Skip to main content
NCBI home page
Human Molecular Genetics logoLink to Human Molecular Genetics
. 2018 Jun 22;27(21):3651–3668. doi: 10.1093/hmg/ddy241

Dissecting KMT2D missense mutations in Kabuki syndrome patients

Dario Cocciadiferro 1,2,✉, Bartolomeo Augello 1, Pasquelena De Nittis 3, Jiyuan Zhang 4, Barbara Mandriani 5, Natascia Malerba 1,2, Gabriella M Squeo 1, Alessandro Romano 6, Barbara Piccinni 7, Tiziano Verri 7, Lucia Micale 1, Laura Pasqualucci 4, Giuseppe Merla 1,✉
PMCID: PMC6488975  PMID: 30107592

Abstract

Kabuki syndrome is a rare autosomal dominant condition characterized by facial features, various organs malformations, postnatal growth deficiency and intellectual disability. The discovery of frequent germline mutations in the histone methyltransferase KMT2D and the demethylase KDM6A revealed a causative role for histone modifiers in this disease. However, the role of missense mutations has remained unexplored. Here, we expanded the mutation spectrum of KMT2D and KDM6A in KS by identifying 37 new KMT2D sequence variants. Moreover, we functionally dissected 14 KMT2D missense variants, by investigating their impact on the protein enzymatic activity and the binding to members of the WRAD complex. We demonstrate impaired H3K4 methyltransferase activity in 9 of the 14 mutant alleles and show that this reduced activity is due in part to disruption of protein complex formation. These findings have relevant implications for diagnostic and counseling purposes in this disease.

Introduction

Kabuki syndrome (KS, MIM #147920, MIM #300867) is a rare autosomal dominant condition characterized by striking facial features such as elongated palpebral fissures with eversion of the lateral third of the lower eyelid, short columella with depressed nasal tip, skeletal anomalies, dermatoglyphic abnormalities, mild to moderate intellectual disability and postnatal growth deficiency (1,2). Additional findings include congenital heart defects, genitourinary anomalies, cleft lip and/or palate, susceptibility to infections, gastrointestinal abnormalities, ophthalmologic defects, ptosis and strabismus, dental anomalies (including widely spaced teeth and hypodontia), ear pits, visceral abnormalities and premature thelarche (3). In 2010, whole-exome sequencing successfully identified heterozygous loss of function mutations in the KMT2D gene (MIM #602113, NM_003482.3, also known as MLL2 and MLL4) as the major cause of KS (4). KMT2D encodes a conserved member of the SET1 family of histone lysine methyltransferases (KMTs), which catalyzes the methylation of lysine 4 on histone H3 (H3K4), a modification associated with active transcription (5–7). The enzymatic function of KMT2D depends on a cluster of conserved C-terminal domains, including plant homeodomains (PHD), two phenylalanine and tyrosine (FY)-rich motifs [FY-rich, N-terminal (FYRN) and FY-rich, C-terminal (FYRC)] and a catalytic Su(var)3–9, Enhancer-of-zeste, Trithorax (SET) domain. In mammalian cells, KMT2D functions as a major histone H3K4 mono-/di-/trimethyltransferase (8–15) that is required for the epigenetic control of active chromatin states in a tissue-specific manner (16).

In addition to KMT2D mutations, a subset of KS individuals has been identified with either point mutations or microdeletions encompassing the X-linked gene, KDM6A (MIM #300128, NM_021140.3, also known as UTX) (17–19), which encodes for a Histone H3 lysine-27 demethylase. KDM6A plays a crucial role in chromatin remodeling (20,21) and interacts with KMT2D in a conserved SET1-like complex (16).

More than 600 KMT2D mutations have been identified so far in KS patients; roughly 84% of them are truncating events (22) including non-sense, indels, small duplications, splice-site, and frameshift mutations, while the remaining 16% are represented by missense variants whose pathogenicity has not been investigated. As a consequence, the interpretation of missense mutations has remained a challenging problem in genetic diagnosis and counseling.

Here we surveyed a cohort of 505 KS patients to expand the mutation spectrum of KMT2D and KDM6A, and investigated the functional impact of missense mutations in the pathogenesis of the disease.

Results

Mutation screening of KMT2D and KDM6A

To expand the spectrum of mutations targeting KMT2D and KDM6A in KS, we integrated our mutation database with mutational screening of 202 newly diagnosed KS patients for a total of 505 cases, by using Sanger sequencing and/or multiplex ligation-dependent probe amplification (MLPA) of the KMT2D coding sequence, followed by KDM6A analysis in patients resulted as KMT2D-negative. We identified a total of 208 KMT2D variants distributed in 196/505 (39%) patients, including 37 that have not been described before (Table 1). These included 54 non-sense mutations (26%), 59 frameshift mutations (28%), 69 missense mutations (33%), 13 splice site variants (6%), 12 indels (6%) and 1 gross deletion (Table 1). Missense mutations were distributed across the entire length of the KMT2D gene (Fig. 1A) and were represented by 8 missense variants (11%) localized within the PHD 1–6 domains, one change (1%) in the Coiled Coil/Poly Q region, 25 variants (36%) localized within the C-terminal of the protein (amino acid 4507–5537) and 36 (52%) variants localized outside of known domains and/or in uncharacterized portions of the protein. Among the KMT2D variants identified, 66 occurred de novo and 32 were inherited from an apparently asymptomatic parent, whereas for the remaining 110 variants we had no access to parental DNA.

Table 1.

