Meiotic Arrest
Qiaoning Guan
Genetics Journal Club
01/28/2016

2. Why This Paper? Reproduction is important
Infertility is a highly prevalent health issue (~15% couples of childbearing age) with a major genetic component (~50% of idiopathic infertilities)
Underlying genetic causes are largely unknown
Colorful paper, multiple pedigrees, functional assays in multi-tissues and organisms
Cutting edge technologies

3. Human Reproduction MITOSIS MEIOSIS Primary oocyte Germinal Vesicle
3-5 mo. Fetus:
Prophase I arrest
Primary oocyte
Puberty:
Continue Meiosis I
Human Reproduction
Germinal Vesicle
When females are still fetuses, the primordial oocytes begins meiosis I, however the meiosis is arrested at the prophase I as a primary oocyte, at around 3-5 month of fetal life, through childhood until puberty. Then at puberty, the lutenizing hormone (LH) surges stimulate the resumption and completion of meiosis I, the day before ovulation. One cell becomes the secondary oocyte the other cell forms the first polar body.
The secondary oocyte then commences meiosis 2 which arrests at metaphase and will not continue without fertilization. At fertilization meiosis 2 completes, forming a second polar body. Note that the first polar body may also undergo this process forming a third polar body.
The hallmark of successful human reproduction is the fusion between a haploid sperm and a metaphase II oocyte.
Without fertilization:
Metaphase II arrest (N)
1 day before ovulation:
Meiosis I completes
Secondary oocyte
Meiosis II resumes upon fertilization

4. Methods Candidate Mutation OR
Patients with oocyte maturation arrest from 24 families
Exome sequencing of 3 affected & 2 unaffected women (family 1)
Not previously reported
Candidate Mutation
Absent in 2 unaffected women
MAF <0.1% in 1000G, EVS, ExAC OR
Present in all 3 affected women
Present in all 3 affected woman
Agilent SureSelect Whole Exome capture and Illumina sequencing
Sanger sequencing of the candidate gene in 24 families
1000G: 1000 Genomes Database
EVS: NHLBI Exome Sequencing Project Exome Variant Server
ExAC Exome Aggregation Consortium Beta version

5. Family 1: Exome Sequencing Result
TUBB8 c.686T>C; p.V229A:
Co-segregated with female infertility in the family
Paternally inherited
Sanger confirmed

6. Family 1: Clinical Characteristics
Patient III-4
Intracytoplasmic sperm injection yielded 3 oocytes in metaphase I and one with abnormal morphologic features, which later failed to become fertilized.
Patient III-5
Tempted IVF resulted in 21 oocytes arrested in metaphase I and failed to mature even after extended culture in vitro.

7. Family 1: Phenotypes of Oocytes
Normal oocyte:
First polar body
Visible spindles
Individual III-4 (V229A):
No first polar body
No visible spindle under highest possible input intensity

8. Inherited TUBB8 Mutations in 4 Other Families
All paternally inherited
Sanger confirmed

9. De Novo TUBB8 Mutations in 2 Families

10. Clinical Characteristics of all Families

11. TUBB8 Mutations are Likely Responsible for Oocyte Maturation Arrest1 3 4 2 6
Abnormal spindle or no detectable spindle
Mutant allele in the S176L oocyte is expressed (58% mutant vs. 42% WT; Fig. S2)

12. TUBB8: Tubulin Beta 8 Class VIII (Primate Specific, Function Unknown)
No information in OMIM and HGMD
All predicted to be deleterious/probably damaging/disease causing by in silico methods
Amino acids highly conserved in primate species (no homolog of TUBB8 in non-primate species)

13. Hypothesis: TUBB8 Influences Microtubule Behavior in Oocytes

14. Heterodimers (α/β-tubulin)
Microtubules
Heterodimers (α/β-tubulin)
Nine β-tubulin isotypes expressed in mammals
Mutations reported
Microtubules are assembled from heterodimers consisting one alpha-tubulin polypeptide and one beta-tubulin polypeptide
Nine β-tubulin isotypes expressed in mammals, primarily distinguished in the C-terminal tails. Mutations have been reported in 6 of them.

15. TUBB8 is the Only β-tubulin Expressed in Oocytes,
and is Localized to the Spindle (Fig. S5)
TUBBs Relative Expression
β-tubulin isotypes

16. Protein Structural Implications
Inhibits microtubule assembly (S176L)
Causes microtubule instability (M363T)
Affects α/β-dimer assembly and stability (R2K)
Destabilize β-tubulin folding (M300I, V229A)
Abolish microtubule-kinesin interaction (D147N, R262Q)
Also, in vitro data showed heterodimer-assembly defects caused by TUBB8 mutations (Fig. S7, Table S2)

17. TUBB8 Mutations Cause Microtubule Disruption In Vivo
Obligated: complete loss of the microtubule network
FLAG-tagged TUBB8 constructs
Cultured Hela cells
Immunostain anti-FLAG,
anti-α tubulin
They introduced three TUBB8 mutations into TUBB5, which is broadly expressed in mammalian tissues especially in the CNS, and found similar abnormal microtubule phenotypes to the TUBB8-FLAG experiment.

