Mutations in CENPE define a novel kinetochore-centromeric mechanism for Microcephalic Primordial Dwarfism in humans.

Ghayda M. Mirzaa1, Benjamin Vitre2, Gillian Carpenter3,Iga Abramowicz3, Joseph G. Gleeson4, Alex R. Paciorkowski5, Don W. Cleveland2, William B. Dobyns1, Mark O’Driscoll3*

1Division of Genetic Medicine, Department of Pediatrics, University of Washington and Center for Integrative Brain Research, Seattle Children’s Research Institute, Seattle, WA, USA.

2Ludwig Institute for Cancer Research, Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA, USA.

3Human DNA Damage Response Disorders Group, Genome Damage & Stability Centre, University of Sussex, Falmer, Brighton, BN1 9RQ, United Kingdom.

4Department of Neurosciences and Pediatrics, University of California, San Diego, La Jolla, CA, USA.

5Departments of Neurology, Pediatrics & Biomedical Genetics, Center for Neural Development & Disease, University of Rochester Medical Center, Rochester, NY, USA.

*Correspondance: .

Ph. 0044(0)1273 678 515

Fax. 0044(0)1273 678 121

Online Resource Tables.

Sample
Designation / Phenotype / Workup
MPD-001 / MPD IUGR MSG / PCNT sequencing
MPD-002 / MPD IUGR MSG / PCNT sequencing
MPD-003 / MPD IUGR MIC PMG / PCNT sequencing.
ATR-dependent G2-M checkpoint normal
MPD-004 / MPD IUGR MSG / WES – known MPD genes negative.
MPD-005 / MPD IUGR MSG Seckel-like / ATR-dependent G2-M checkpoint normal
MPD-006 / MPD IUGR MSG / PCNT, ASPM, CENPJ
MPD-007 / MPD IUGR MSG / WES – known MPD genes negative
MPD-008 / MPD IUGR MSG ISS / –
MPD-009 / MPD IUGR MSG / Targeted sequencing for all MIC and MPD genes
MPD-010 / MIC MPD-like / CENPJ, MCPH1, ASPM, PCNT, STIL

Supplementary Table 1. MIC MPD cohort screened (negative) for CENPE mutations (n=10).

All 49 coding CENPE exons were sequenced. ISS, infantile spasms; MIC, microcephaly; MPD, microcephalic-osteodysplastic primordial dwarfism; MSG, microcephaly with simplified gyri; WES, whole exome sequencing.

Algorithim / Mutation / Score / Interpretation
PolyPhen-2 / p.D933N
P.K1355E / 0.055
0.956 / Benign
Possibly damaging
SIFT / p.D933N
P.K1355E / 0.49
0.04 / Tolerated
Damaging
ALIGN-GVGD / p.D933N
P.K1355E / C15
C55 / Unlikely to impact.
Very likely to impact.
Mutation Taster / p.D933N
P.K1355E / 23
56 / Protein features might be affected (both variants)

Supplementary Table 2. Bioinformatic analysis of CENPE variants.

PolyPhen:

SIFT:

ALIGN-GVGD pathogenicity prediction:

Class C0 (no impact) to Class C65 (most likely to impact).

Mutation Taster:

Grantham matrix 0-215.

PCNT / CENPE
Primary phenotype / MPD
(MOPD type II*) / MPD
Common phenotypic features / Severe intra-uterine and postnatal growth restriction, MIC, ID, typical facies (prominent nose micrognathia), delayed ossification, abnormal teeth
Distinct phenotypic features / Skeletal anomalies*
High juvenile mortality due to cerebral vessel insults, cardiomyopathy, early onset type 2 diabetes, dislipidemia. / Severe MIC with simplified gyri
(OFC -5 to -9 SD)
Congenital restrictive cardiomyopathy (1 sibling)
Function / Centrosome-structural role? / Kinetochore-associated; spindle
microtubule capture and attachment
Mutations / Recessive
(Nonsense, splice site, frameshift, missense) / Recessive
(Missense)
Patient LCL Characteristics / PCNT / CENPE
Mitotic spindles / Disorganized, multipolar spindles / Disorganized, monopolar and multipolar spindles
Mitotic segregation / Impaired; elevated levels of bi-nucleated cells (with
nuclei of equal size) / Impaired, elevated levels of bi-nucleated cells (with 50% nuclei unequal in size)
Mitotic progression / Delayed / Delayed
ATR-dependent DDR / Defective / Functional
Centromeric accumulation of CENP-E in mitotic cells / Reduced / Reduced
CENPE-dependent BubR1-autophos / Impaired following colcemid
treatment / Impaired following colcemid treatment

Supplementary Table 3. Comparison between the clinical and biochemical characteristics of CENPE-related MPD and PCNT-related MOPD type II patients.

*Skeletal anomalies in MOPD type II include: (small iliac wings, coxa vara, V-shaped distal femoral metaphyses, triangular distal femoral epipyses, pseudoepiphyses, brachymesophalangy V, metaphyseal flaring of the radius and ulna, relatively short arms). These features often develop in an age-dependent manner. BubR1; kinetochore associated kinase; DDR; DNA damage response.