KMT2D and KDM6A variants identified in our cohort

ID Inheritance Exon/intron Variant AA change Reference ACMG classification
 KMT2D
 Non-sense
  KB49 NA ex 5 c.669T>G p.(Tyr223*) (46) P
  KB343 NA ex 8 c.1016G>A p.(Trp339*) This study P
  KB35 NA ex 10 c.1921G>T p.(Glu641*) (46) P
  KB33 NA ex 16 c.4419G>A p.(Trp1473*) (46) P
  KB63 NA ex 19 c.4895delC p.(Ser1632*) (46) P
  KB317 NA ex 22 c.5212G>T p.(Glu1738*) (19) P
  KB336 De novo ex 22 c.5269C>T p.(Arg1757*) (18) P
  KB262 NA ex 26 c.5674C>T p.(Gln1892*) (19) P
  KB429 NA ex 26 c.5707C>T p.(Arg1903*) (18,22,73,74) P
  KB26 NA ex 31 c.6295C>T p.(Arg2099*) (4,22,46) P
  KB502 De novo ex 31 c.7228C>T p.(Arg2410*) (9,73,75,76) P
  KB66 NA ex 31 c.7246C>T p.(Gln2416*) (46) P
  KB59 NA ex 31 c.7903C>T p.(Arg2635*) (22,46) P
  KB153 De novo ex 31 c.7903C>T p.(Arg2635*) (22,46) P
  KB226 De novo ex 31 c.7903C>T p.(Arg2635*) (22,46) P
  KB338 De novo ex 31 c.7933C>T p.(Arg2645*) (77) P
  KB198 De novo ex 31 c.7936G>T p.(Glu2646*) (19) P
  KB352 NA ex 32 c.8227C>T p.(Gln2743*) This study P
  KB323 NA ex 33 c.8311C>T p.(Arg2771*) (76,77) P
  KB289 NA ex 34 c.8743C>T p.(Arg2915*) (22,77–79) P
  KB422 De novo ex 34 c.9396C>A p.(Cys3132*) This study P
  KB186 De novo ex 34 c.9961C>T p.(Arg3321*) (4,75,76,79) P
  KB56 De novo ex 34 c.10135C>T p.(Gln3379*) (46) P
  KB168 De novo ex 39 c.10750C>T p.(Gln3584*) (19) P
  KB46 De novo ex 39 c.10841C>G p.(Ser3614*) (46) P
  KB41 NA ex 39 c.11119C>T p.(Arg3707*) (46) P
  KB44 NA ex 39 c.11119C>T p.(Arg3707*) (46) P
  KB42 De novo ex 39 c.11269C>T p.(Gln3757*) (22,46) P
  KB25 NA ex 39 c.11434 C>T p.(Gln3812*) (46) P
  KB244 De novo ex 39 c.11674 C>T p.(Gln3892*) (76) P
  KB178 NA ex 39 c.11704C>T p.(Gln3902*) (19) P
  KB425 De novo ex 39 c.11731C>T p.(Gln3911*) This study P
  KB461a NA ex 39 c.11749C>T p.(Gln3917*) This study P
  KB463 NA ex 39 c.11845C>T p.(Gln3949*) This study P
  KB181 NA ex 39 c.11869C>T p.(Gln3957*) (19) P
  KB358 NA ex 39 c.11944C>T p.(Arg3982*) (18,22,77) P
  KB40 NA ex 39 c.12274C>T p.(Gln4092*) (18,46) P
  KB114 De novo ex 39 c.12274C>T p.(Gln4092*) (18,46) P
  KB65 NA ex 39 c.12076C>T p.(Gln4026*) (46) P
  KB333 NA ex 39 c.12703C>T p.(Gln4235*) (4) P
  KB410 NA 39 c.12760C>T p.(Gln4254*) (22) P
  KB82 De novo ex 39 c.12844C>T p.(Arg4282*) (19) P
  KB350 De novo ex 39 c.12844C>T p.(Arg4282*) (19) P
  KB189 De novo ex 39 c.12955A>T p.(Arg4319*) (19,22) P
  KB183 De novo ex 39 c.13450C>T p.(Arg4484*) (9,22,74,77) P
  KB450 NA ex 39 c.13450C>T p.(Arg4484*) (9,22,74,77) P
  KB175 De novo ex 39 c.13507C>T p.(Gln4503*) (19) P
  KB73 De novo ex 40 c.13666A>T p.(Lys4556*) (46) P
  KB83 NA ex 48 c.15022G>T p.(Glu5008*) (19) P
  KB377 NA ex 48 c.15061C>T p.(Arg5021*) (18,76) P
  KB45 NA ex 48 c.15079C>T p.(Arg5027*) (22,46,77) P
  KB72 NA ex 48 c.15079C>T p.(Arg5027*) (22,46,77) P
  KB362 NA ex 50 c.16018C>T p.(Arg5340*) (77) P
  KB130 NA ex 52 c.16360C>T p.(Arg5454*) (4,22,75,77) P
 Frameshift
  KB454 NA ex 3 c.234_235delGC p.(Gln79Alafs*7) This study P
  KB469 NA ex 3 c.345dupA p.(Ser116Ilefs*7) This study P
  KB337 NA ex 4 c.446_449delTATG p.(Val149Glyfs*58) This study P
  KB75 De novo ex 4 c.472delT p.(Cys158Valfs*50) (46) P
  KB8 De novo ex 5 c.588delC p.(Cys197Alafs*11) (45) P
  KB58 NA ex 6 c.705delA p.(Glu237Serfs*24) (46) P
  KB57 NA ex 8 c.1035_1036delCT p.(Cys346Serfs*17) (46) P
  KB89 NA ex 10 c.1345_1346delCT p.(Leu449Valfs*5) (46,74) P
  KB156 De novo ex 10 c.1503dupT p.(Pro502Serfs*7) (19) P
  KB116 NA ex 10 c.1634delT p.(Leu545Argfs*385) (76) P
  KB349 NA ex 10 c.1634delT p.(Leu545Argfs*385) (76) P
  KB545 NA 10 c.2091dupC p.(Thr698Hisfs*6) This study P
  KB369 NA ex 11 c.3596_3597del p.(Leu1199Hisfs*7) This study P
  KB48 De novo ex 11 c.2993dupC p.(Met999Tyrfs*69) (46) P
  KB203 NA ex 11 c.3161_3171delCGTTGAGTCCC p.(Pro1054Hisfs*10) (18,19,43) P
  KB309 NA ex 11 c.3730delG p.(Val1244Serfs*86) (19) P
  KB142 De novo ex 13 c.4021delG p.(Val1341Leufs*35) (19) P
  KB311 NA ex 14 c.4135_4136delAT p.(Met1379Valfs*52) (19,22,80) P
  KB524 NA 14 c.4135_4136delAT p.(Met1379Valfs*52) (19,22,80) P
  KB188 De novo ex 16 c.4454delC p.(Pro1485Leufs*21) (19) P
  KB159 NA ex 19 c.4896_4905delAGATGCCCTT p.(Asp1633Alafs*86) (19) P
  KB443a De novo ex 25 c.5575delG p.(Asp1859Thrfs*17) This study P
  KB3 NA ex 26 c.5652dup p.(Lys1885Glnfs*18) (45) P
  KB84 NA ex 26 c.5779delC p.(Gln1927Lysfs120*) (46) P
  KB146 De novo ex 27 c.5857delC p.(Leu1953Trpfs*94) (19) P
  KB208 NA ex 28 c.5954delC p.(Thr1985Lysfs*62) (19) P
  KB221 De novo ex 29 c.6149_6150delGA p.(Arg2050Lysfs*6) (19) P
  KB525 NA 30 c.6212_6213delAC p.(His2071Profs*10) This study P
  KB152 De novo ex 31 c.6583delA p.(Thr2195Profs*69) (19) P
  KB267 NA ex 31 c.6594delC p.(Tyr2199Ilefs*65) (75) P
  KB79 De novo ex 31 c.6595delT p.(Tyr2199Ilefs*65) (4,20,22,45,46,75–77,81) P
  KB102 De novo ex 31 c.6595delT p.(Tyr2199Ilefs*65) (4,20,22,45,46,75–77,81) P
  KB342 NA ex 31 c.6595delT p.(Tyr2199Ilefs*65) (4,20,22,45,46,75–77,81) P
  KB67 De novo ex 31 c.6638_6641delGCGC p.(Gly2213Alafs*50) (46) P
  KB176 NA ex 31 c.6738delA p.(Lys2246Asnfs*18) (19) P
  KB253 NA ex 31 c.6794delG p.(Gly2265Glufs*21) (19,22) P
  KB278 NA ex 31 c.7481dupT p.(Ala2496Serfs*10) (19,79) P
  KB313 De novo ex 32 c.8196delG p.(Ser2733Valfs*24) (19) P
  KB80 NA ex 33 c.8273delG p.(Gly2758Alafs*29) (46) P
  KB243 De novo ex 34 c.8430_8431insAA p.(Gln2811Asnfs*41) (19) P
  KB182 NA ex 34 c.9203delA p.(Gln3068Glyfs*3) (19) P
  KB30 NA ex 38 c.10606delC p.(Arg3536Alafs*122) (46) P
  KB101 De novo ex 39 c.11066_11078delCT GGATCCCTGGC p.(Ala3689Valfs*56) (46) P
  KB504 De novo ex 39 c.11093dupG p.(Phe3699Leufs*14) This study P
  KB495 De novo ex 39 c.11715delG p.(Gln3905Hisfs*74) This study P
  KB172 NA ex 39 c.12647delC p.(Pro4216Leufs*62) (19) P
  KB192 De novo ex 39 c.12966delA p.(Gln4322Hisfs*62) (19) P
   KB54 NA ex 39 c.13129dupT p.(Trp4377Leufs*33) (46) P
  KB121 De novo ex 39 c.13277dupT p.(Ala4428Serfs*59) (19) P
  KB540 NA 41 c.13780delG p.(Ala4594Profs*23) (22) P
  KB123 De novo ex 42 c.13884dupC p.(Thr4629Hisf*18) (19) P
  KB481 NA ex 42 c.13895dupC p.(Ser4633Ilefs*14) This study P
  KB197 De novo ex 47 c.14592dupG p.(Pro4865Alafs*48) (19) P
  KB125 NA ex 48 c.15031delG p.(Glu5011Serfs*40) (19) P
  KB16 De novo ex 48 c.15374dupT p.(Phe5126Leufs*12) (45) P
  KB535 NA 50 c.16043_16044delAC p.(His5348Leufs*14) This study P
  KB355 NA ex 53 c.16438_16441delAACT p.(Asn5480Val*6) (75) P
  KB64 NA ex 53 c.16469_16470delAA p.(Lys5490Argfs*21) (46) P
  KB533 NA 53 c.16469_16470delAA p.(Lys5490Argfs*21) (46) P
Missense
  KB21a inherited M ex 3 c.346T>C p.(Ser116Pro) (19) LB
   KB21a,b inherited M ex 4 c.510G>C p.Gln170His (19,45) P