18. “Humanized” Yeast Supports the Disruption of Microtubule Function by TUBB8 Mutations
V229A, R262Q: Inviable, consistent with disruption of microtubule function
R2K, M300I, M363T: Viable, but with varying degrees of growth impairment
Embedded human TUBB8 mutations (on URA3+ vector)
into yeast TUB2 gene
Mate with TUB2 cells, sporulation
Dissect tetrads and examine viability

19. TUBB8 Mutations Impair Spindle Assembly in Mouse and Human Oocytes
Mouse oocyte
Human oocyte
Microinject WT or mutated TUBB8 RNA into oocytes
Allow for oocyte maturation
(~12-16 hrs)
Immunostain; Count 1st polar body extrusion
To establish the causal relationship between mutations in TUBB8 and the infertility phenotype, they microinjected WT and mutated TUBB8 RNA in mouse oocytes.

20. Human Oocytes with IntroducedTUBB8 Mutations vs
Human Oocytes with IntroducedTUBB8 Mutations vs. Oocytes from the Original Patients
Patient oocyte TUBB8 RNA  Human oocyte 3 2

21. Summary
DNA sequencing identified TUBB8 mutations in 7 of 24 families (29% sensitivity) with female infertility (MI arrest)
Paternally inherited autosomal dominant (5), or de novo (2)
TUBB8 is primarily expressed in human oocytes and is major component of the oocyte spindle
TUBB8 mutations are predicted to affect α/β-tubulin heterodimer folding, assembly, or stability (protein structure)
TUBB8 mutations caused disruption of the microtubule network in vivo (Hela cells, Yeast, mouse and Human oocytes)

22. Exacting Requirements for Development of the Egg
Dean J, NEJM 2016 Jan 21;374(3):279-80
gonadotrophin

23. Therapeutic Debates Gene editing “Humanized” mouse model
Oocyte regeneration (germline stem cells)

24. Because ethical concerns preclude studies of gene editing in normal embryos, the authors used tripronuclear (3PN) zygotes, which have one oocyte nucleus and two sperm nuclei. The polyspermic zyotes are discarded zygotes in clinics, for instance from in vitro fertilization. They will generate blastocysts in vitro but will invariably fail to develop normally in vivo.
CRISPR/Cas9 has been widely used to modify genes in model systems, to study gene function, and perform gene therapy in animals and human cells. It holds tremendous promise for both basic research and clinical applications.

25. “…Despite great progress in understanding the utilization of CRISPR/Cas9 in a variety of model organisms, much remains to be learned regarding the efficiency and specificity of CRISPR/Cas9-mediated gene editing in human cells, especially in embryos.”
“…Furthermore, mosaicism and mutations at non-target sites are apparent
in the edited embryos. Taken together, our data underscore the need to more comprehensively understand the mechanisms of CRISPR/Cas9-mediated genome editing in human cells, and support the notion that clinical applications of the CRISPR/Cas9 system may be premature at
this stage.”

26. Mice bearing “humanized” alleles of four essential meiosis genes, each predicted to be deleterious by most of the commonly used algorithms… Only a Cdk2 allele mimicking SNP rs …, caused infertility and revealed a novel function in regulating spermatogonial stem cell maintenance. Our data indicate that… whereas computational prediction of SNP effects is useful for identifying candidate causal mutations for diverse diseases, this study underscores the need for in vivo functional evaluation of physiological consequences. This approach can revolutionize personalized reproductive genetics by establishing a permanent reference of benign vs. infertile alleles.

27. Proc Natl Acad Sci U S A. 2014 Dec 16;111(50):17983-8.
Nature Mar 11;428(6979):
It has been generally accepted for a long time that in most mammalian species oocytes cannot renew themselves in postnatal or adult life.
Studies from a decades ago proposed that a group of GSCs, which had originated from the epithelium of the ovarian surface, can serve as the source of oocyte regeneration.
The same group reported a year later that the GSCs had actually originated from the bone marrow

28. According to The Guardian, after standard treatment at a US clinic a Philadelphia couple had 13 in vitro fertilization embryos to choose from. Tests performed using NGS on a Ion Torrent platform showed that while most of the embryos looked healthy, only three had the right number of chromosomes. US doctors transferred one of the healthy embryos into the mother. The single embryo implanted, and on 18 May 2013 a healthy boy, named Connor, was born. Apparently the Oxford team has used NGS for testing for aneuploidy, mutations in the cystic fibrosis gene and mtDNA.