ABCC9ACTCACTC1ACTN2ANKRD1BAG3

BRAFCAV3CRYABCSRP3DESDMD

DSC2DSG2DSPDTNAEMDFKTN

GATAD1GLAHRASILKJPH2JUP

KRASLAMA4LAMP2LDB3ZASPLMNA

MAP2K1MAP2K2MTND1MTND5MTND6MTTD

MTTGMTTHMTTIMTTKMTTL1MTTL2

MTTMMTTQMTTS1MTTS2MYBPC3MYH7

MYL2MYL3MYLK2MYOZ2MYPNNEBL

NEXNNRASPDLIM3PKP2PLNPRKAG2

PTPN11RAF1RBM20RYR2SCN5ASGCD

SOS1TAZTCAPTMEM43TMPOTNNC1

TNNI3TNNT2TPM1TTNTTRVCL

Supplementary Table 4. Cardiomyopathy-associated genes. A list of the known cardiomyopathy-related genes that were interrogated for variants in the exome sequence of LR05-054a1.

Supplementary Table 5. A list of the variants identified in the cardiomyopathy-related genes from the exome sequence of LR05-054a1.

Of the 218 variants identified, all but one are commonly represented in dbSNP. The one exception is the intronic variant chr2:g.179549378G>C in the gene TTN, that is most likely not pathogenic.