  KB256 NA ex 5 c.626C>T p.(Thr209Ile) (19) VOUS
  KB458 NA ex 8 c.1076G>C p.(Arg359Pro) This study VOUS
  KB269 inherited M ex 10 c.1940C>A p.(Pro647Gln) (19,45,77) LB
  KB126 NA ex 10 c.2074C>A p.(Pro692Thr) (77) LB
  KB374c inherited M ex 10 c.2074C>A p.(Pro692Thr) (77) LB
  KB487 NA ex 10 c.2654C>T p.(Pro885Leu) This study VOUS
  KB370a Inherited P–M ex 11 c.2837C>G p.(Ala946Gly) This study LB
  KB215 Inherited M ex 11 c.3392C>T p.(Pro1131Leu) (19) LB
  KB341 Inherited M ex 11 c.3392C>T p.(Pro1131Leu) (19) LB
  KB222 Inherited P ex 11 c.3572C>T p.(Pro1191Leu) (19) LB
  KB32 Inherited P ex 11 c.3773G>A p.(Arg1258Gln) (46) LB
  KB307 De novo ex 14 c.4171G>A p.(Glu1391Lys) (19,22) LP
  KB28a Inherited M ex 15 c.4249A>G p.(Met1417Val) (46) LB
  KB28a Inherited M ex 15 c.4252C>A p.(Leu1418Met) (46) LB
  KB174 Inherited M ex 15 c.4283T>C p.(Ile1428Thr) (19) LB
  KB138 NA ex 16 c.4427C>G p.(Ser1476Cys) (19) VOUS
  KB34 Inherited P ex 16 c.4565A>G p.(Gln1522Arg) (46) LB
  KB535 NA 25 c.5549G>A p.(Gly1850Asp) This study LB
  KB119 Inherited M ex 31 c.6638G>A p.(Gly2213Asp) (19) LB
  KB204c Inherited M ex 31 c.6638G>A p.(Gly2213Asp) (19) LB
  KB330a NA ex 31 c.6733C>G p.(Leu2245Val) This study VOUS
  KB326c Inherited M ex 31 c.6811C>T p.(Pro2271Ser) (19) LB
  KB107a NA ex 31 c.6970C>A p.(Pro2324Thr) (19) VOUS
  KB430 NA ex 31 c.7378C>T p.(Arg2460Cys) (77) LB
  KB122 Inherited M ex 31 c.7829T>C p.(Leu2610Pro) (19,73) LB
  KB287 Inherited M ex 31 c.7829T>C p.(Leu2610Pro) (19,73) LB
  KB27 NA ex 34 c.8521C>A p.(Pro2841Thr) (46) VOUS
  KB330a NA ex 34 c.8774C>T p.(Ala2925Val) This study VOUS
  KB443a Inherited M ex 34 c.9971G>T p.(Gly3324Val) This study LB
  KB326c Inherited P ex 34 c.10192A>G p.(Met3398Val) (19) LB
  KB357 Inherited P ex 34 c.10192A>G p.(Met3398Val) (19) LB
  KB297 NA ex 35 c.10256A>G p.(Asp3419Gly) (77) LB
  KB378 NA ex 35 c.10256A>G p.(Asp3419Gly) (77) LB
  KB292 De novo ex 37 c.10499G>T p.(Gly3500Val) (19) LP
  KB86a NA ex 39 c.10966C>T p.(Arg3656Cys) (19) VOUS
  KB374c Inherited P ex 39 c.11380C>T p.(Pro3794Ser) This study LB
  KB293 Inherited P ex 39 c.11794C>G p.(Gln3932Glu) (19) LB
  KB204c Inherited P ex 39 c.12070A>G p.(Lys4024Glu) (19) LB
  KB385 NA ex 39 c.12302A>C p.(Gln4101Pro) This study VOUS
  KB247 NA ex 39 c.12485G>A p.(Arg4162Gln) (19) VOUS
  KB107a NA ex 39 c.12488C>T p.(Pro4163Leu) (19) VOUS
  KB170 Inherited M ex 39 c.13256C>T p.(Pro4419Leu) (19) LB
  KB512 NA 45 c.14381A>G p.(Lys4794Arg) This study VOUS
  KB86a NA ex 48 c.14893G>A p.(Ala4965Thr) (19) VOUS
  KB38a De novo ex 48 c.15084C>G p.(Asp5028Glu) (46) LP
  KB154 NA ex 48 c.15088C>T p.(Arg5030Cys) (18,45) VOUS
  KB185 De novo ex 48 c.15088C>T p.(Arg5030Cys) (18,45) LP
  KB423 Inherited P ex 48 c.15089G>A p.(Arg5030His) This study LB
  KB38a De novo ex 48 c.15100T>G p.(Phe5034Val) (46) LP
  KB76 De novo ex 48 c.15176A>C p.(His5059Pro) (46) LP
  KB129 NA ex 48 c.15292A>C p.(Thr5098Pro) (19) VOUS
  KB462 De novo ex 48 c.15310T>C p.(Cys5104Arg) This study LP
  KB171 Inherited M ex 48 c.15565G>A p.(Gly5189Arg) (18,22,46) VOUS
  KB264 NA ex 48 c.15640C>T p.(Arg5214Cys) (22,45,75,76) LP
  KB376 NA ex 48 c.15640C>T p.(Arg5214Cys) (22,45,75,76) LP
  KB24 De novo ex 48 c.15641G>A p.(Arg5214His) (4) LP
  KB219 NA ex 48 c.15641G>A p.(Arg5214His) (4,75) LP
  KB408 De novo ex 48 c.15641G>A p.(Arg5214His) (4,75) LP
  KB109 De novo ex 48 c.15649T>C p.(Trp5217Arg) (19) LP
  KB17 De novo ex 50 c.16019G>A p.(Arg5340Gln) (22,46) LP
  KB169 De novo ex 51 c.16273G>A p.(Glu5425Lys) (19,22,78) P
  KB90 NA ex 51 c.16295G>A p.(Arg5432Gln) (22,82,83) LP
  KB467 NA ex 51 c.16295G>A p.(Arg5432Gln) (22,82,83) LP
  KB480 NA ex 52 c.16385A>G p.(Asp5462Gly) This study VOUS
  KB177 De novo ex 52 c.16412G>T p.(Arg5471Met) (19) LP
  KB489 NA ex 53 c.16498C>T p.(Arg5500Trp) (78) P
  KB120 De novo ex 54 c.16528T>G p.(Tyr5510Asp) (19) LP
 Indel  
  KB404 Inherited M ex 10 c.2283_2309del p.(Ala765_Gln773del) This study LB
  KB274 NA ex 10 c.2532_2591del p.(Arg845_Pro864del) (19) VOUS
  KB370d De novo ex 14 c.4202_4210del p.(Ser1401_Cys1403del) This study LP
  KB384 NA ex 39 c.11220_11222dup p.(Gln3745dup) This study LB
  KB461a,d NA ex 39 c.11220_11222dup p.(Gln3745dup) This study LB
  KB281 Inherited M ex 39 c.11714_11716dup p.(Gln3905dup) (19) LB
  KB71 Inherited M ex 39 c.11819_11836dup p.(Leu3940_Gln3945dup) (46) LB
  KB227 Inherited P ex 39 c.11843_11860del p.(L3948_Q3953del) (19) LB
  KB228 Inherited P ex 39 c.11854_11874dup p.(Q3952_Q3958dup) (19) LB
  KB77 NA ex 48 c.15163_15168dup p.(Asp5055_Leu5056dup) (18,46) VOUS
  KB403 De novo ex 53 c.16489_16491del p.(Ile5497del) (22,46,75,76) LP
  KB53 NA ex 53 c.16489_16491del p.(Ile5497del) (22,46,75,76) LP
 Splice site      
  KB286 De novo int 2–3 c.177-2A>C r.177_400del224; p.S59Rfs*86 (19) P
  KB31 NA int 3–4 c.400 + 1 G>A r.177_400del224; p.Ser59Argfs*86 (46) P
  KB20 De novo int 3–5 c.401-3 A>G r.400_401insAG; p.Gly134Glufs*75 (46) P
  KB442 NA int 6–7 c.840-6delC r.? This study VOUS
  KB519 NA int 13–14 c.4132-2A>G r.? This study P
  KB529 NA int 16–17 c.4584-6C>G r.? This study VOUS
  KB210 De novo in 17–18 c.4693 + 1G>A r.4681_4693del13; p.Val1561Argfsplice*11 (48) P
  KB29 NA int 42–43 c.13999 + 5 G>A r.13840_13999del160; p.Asn4614Ilefs*5 (46,77) P
  KB290 De novo int 47–48 c.14643 + 1G>A r.14644_14875del232; p.Glu4882Profs*36 (19) P
  KB195 De novo int 47–48 c.14644-3C>G r.14644_14875del232; p.Gln4882Profs*36 (19) P
  KB7 De novo int 44–45 c.14252-6_14252-5insGAAA r.14252_14382del131; p.Val4751_Glufsplice*22 (45) P
  KB360 NA int 47–48 c.14644-2A>T r.? (77) P
  KB496 NA int 53–54 c.16520_16521 + 1delAGG r.? This study P
 Gross deletion
  KB43e NA ex 48–51 c.15785-238_16168delins p.? (19) P
KDM6A
 Non-sense
  KB215f De novo ex 6 c.514C>T p.(Arg172*) (19,22,84) P
  KB341 De novo ex 6 c.514C>T p.(Arg172*) (22,84) P
 Frameshift      
  KB39 NA ex 16 c.1846_1849del p.(Thr616tyrfs*8) (19) P
  KB141 NA ex 17 c.2118_2119ins p.(G707Hfs*13) This study P
  KB434 NA ex 17 c.2515_2518del p.(Asn839Valfs*27) (85) P
  KB381 NA ex 20 c.3044delC p.(Thr1015Metfs*33) This study P
 Missense      
  KB415 NA ex 16 c.1843C>T p.(Leu615Phe) This study VOUS
  KB272 NA ex 17 c.2326G>T p.(Asp776Tyr) This study VOUS
  KB131 De novo ex 20 c.2939A>T p.(Asp980Val) (19) P
  KB380 De novo ex 26 c.3743A>G p.(Gln1248Arg) This study P
 Gross deletions  
  KB11 NA ex 1–2   p.? This study P
  KB50 De novo ex 5–9   p.? (17) P
 Splice site      
  KB314 De novo int 11–12 c.975-1G>A r.876_1320del; p.Cys293IlefsX26 This study P
  KB127 De novo int 22–23 c.3384 + 3_3384 + 6del r.3210_3284del; p.Asn1070_Lys1094del (19) P
Open in a new tab
a

Detected together with pathogenic mutation.

b

Falls in the last base of exon, predicted to disrupt the donor splice site.

c

Patients with compound heterozygous variants.

d

Detected together with other variant.

e

Identified by MLPA.

f

Detected together with missense in KMT2D.

P, pathogenic; LP, likely pathogenic; LB, likely benign; VOUS variant of unknown significance.

Figure 1.

Figure 1.

Open in a new tab

Missense variants distribution across the entire length or FUSION–KMT2D gene. (A) Schematic representation of the KMT2D protein (PHD, plant homeodomain; HMG, high-mobility group; Coiled Coil domain; LXXLL domain, motifs with the consensus sequence L–X–X–L–L motif; Poli Q region, Poli Q reach region identified in this study; ZF domain, Zinc Finger Domain; FYRN, FY-rich, N-terminal; FYRC, FY-rich, C-terminal; WIN, WDR5 Interaction domain, SET, Su(var)3-9, Enhancer-of-zeste, Trithorax). In black, missense variants found in our cohort of KS patients. De novo variants are indicated in bold, pathogenic variants with P, likely pathogenic with LP, likely benign with LP, and VOUS with V. (B) Distribution of the functionally analyzed KMT2D missense variants across the FUSION–KMT2D construct composed of PHD4–5–6 (amino acids 1358–1572) and ZF-PHD7–FYRN–FYRC–WIN–SET–post-SET domains (amino acids 4507–5537). De novo variants are indicated in bold, pathogenic variants are indicated with P, likely pathogenic with LP, likely benign with LB and VOUS with V.

Moreover, we identified 14 KDM6A variants; 12 of those were predicted to be pathogenic based on publicly available algorithms (see Materials and Methods) and 7 were never described previously. Seven of the variants were de novo, while for the remaining ones the inheritance was unknown. Thus, 210/505 (41%) KS patients in our cohort carried genetic alterations in one of these two genes; the underlying event in the remaining 295 cases remains unknown (see Discussion).

Pathogenic assessment of missense mutations by bioinformatics tools

Analogous to previous studies, most KMT2D variants in KS are inactivating truncating events that render the protein functionally defective due to the loss of the catalytic SET activity. However, ∼30% (69/208) of the mutations found in our data set, and 100 of 621 (16.1%) mutations from a recently published mutational analysis review (22), are in-frame amino acid changes that affect various residues along the KMT2D protein. To begin to elucidate the functional consequences of KMT2D missense mutations in KS, we first applied published bioinformatics tools to the 58 unique missense variants identified in the 69 KS patients (see Materials and Methods). These prediction algorithms classified 16/58 variants (identified in 20/69 patients) as pathogenic or likely pathogenic, while 24/58 variants (30/69 patients) were scored as likely benign, and 18/58 (19/69 patients) as variants of uncertain significance (VOUS, Table 2).

Table 2.