GeneChrCoordinatersIDRefSubject

ABCC91221958399rs829060GC

ABCC91221970019rs2638441CT

ABCC91222005003rs2307024TG

ABCC91222017422rs697250AG

ABCC91222017486rs697251CG

ABCC91222047151rs704216GT

ABCC91222047174rs704217CT

ABCC91222063115rs10770865AG

ABCC91222063337rs4148656AG

ABCC91222063737rs4762719GT

ABCC91222063749rs4762720TC

ABCC91222063971rs4148654AG

ABCC91222068849rs3759236GT

ABCC9 1222078838rs2277405TG

ACTN21236849952rs138279482CT

ACTN21236882303rs1341864TC

ACTN21236883421rs1341863CT

ACTN21236899042rs2288600GA

ACTN21236902594rs2288601CG

ACTN21236924506rs2282366AG

BAG310121435955rs196294AC

BAG310121436362rs196295AG

BRAF7140449071rs3789806CG

BRAF7140449150rs9648696TC

BRAF7140487202rs1267632CT

DES2220285309rs1058261CT

DES2220286142rs1058284GA

DSG21829104714rs2230234AG

FKTN9108366734rs34787999GA

FKTN9108380355rs17309806CA

HRAS11534242rs12628AG

ILK116629665rs1043388CT

ILK116630833rs1043390GA

JPH22042747247rs3810510CT

JPH22042814931rs6031442TC

JPH22042815190rs1883790GA

JUP1739912145rs1126821TA

JUP1739912581rs9890858AG

GeneChrCoordinatersIDRefSubject

JUP1739913645rs7216034TC

JUP1739914070rs8067890GT

JUP1739923614rs12942034AG

JUP1739925713rs41283425CT

KRAS1225362777rs1137282AG

KRAS1225368462rs4362222CT

KRAS1225378456rs76433096TC

LAMA46112451367rs34752945GT

LAMA46112454185rs764587AG

LAMA46112457390rs2032567CT

LAMA46112457471rs2032568GA

LAMA46112493872rs1050348AG

LAMA46112506375rs9374309GA

LAMA46112506583rs6908219GT

LAMA46112508769rs9387061TG

LAMA46112508770rs9400522GT

LAMA46112522852rs11757455GA

LAMA46112537682rs78871662AG

LMNA1156104292rs12117552GA

MYH71423888617rs200234670GT

MYH71423894565rs139882431GA

MYH71423902753rs2069540GA

NEBL1021074724rs2296614TC

NEBL1021104694rs4748727AT

NEBL1021108247rs1006362CT

NEBL1021115332rs1409348TC

NEBL1021120116rs10491056AG

NEBL1021129588rs41277368TC

NEBL1021134282rs41277370CG

NEBL1021139389rs4025981TC

NEBL1021141469rs703089TC

NEBL1021148584rs4748729CA

NEBL1021157492rs2296609TC

NEBL1021176945rs703100GA

NEBL1021185821rs788971AC

NEBL1021399813rs3864841CG

NEBL1021401765rs4748749TC

NEBL1021414892rs625223CG

NEBL1021415006rs11591355TC

NEBL1021461232rs35856892GA

PDLIM34186423637rs4635850GA

PDLIM34186423655rs12644280GA

PDLIM34186423677rs10866276GA

PDLIM34186427841rs2306705TC

RYR21237617757rs3765097CT

RYR21237620049rs2045955TC

RYR21237711797rs2253273AG

RYR21237730124rs2805390AG

GeneChrCoordinatersIDRefSubject

RYR21237730169rs2779401CT

RYR21237753364rs2805409AC

RYR21237753390rs2805410AG

RYR21237796837rs10802626GA

RYR21237797082rs1967579TG

RYR21237801770rs707189TC

RYR21237813126rs625006AG

RYR21237814783rs684923CT

RYR21237881770rs2797441CT

RYR21237890437rs2685301CT

RYR21237923053rs2253831CT

RYR21237946964rs790889TC

RYR21237951451rs2256242AG

RYR21237955680rs146659498TC

RYR21237955684rs78394746TC

RYR21237957146rs790902GA

RYR21237957161rs790901AG

RYR21237957309rs790900AC

RYR21237965043rs2790347AG

RYR21237965094rs2794820GT

RYR21237965131rs9428384GA

RYR21237969638rs790879AG

SCN5A338592406rs1805126AG

SCN5A338598669rs41312393AG

SCN5A338622467rs7430407TC

SCN5A338645420rs1805124TC

SCN5A338646423rs7428779CT

SCN5A338674712rs6599230TC

SGCD5155756414rs11740347GA

SGCD5155771579rs1801193TC

SGCD5155771773rs4434364AG

SGCD5155885469rs11954391TC

TCAP1737822311rs1053651AC

TNNT21201331554rs1104859TG

TNNT21201331664rs2365652CA

TNNT21201334382rs3729547GA

TNNT21201335899rs1573230CT

TNNT21201337170rs3729842AG

TNNT21201338896rs10920184TC

TNNT21201339043rs12049476CT

TNNT21201341341rs868407CT

TPM11563349096rs4775613GA

TPM11563351687rs4775614AG

TPM11563351840rs1071646CA

TTN2179392080rs16866373AT

TTN2179395958rs3813250TC

TTN2179396354rs3829748GA

TTN2179397561rs3829747CT

GeneChrCoordinatersIDRefSubject

TTN2179398823rs2857265GA

TTN2179403593rs2303539GA

TTN2179404786rs2288325AT

TTN2179406191rs3731749CT

TTN2179411665rs2288327AG

TTN2179412966rs35445420GA

TTN2179413110rs3731748GA

TTN2179414633rs890578CA

TTN2179421694rs9808377AG

TTN2179427186rs2366751AG

TTN2179427536rs3829746TC

TTN2179430997rs3731746GA

TTN2179436020rs744426GA

TTN2179444137rs2288571AG

TTN2179444768rs4145333CG

TTN2179444939rs2303838CT

TTN2179447848rs4894029TC

TTN2179448911rs16866400GA

TTN2179451420rs2042996GA

TTN2179454394rs1560221AG

TTN2179455207rs2163009TC

TTN2179457147rs16866406GA

TTN2179458591rs2288569CT

TTN2179464527rs1001238TC

TTN2179514433rs2562845TC

TTN2179523590rs146110527CA

TTN2179539903rs2472751CA

TTN2179542674rs72650048CT

TTN2179543217rs35112591CT

TTN2179549378.GC

TTN2179558366rs2042995TC

TTN2179576596rs2742331AT

TTN2179578730rs2562839GA

TTN2179579093rs12693164TC

TTN2179579212rs2562838TC

TTN2179579822rs2562836TA

TTN2179580093rs12622914AC

TTN2179582162rs62178977CT

TTN2179582327rs13390491CT

TTN2179582537rs2627043GT

TTN2179582605rs6750145AG

TTN2179582853rs72648982TC

TTN2179583398rs62178978TC

TTN2179583496rs16866465TG

TTN2179585266rs2562831CT

TTN2179585393rs2562830AG

TTN2179587130rs12693166CG

TTN2179587687rs2742327TC

GeneChrCoordinatersIDRefSubject

TTN2179591708rs114742263CT

TTN2179595117rs17076CG

TTN2179598228rs12993099AG

TTN2179600563rs2742348GA

TTN2179605180rs746578CT

TTN2179614952rs10803917AG

TTN2179615887rs922984TC

TTN2179615931rs922985CG

TTN2179615994rs922986TC

TTN2179620951rs7585334CT

TTN2179621477rs6433728CT

TTN2179623758rs2291310CT

TTN2179623939rs6705594TC

TTN2179629363rs4894043TC

TTN2179629461rs2291311CT

TTN2179632710rs2291313TC

TTN2179633298rs4471922GT

TTN2179634392rs13388274AT

TTN2179634421rs200875815TG

TTN2179634961rs33917087CA

TTN2179635919rs13011633CT

TTN2179642425rs719201GA

TTN2179643886rs2291302AG

TTN2179643934rs2291301AG

TTN2179644035rs1552280GA

TTN2179644855rs10497520TC

TTN2179650408rs35813871GA

TTN2179650701rs6715406CT

TTN2179667090rs3816849CT

TTR1829175210rs121918074CA

VCL1075865065rs767809GA

VCL1075866929rs2131957CA

VCL1075871735rs2131956CG

VCL1075873892rs10824072CT

VCL1075874192rs28639484T C

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