Bioinformatics analysis of KMT2D missense variants

        Prediction softwares
Code Variant AA change ACMG Variant Class Polyphen
 Align GVGD
 Provean
 SIFT
 UMD predictor
 Mutation Taster
Score Result Score Result Score Result Score Result Score Result Score Result
KB21a c.346T>C p.(Ser116Pro) LB PD 0.997 73.35 Class C65 −3.35 D 0.001 D 90 P DC 0, 994
KB21a,b c.510G>C p.Gln170His P PD 1.00 24.08 Class C15 −2.32 N 0.016 D 100 P DC 0.998
KB256 c.626C>T p.(Thr209Ile) VOUS B 0.096 89.28 Class C65 −2.23 N 0.009 D 75 P PM 0.980
KB458 c.1076G>C p.(Arg359Pro) VOUS B 0.011 102.71 Class C65 0.04 N 0.2056 T 33 PM PM 0.999
KB269 c.1940C>A p.(Pro647Gln) LB PD 0.59375 75.14 Class C65 −0.90 N 0.0771 T 78 P PM 0.999
KB126, KB374a c.2074C>A p.(Pro692Thr) LB B 0.008 37.56 Class C35 −0.92 N 0.011 D 66 PP PM 0.999
KB487 c.2654C>T p.(Pro885Leu) VOUS B 0.000 97.78 Class C65 −1.53 N 0.003 D 60 PPM PM 0.999
KB370a c.2837C>G p.(Ala946Gly) LB B 0.000 60.00 Class C55 −0.78 N 0.032 D 30 PM PM 0.999
KB215, KB341 c.3392C>T p.(Pro1131Leu) LB B 0.196 97.78 Class C65 −0.30 N 0.001 D 39 PM PM 0.989
KB222 c.3572C>T p.(Pro1191Leu) LB PD 0.764  97.78 Class C65 −2.50 D 0.001 D 35 PM DC 0.999
KB32 c.3773G>A p.(Arg1258Gln) LB PD 0.997 42.81 Class C35 −1.37 N 0.002 D 75 P PM 0.691
KB307 c.4171G>A p.(Glu1391Lys) LP PD 1.000 56.87 Class C55 −3.29 D 0.001 D 78 P DC 0.999
KB28a c.4249A>G p.(Met1417Val) LB PD 0.476 20.52 Class C15 00: 19 N 0.4097 T 60 PPM DC 0.559
KB28a c.4252C>A p.(Leu1418Met) LB PD 1.000 14.30 Class C0 −1.71 N 0.004 D 69 PP DC 0.999
KB174 c.4283T>C p.(Ile1428Thr) LB PD 0.889 89.28 Class C65 01: 14 N 0.5847 T 93 P DC 0.996
KB138 c.4427C>G p.(Ser1476Cys) VOUS B 0.005 111.67 Class C65 0.05 N 0.1771 T 99 P PM 0.885
KB34 c.4565A>G p.(Gln1522Arg) LB PD 0.997 42.81 Class C35 −3.43 D 0.021 D 100 P DC 0.999
KB535a c.5549G>A p.(Gly1850Asp) LB PD 0.535 93.77 Class C65 −0.72 N 0.103 T 87 P PM 0.997
KB119, KB204a c.6638G>A p.(Gly2213Asp) LB PD 0.454 93.77 Class C65 −0.70 N 0.004 D 42 PM DC 0.917
KB330a c.6733C>G p.(Leu2245Val) VOUS PD 0, 69 30.92 Class C25 −0.46 N 0.046 D 69 PP PM 0.996
KB326a c.6811C>T p.(Pro2271Ser) LB B 0.024 73.35 Class C65 −2.00 N 0.004 D 75 P DC 0.850
KB107a c.6970C>A p.(Pro2324Thr) VOUS PD 0.985 37.56 Class C35 −2.47 N 0.003 D 81 P PM 0.998
KB430 c.7378C>T p.(Arg2460Cys) LB PD 1.000 179.53 Class C65 −2.22 N 0.023 D 96 P DC 0.999
KB122, KB287 c.7829T>C p.(Leu2610Pro) LB B 0.076 97.78 Class C65 −1.20 N 0.0819 T 72 PP DC 0.998
KB27 c.8521C>A p.(Pro2841Thr) VOUS B 0.009 37.56 Class C35 −1.45 N 0.010 D 69 PP DC 0.997
KB330a c.8774C>T p.(Arg2925Val) VOUS B 0.004 65.28 Class C65 −1.07 N 0.004 D 84 P PM 0.958
KB443a c.9971G>T p.(Gly3324Val) LB PD 0.988 109.55 Class C65 −2.24 N 0.011 D 78 P DC 0.999
KB326a, KB357 c.10192A>G p.(Met3398Val) LB B 0.000 20.52 Class C15 −2.84 D 0.002 D 41 PM PM 0.958
KB297, KB378 c.10256A>G p.(Asp3419Gly) LB PD 1.000 93.77 Class C65 −1.81 N 0.000 D 100 P DC 0.999
KB292 c.10499G>T p.(Gly3500Val) LP PD 0.6875 109.55 Class C65 −8.18 D 0.000 D 100 P DC 0.999
KB86a c.10966C>T p.(Arg3656Cys) VOUS PD 1.000 179.53 Class C65 −1.87 N 0.001 D 84 P DC 0.999
KB374a c.11380C>T p.(Pro3794Ser) LB B 0.016 73.35 Class C65 −1.04 N 0.029 D 66 PP PM 0.993
KB293 c.11794C>G p.(Gln3932Glu) LB B 0.002 29.27 Class C25 −0.68 N 0.047 D 45 PM PM 0.999
KB204a c.12070A>G p.(Lys4024Glu) LB B 0.296 56.87 Class C55 00: 08 N 0.000 D 63 PPM PM 0.997
KB385 c.12302A>C p.(Gln4101Pro) VOUS B 0.000 75.14 Class C65 −0.03 N 0.2014 T 66 PP PM 0.939 
KB247 c.12485G>A p.(Arg4162Gln) VOUS B 0.003 42.81 Class C35 00: 30 N 0.000 D 72 PP PM 0.995
KB107a c.12488C>T p.(Pro4163Leu) VOUS PD 0.991 97.78 Class C65 −2.29 N 0.000 D 72 PP DC 0.983
KB170 c.13256C>T p.(Pro4419Leu) LB PD 1.000 97.78 Class C65 −4.16 D 0.000 D 87 P DC 0.999
KB512 c.14381A>G p.(Lys4794Arg) VOUS PD 0.999 26.00 Class C25 −1.43 N 0.005 D 69 PP DC 0.999
KB86a c.14893G>A p.(Ala4965Thr) VOUS PD 0.941 58.02 Class C55 −1.17 N 0.0847 T 75 P DC 0.997 
KB38a c.15084C>G p.(Asp5028Glu) LP PD 0.999 44.60 Class C35 −3.83 D 0.001 D 66 PP DC 0.999 
KB154, KB185 c.15088C>T p.(Arg5030Cys) VOUS PD 1.000 179.53 Class C65 −7.56 D 0.000 D 99 P DC 0.999
KB423 c.15089G>A p.(Arg5030His) LB PD 1.000 28.82 Class C25 −4.79 D 0.000 D 81 P DC 0.999
KB38a c.15100T>G p.(Phe5034Val) LP PD 0.999 48.95 Class C45 −6.17 D 0.001 D 84 P DC 0.999
KB76 c.15176A>C p.(His5059Pro) LP PD 1.000 76.28 Class C65 −9.60 D 0.001 D 93 P DC 0.999
KB129 c.15292A>C p.(Thr5098Pro) VOUS PD 0.992 37.56 Class C35 −4.21 D 0.1035 T 93 P DC 0.999
KB462 c.15310T>C p.(Cys5104Arg) LP PD 1.000 179.53 Class C65 −11.51 D 0.000 D 96 P DC 0.999
KB171 c.15565G>A p.(Gly5189Arg) VOUS PD 1.000 125.13 Class C65 −7.68 D 0.000 D 100 P DC 0.999
KB264, KB376 c.15640C>T p.(Arg5214Cys) LP PD 1.000 179.53 Class C65 −7.68 D 0.000 D 96 P DC 0.999
KB24, KB219, KB408 c.15641G>A p.(Arg5214His) LP PD 1.000 28.82 Class C25 −4.80 D 0.000 D 78 P DC 0.999
KB109 c.15649T>C p.(Trp5217Arg) LP PD 1.000 101.29 Class C65 −13.43 D 0.000 D 96 P DC 0.999
KB17 c.16019G>A p.(Arg5340Gln) LP PD 1.000 42.81 Class C35 −3.84 D 0.000 D 81 P DC 0.999
KB169 c.16273G>A p.(Glu5425Lys) P PD 1.000 56.87 Class C55 −3.70 D 0.000 D 75 P DC 0.999
KB90, KB467 c.16295G>A p.(Arg5432Gln) LP PD 1.000 42.81 Class C35 −3.70 D 0.000 D 84 P DC 0.999
KB480 c.16385A>G p.(Asp5462Gly) VOUS PD 1.000 93.77 Class C65 −6.69 D 0.000 D 90 P DC 0.999
KB177 c.16412G>T p.(Arg5471Met) LP PD 1.000 91.64 Class C65 −5.72 D 0.000 D 100 P DC 0999
KB489 c.16498C>T p.(Arg5500Trp) P PD 1.000 101.29 Class C65 −7.44 D 0.000 D 99 P DC 0.999
KB120 c.16528T>G p.(Tyr5510Asp) LP PD 1.000 159.94 Class C65 −9.52 D 0.000 D 96 P DC 0.999
Open in a new tab

Bold values indicate de novo variants.

a

Detected together with other variant.

b

Variant that falls in the last base of the exon.

P, pathogenic; LP, likely pathogenic; LB, likely benign; VOUS, variant of unknown significance; PD, probably damaging; B, benign; D, deleterious/damaging; N, neutral; T, tolerated; PP, probably pathogenic; PPM, probable polymorphism; PM, polymorphism; DC, disease causing.

To further assess how KMT2D missense variants may affect protein function/activity, a combination of structure-based methods was employed. Since KMT2D is a multi-domain protein too large to be accurately modeled using computational methods, we aimed to predict and analyze the individual structures of single KTM2D domains. Three-dimensional models were obtained for the ZF-7, FYR and SET domains (QMEAN scores of 0.63, 0.68 and 0.73, respectively) of the human KMT2D (see Materials and Methods for details). Moreover, a crystal structure at 1.4 Å resolution of the WIN domain was retrieved from the Protein Data Bank. Eight missense variants were analyzed to test their effects on protein stability (ΔΔG) and change in total charge (ΔCharge). The analysis showed that all eight variants target highly conserved regions/residues within these domains (Supplementary Material, Fig. S1) and are expected to significantly alter their structure (Supplementary Material, Fig. S2). In particular, with one exception (p.H5059P, which falls on the N-terminal residue of the predicted ZF-PHD7 and can be poorly analyzed), all variants were predicted to alter the total charge of the domain and/or the protein stability (Supplementary Material, Fig. S3). These data suggest that the missense variants located in the ZF-PHD7, FYR, WIN and SET domains affect the normal structure of the KMT2D protein and may thus potentially alter/impair the protein function.

Missense variants impair KMT2D methyltransferase activity

To experimentally test the functional impact of KS-associated KMT2D missense variants, we generated FLAG-tagged versions of 14 representative KMT2D mutant alleles that harbor amino acid changes in both functional N-terminal and C-terminal domains of the protein including PHD 4–5–6–7, FYRN, WIN and SET domains, using the FUSION–KMT2D construct as template (see Materials and Methods) (Fig. 1B).

The 14 tested missense variants have been selected as representative of the entire set of variants identified in our KS cohort that harbor amino acid changes in both N-terminal and C-terminal domains of the protein including PHD 4–5–6–7, FYRN, WIN and SET domains. This construct was selected because the protein encoded by the FUSION–KMT2D wild-type vector retains dose-dependent mono, di and tri methyltransferase activity in vitro to the same extent of a full-length wild-type protein (Supplementary Material, Fig. S4), while guaranteeing higher transfection efficiency (not shown).

We measured the ability of the mutated proteins to catalyze mono-, di- and tri-methylation of H3K4 (H3K4me1, me2 and me3) in vitro, using purified nucleosomal histones as substrate and western blot analysis with antibodies directed against these three histone marks. In this assay, 9/14 missense variants led to heterogeneous but significant impairment in H3K4 monomethylation, compared with wild-type KMT2D (range 30–80%), with two additional mutants showing borderline effects (Fig. 2A and B). Additionally, six of the nine mutations were associated with reduced H3K4me2, and 6 with reduced H3K4me3 levels (Fig. 2A and B).

Figure 2.

Figure 2.

Open in a new tab

KMT2D missense variants are associated with defective methyltransferase activity and diminished H3K4 methylation. (A) H3K4 Lysine methyltransferase activity of mutated FLAG-KMT2D proteins on HeLa nucleosomes. (B) Relative H3K4 methyltransferase activity of semi-purified FLAG-KMT2D proteins on HeLa nucleosomes (mean±SD; n=two independent biological replicates). Data are expressed as fold differences compared with the activity of the wild-type control, arbitrarily set as 1. Pathogenic variants are indicated with P, likely pathogenic with LP, likely benign with LB, and VOUS with V. Student’s T-test is indicated as *P<0.05; **P<0.01; ***P<0.001. (C) Schematic representation of the epigenetic reporter allele encoding the two halves of GFP separated by a flexible linker region with a histone tail (H3) and a histone reader (TAF3 PHD) at the N and C termini, respectively (23). Modification of the histone tail by (KMT2D-mediated) trimethylation leads to reconstitution of the GFP structure and function, which can be measured by fluorescence. (D) The H3K4me3 indicator demonstrated a decreased H3K4me3 activity for the 6 tested missense variants compared with the WT. Pathogenic variants are indicated with P, likely pathogenic with LP, likely benign with LB, and VOUS with V. Student’s t-test is indicated as *P<0.001.

To independently confirm the reduced H3K4 tri-methyltransferase activity, we used a more sensitive epigenetic EGFP reporter system that specifically monitors this modification (23) (Fig. 2C). Fluorescence data confirmed the significantly reduced ability to catalyze H3K4me3 for all 6 missense variants tested (on average, ∼50% compared with the KMT2D wild-type counterpart) (Fig. 2D). Together, these data indicate that, to various extents, a subset of KS-associated missense mutations can inactivate the function of KMT2D, analogous to truncating mutations.

KMT2D-complex protein interaction

ASH2L and RbBP5 interact with KMT2D co-regulating the expression of target genes (24,25). To determine whether KMT2D missense variants affect its function by altering the interaction with ASH2L and RbBP5, we immunoprecipitated protein lysates from HEK 293T cells co-transfected with Flag-tagged FUSION–KMT2D constructs and vectors expressing either ASH2L or RbBP5. Western blot analysis of immunoprecipitated cell lysates showed that the missense variants located at the C-terminal of the protein (p.H5059P, p.T5098P, p.G5189R, p.W5217R, p.R5340Q, p.E5425K p.R5471M and Y5510D) exhibit a weaker interaction with both ASH2L and RbBP5, while p.E1391K and p.F5034V only distressed the interaction between KMT2D and ASH2L, when compared with the wild-type construct. The missense variants p.I1428T and p.Q1522R exhibited an increased interaction with both ASH2L and RbBP5, while the p.M1417V and p.S1476C only increased the interaction of KMT2D with ASH2L (Fig. 3A).

Figure 3.

Figure 3.

Open in a new tab

Interaction of KMT2D missense mutants with ASH2L, RbBP5 and WDR5. (A) On the left, immunoblot analysis of KMT2D, ASH2L and RbBP5 in HEK293T cell line transfected with wild-type KMT2D or KMT2D missense mutants followed by immunoprecipitation with anti-Flag antibody. On the right, relative interaction of the indicated KMT2D missense mutants with ASH2L (top) and RbBP5 (bottom) (mean±SD, n=2 independent replicates). Pathogenic variants are indicated with P, likely pathogenic with LP, likely benign with LB, and VOUS with V. Student’s T-test is indicated as *P<0.05, **P<0.01 and ***P<0.001. (B) On the left, the predicted amino acid tolerance for KMT2D p.5340: the presence of Methionine (M) is very slightly tolerated. On the right, immunoblot analysis of KMT2D and WDR5 in HEK293 cell line transfected with wild-type WDR5, wild-type C-Ter KMT2D or the p.R5340Q KMT2D missense mutant before (input) and after immunoprecipitation with the anti-Flag antibody. EV, empty vector.

Combined conformational 3D modeling and free energy interaction variations (see Supplementary Material, Fig. S2) indicated that the p.R5340Q missense variant—in the WIN motif of KMT2D—alters the protein domain structure, changing an evolutionarily conserved amino acid position expected to tolerate Arginine and minimally Methionine (Fig. 3B, left). Since the KMT2D WIN motif (5337–5342) is necessary for the interaction with the WDR5 protein (26), we used transient transfection/co-immunoprecipitation assays to assess whether p.R5340Q alters the ability of KMT2D [expressed as an Myc-tagged green fluorescent protein (GFP) fusion] to physically bind a Flag-tagged WDR5 protein.

As shown in Figure 3B, western blot analysis of FLAG-immunoprecipitates using anti-Myc antibodies showed efficient co-immunoprecipitation of the wild-type KMT2D, but not of the R5340 mutant, with WDR5. This was not due to differences in expression levels/protein stability between the two KMT2D proteins, as documented by western blot analysis of EGFP in the total cell lysates, indicating that this variant completely abrogates the interaction with WDR5 (Fig. 3B).

Discussion

Here we report the mutation pattern of KMT2D and KDM6A in a cohort of 505 patients clinically diagnosed as KS-affected, and document the functional role of a subset of KMT2D missense mutations in impairing the protein enzymatic activity.

Overall, the mutation detection rate for KMT2D and KDM6A in our cohort (41%) was lower than that reported in a recent survey (22). This does not seem to be due to differences in sensitivity as the same Sanger Sequencing approach was used, and may be derived from the involvement of other causative genes, epigenetics mechanisms or yet unrecognized promoter or deep intronic variants affecting normal splicing. Moreover, we cannot exclude that some patients might have been clinically misdiagnosed and present conditions partially overlapping with KS. In view of multiple studies highlighting the clinical and molecular overlap of KS (27–31), these samples should be interrogated by using NGS targeted-genes panels focused on components of the histone methylation machinery that are associated to diseases in differential diagnosis with KS.

Our screening revealed 58 different missense variants distributed across the entire length of the KMT2D gene. Indeed, 9 of the 14 representative mutations that were tested in our study had variably reduced histone methylation activity in vitro, including H3K4 mono-, di- and/or tri-methylation. This effect is consistent with the known function of KMT2D as a major mono/dimethyltransferase, which is enriched at enhancer regions and is required for enhancer activation during cell differentiation, as reported in brown adipose tissue and skeletal muscle development (11,32), or in mature B cells (33,34). Even though recent publications focus on the role of KMT2D as major monomethyltransferase, at least during carcinogenesis (35), KMT2D is also required during oogenesis and early development for bulk histone H3 lysine 4 trimethylation (36). The ability to modulate H3K4(me3), at active promoters/transcription start sites is important for the regulation of actively transcribed genes (14,15), and may reflect in part the formation of promoter-enhancer loops. As a matter of fact, six of the nine KS missense variants with reduced histone methylation activity showed a flawed H3K4(me3) activity in two different assays, emphasizing the role of KMT2D in modulating histone H3K4(me3) in vitro.

Of note, all nine functionally defective missense mutations were localized within the FYRN, WIN and SET domains, and may contribute to the KS phenotypes by interfering with its enzymatic activity in a manner analogous to C-terminal truncating mutations. In contrast, amino acid changes in the N-terminal domain of the protein had minimal effects on H3K4 methylation. Indeed, according to ACMG, most of the amino acid changes in the N-terminal domain of the protein are classified as Likely Benign or Variants Of Unknown Significance (Table 2), suggesting that they may act through different mechanisms or represent neutral events.

The entire KMT2D C-terminal domain (aa 4507–5537) is involved in the interaction with the protein of the WRAD complex (25), albeit the specific amino acid interaction region is not fully characterized yet. Our study showed that missense variants localized in the C-terminal domains impaired the interaction of KMT2D with other components of its multi-subunit complex, including WDR5, RbBP5 and ASH2L, whereas most of the variants localized within the PHD 4–6 do not affect the interaction. The same C-terminal missense variants also showed altered protein domain structure and free energy interaction levels, suggesting that structural changes occurring in KMT2D may reduce the interaction with the proteins of the WRAD complex.

These data corroborate the hypothesis that the reduced methyltransferase activity is a direct consequence of the lack of multi-protein WRAD complex formation. Consistently, the missense variant R5340Q, localized within the WIN domain, fully abrogated the interaction with WDR5, resulting in a significantly reduced methyltransferase activity.

The classification and interpretation of KMT2D missense variants is a significant challenge in molecular diagnostics and genetic counseling. A number of prediction tools have been implemented in the last years, mainly as support for NGS data. However, in silico analyses should not be considered as conclusive evidence in the assessment of variants of unknown clinical significance. The American College of Medical Genetics and Genomics and the Association for Molecular Pathology Standards and Guidelines for the interpretation of sequence variants (37) recommended that these predictions should be used as support to additional findings, including functional evidence that can prove the pathogenicity of the variants found. Recently, different DNA methylation profiling studies showed that KS patients present a highly specific and univocal DNA methylation signature that has the potential to be used as a diagnostic method for differentiating samples with pathogenic mutations from those with benign variants, and therefore enabling the functional assessment of genetic variants of unknown clinical significance (31,38–41).

Although additional studies will be required to dissect the precise mechanisms underlying the pathogenic role of the missense variants in KS, our data indicate that a subset of them may influence the disease status by affecting: (i) the methyltransferase activity of the protein and (ii) the interaction with the WRAD complex.

The biochemical approaches presented here may provide the basis for the development of diagnostic assays that could be of support to in silico prediction tools, with the advantage of addressing the functional effect of the variant.

Conclusions

This study expands the number of KMT2D and KDM6A mutations that are associated with KS and provides evidence that a number of naturally occurring missense mutations in KMT2D effectively impact KMT2D interaction and H3K4 methylation activity. These data have direct relevance for diagnostic and counseling purposes.

Materials and Methods

Patients and samples preparation

Our study cohort comprised a total of 505 index patients clinically diagnosed as affected by KS (Table 1), including 202 new cases that were referred to our institution between 2014 and 2017, and 303 patients from previous studies (19,42–48).

Patients were enrolled after obtaining appropriate informed consent by the physicians in charge, and approval by the local ethics committees. KS patients were included following the inclusions criteria as reported (46). Genomic DNA was extracted from fresh and/or frozen peripheral blood leukocytes of patients and their available family members using an automated DNA extractor and commercial DNA extraction Kits (Qiagen, Germany). Total RNA was extracted from peripheral blood leukocytes using TRIzol reagent (ThermoFisher Scientific, USA) and reverse transcribed using the QuantiTect Transcription kit (Qiagen), according to the manufacturer’s instructions.

Sequencing and MLPA of KMT2D and KDM6A

Mutation screening of all 54 coding exons of the KMT2D (MIM #602113, NM_003482.3) gene and 29 coding exons of the KDM6A (MIM #300128, NM_021140.3) gene was performed by PCR amplification and direct sequencing as reported (46). MLPA analysis was performed as in (47).

In silico analysis of KMT2D and KDM6A variants

The putative causal and functional effect of missense variants was estimated by using the following in silico prediction tools: Polyphen-2 version 2.2.2 (http://genetics.bwh.harvard.edu/pph; date last accessed 5 July 2018) (49), Align GVGD (http://agvgd.hci.utah.edu; date last accessed 5 July 2018) (50), PROVEAN v1.1 (http://provean.jcvi.org/index.php; date last accessed 5 July 2018) (51), SIFT v1.03 (http://sift.jcvi.org/; date last accessed 5 July 2018) (52), UMD-predictor (http://www.umd.be/; date last accessed 5 July 2018) (53) and Mutation Taster (http://www.mutationtaster.org/; date last accessed 5 July 2018) (54) using default parameters. Splice-site variants were evaluated for putative alteration of regulatory process at the transcriptional or splicing level with Human Splice Finder (http://www.umd.be/HSF3/; date last accessed 5 July 2018) (55), NNSPLICE (http://www.fruitfly.org/seq_tools/splice.html; date last accessed 5 July 2018) (56) and NetGene2 (http://www.cbs.dtu.dk/services/NetGene2; date last accessed 5 July 2018) (57). Frequency variants were checked on dbSNP (http://www.ncbi.nlm.nih.gov/projects/SNP/; date last accessed 5 July 2018), 1000 Genomes Project (http://www.internationalgenome.org/; date last accessed 5 July 2018), EVS (http://evs.gs.washington.edu/EVS/; date last accessed 5 July 2018) and ExAC (http://exac.broadinstitute.org/; date last accessed 5 July 2018).

Protein modeling

Three-dimensional models of the ZF-PHD7 (residues 5059–5137), FYRN (residues 5180–5327) and SET (residues 5398–5537) domains of the human KMT2D protein were obtained employing a combination of threading and homology methods. For each domain, different models were generated using publicly available web servers (58–61). Final KMT2D model domains were obtained using the Modeller program and, as templates, the server models (62). A total of 200 unique models were generated by Modeller for each KMT2D domain. Qmean server (63) was used to assess the quality of the predicted models and KoBaMIN web server (64) was used to carry out the protein energy minimization. The crystal structure of the WIN domain bound to WDR5 was obtained from the RCSB Protein Data Bank (PDB code: 3UVK; residues 5337–5347) (65). The effects of missense variants on KMT2D domains structure and stability were predicted by the Rosetta Backrub server (66) and FoldX program (67). Total charge of domains was calculated using the PROPKA program (68) and the electrostatic surface potentials were computed using the Adaptive Poisson-Boltzmann Solver (APBS) software (69). Models’ inspection and model figures were performed using UCSF Chimera (version 1.11) (70).

Plasmids, cell lines and transfection assays

The pFlag-CMV2 FUSION–KMT2D vector [cDNA spanning the PHD4–5–6 domains (amino acids 1358–1572)] and ZF-PHD7-FYRN-FYRC-WIN-SET-post SET domains (amino acids 4507–5537) was a gift of Professor Min Gyu Lee, Department of Molecular and Cellular Oncology, The University of Texas (25). We sub-cloned this partial KMT2D sequence into the p3XFlag-CMV14 vector (Sigma) using standard procedures. Fourteen representative KMT2D variants identified in our KS cohort that harbor amino acid changes in both N-terminal and C-terminal domains of the protein, including PHD 4–5–6–7, FYRN, WIN and SET domains, have been selected for functional assays: p.E1391K (Likely Pathogenic), p.M1417V (Likely Benign), p.I1428T (Likely Benign), p.S1476C (VOUS), p.Q1522R (Likely Benign), p.F5034V (Likely pathogenic), p.H5059P (Likely Pathogenic), p.T5098P (VOUS), p.G5189R (VOUS), p.W5217R (Likely pathogenic), p.R5340Q (Likely Pathogenic), p.E5425K (Pathogenic), p.R5471M (Likely Pathogenic) and p.Y5510D (Pathogenic). Expression plasmids harboring patient-derived KMT2D missense variants were generated by site-directed mutagenesis according to standard methods.

HEK 293 and 293T cells were cultured in Dulbecco’s Modified Eagle Medium with 10% FBS, penicillin (100 U/ml) and streptomycin (100 μg/ml) (Life Technologies). The KMT2D C-terminal ORF was assembled into the pcDNA3-Myc-EGFP vector (71) by PCR site directed amplification using human cDNA and the pFlag-CMV2 FUSION–KMT2D vector as templates respectively. The WDR5 full length ORF was assembled into the p3XFlag-CMV14 vector (Sigma) by RT-PCR amplification reactions using cDNA from HEK 293T cells as template. HEK 293T cells were transiently transfected using the polyethylenimine method, following the published protocols (72). Cells were harvested 48 h after transfection and used for protein extraction and histone methyltransferase (HMT) assay.

In vitro HMT assay and epigenetic reporter allele

Partially purified FLAG-KMT2D wild-type and mutant derivative proteins were extracted from transfected HEK 293T cells by co-IP buffer (50 mM Tris, pH 7.5, 250 mM NaCl, 1% Triton X-100, 1 mM EDTA), followed by overnight incubation with EZview™ Red Anti-FLAG Affinity Gel (Sigma) at 4°C and final elution in BC100 buffer (20 mM Tris, pH 7.5, 10% glycerol, 0.2 mM EDTA, 1% Triton X-100, 100 mM NaCl) containing FLAG peptide (Sigma). KMT2D protein amounts were quantified by Coomassie staining and immunoblot analysis using mouse monoclonal FLAG antibodies. Enzymatic activity against HeLa nucleosomes was measured following a published method (25). Briefly, equal amounts of wild-type or mutant FLAG-KMT2D proteins were incubated at 37°C for 4 h with HeLa nucleosomes (Reaction Biology) in KMT buffer (50 mM Tris, pH 8.5, 100 mM KCl, 5 mM MgCl2, 10% glycerol, 4 mM DTT) supplemented with S-adenosyl methionine (New England BioLabs). Reactions were stopped by adding equal volumes of 2× Laemmli buffer and heated at 100°C for 5 min before loading onto Tris–glycine 4–20% gradient gels. All assays were performed at least two times independently.

The epigenetic reporter allele, a kind gift from Dr Hans T. Bjornsson (McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University, Baltimore), was used as described (23).

Co-immunoprecipitation assay and western blotting analysis

Co-immunoprecipitations were performed using Dynabeads magnetic beads (ThermoFisher Scientific) and EZview™ Red Anti-FLAG Affinity Gel (Sigma) following the manufacturer’s instructions. Complexes were analyzed by Western blot using the indicated antibodies. Protein extracts were resolved on NuPAGE Tris–acetate 3–8% gels (for KMT2D) or Tris–glycine 4–20% gels (for histone H3) (ThermoFisher Scientific) and transferred to nitrocellulose membranes (GE Healthcare) according to the manufacturer’s instructions. Antibodies used were mouse monoclonal antibody to α-tubulin (clone DM1A, Sigma), rabbit polyclonal anti-H3K4me1 (Abcam), anti-H3K4me2 (Active Motif), anti-H3K4me3 (Abcam), rabbit monoclonal anti-Histone H3 (clone D1H2, Cell Signaling Technology), mouse monoclonal anti-Flag (Sigma cat# F3165), rabbit monoclonal anti GFP (Santa Cruz), rat monoclonal anti-HA (Roche), mouse monoclonal anti Myc (Roche), rabbit polyclonal anti Ash2 (Bethyl) and rabbit polyclonal anti RbBP5 (Bethyl). Horseradish peroxidase conjugated anti-mouse (Santa Cruz) or anti-rabbit (Santa Cruz) antibodies, and the ECL chemiluminescence system (GE Healthcare) were used for detection. ImageJ software (http://imagej.nih.gov/ij/; date last accessed 5 July 2018) was used to quantify band signal intensity. Values are expressed as fold differences relative to the wild-type protein sample, set at 1, after normalization for the loading control.

Supplementary Material

Supplementary Data
Click here for additional data file. (8.9MB, zip)

Acknowledgements

We acknowledge the family that agreed to participate and made this study possible. We also acknowledge Professor Hans T. Bjornsson for providing Epigenetic reporter allele vector. We are grateful to the Genomic Disorder Biobank and Telethon Network of Genetic Biobanks (Telethon Italy Grant GTB12001G) for biobanking biospecimens.

Conflict of Interest statement: The authors declare no conflicts of interest with the exception of G.M. who is a consultant for Takeda Pharmaceutical Company.

Funding

N.I.H. Grant RO1-CA172492 (to L.P.), in part; Lymphoma Research Foundation post-doctoral fellowship to J.Z. Telethon - Italy (Grant no. GGP13231), Italian Ministry of Health, Jérôme Lejeune Foundation, Daunia Plast, Circolo Unione Apricena, Fidapa Apricena, and Associazione Italiana Sindrome Kabuki (AISK) to G.M. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

References

  • 1. Kuroki Y., Suzuki Y., Chyo H., Hata A., Matsui I. (1981) A new malformation syndrome of long palpebral fissures, large ears, depressed nasal tip, and skeletal anomalies associated with postnatal dwarfism and mental retardation. J. Pediatr., 99, 570–573. [DOI] [PubMed] [Google Scholar]
  • 2. Niikawa N., Matsuura N., Fukushima Y., Ohsawa T., Kajii T. (1981) Kabuki make-up syndrome: a syndrome of mental retardation, unusual facies, large and protruding ears, and postnatal growth deficiency. J. Pediatr., 99, 565–569. [DOI] [PubMed] [Google Scholar]
  • 3. Cheon C.K., Ko J.M. (2015) Kabuki syndrome: clinical and molecular characteristics. Korean J. Pediatr., 58, 317–324. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4. Ng S.B., Bigham A.W., Buckingham K.J., Hannibal M.C., McMillin M.J., Gildersleeve H.I., Beck A.E., Tabor H.K., Cooper G.M., Mefford H.C.. et al. (2010) Exome sequencing identifies MLL2 mutations as a cause of Kabuki syndrome. Nat. Genet., 42, 790–793. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5. Shilatifard A. (2012) The COMPASS family of histone H3K4 methylases: mechanisms of regulation in development and disease pathogenesis. Annu. Rev. Biochem., 81, 65–95. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6. Kouzarides T. (2007) Chromatin modifications and their function. Cell, 128, 693–705. [DOI] [PubMed] [Google Scholar]
  • 7. Li B., Carey M., Workman J.L. (2007) The role of chromatin during transcription. Cell, 128, 707–719. [DOI] [PubMed] [Google Scholar]
  • 8. Ang S.Y., Uebersohn A., Spencer C.I., Huang Y., Lee J.E., Ge K., Bruneau B.G. (2016) KMT2D regulates specific programs in heart development via histone H3 lysine 4 di-methylation. Development, 143, 810–821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9. Herz H.M., Mohan M., Garruss A.S., Liang K., Takahashi Y.H., Mickey K., Voets O., Verrijzer C.P., Shilatifard A. (2012) Enhancer-associated H3K4 monomethylation by Trithorax-related, the Drosophila homolog of mammalian Mll3/Mll4. Genes. Dev., 26, 2604–2620. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10. Hu D., Gao X., Morgan M.A., Herz H.M., Smith E.R., Shilatifard A. (2013) The MLL3/MLL4 branches of the COMPASS family function as major histone H3K4 monomethylases at enhancers. Mol. Cell. Biol., 33, 4745–4754. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11. Lee J.E., Wang C., Xu S., Cho Y.W., Wang L., Feng X., Baldridge A., Sartorelli V., Zhuang L., Peng W.. et al. (2013) H3K4 mono- and di-methyltransferase MLL4 is required for enhancer activation during cell differentiation. Elife (Cambridge), 2, e01503.. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12. Guo C., Chen L.H., Huang Y., Chang C.C., Wang P., Pirozzi C.J., Qin X., Bao X., Greer P.K., McLendon R.E.. et al. (2013) KMT2D maintains neoplastic cell proliferation and global histone H3 lysine 4 monomethylation. Oncotarget, 4, 2144–2153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13. Kaikkonen M.U., Spann N.J., Heinz S., Romanoski C.E., Allison K.A., Stender J.D., Chun H.B., Tough D.F., Prinjha R.K., Benner C.. et al. (2013) Remodeling of the enhancer landscape during macrophage activation is coupled to enhancer transcription. Mol. Cell, 51, 310–325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14. Heintzman N.D., Hon G.C., Hawkins R.D., Kheradpour P., Stark A., Harp L.F., Ye Z., Lee L.K., Stuart R.K., Ching C.W.. et al. (2009) Histone modifications at human enhancers reflect global cell-type-specific gene expression. Nature, 459, 108–112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15. Denissov S., Hofemeister H., Marks H., Kranz A., Ciotta G., Singh S., Anastassiadis K., Stunnenberg H.G., Stewart A.F. (2014) Mll2 is required for H3K4 trimethylation on bivalent promoters in embryonic stem cells, whereas Mll1 is redundant. Development, 141, 526–537. [DOI] [PubMed] [Google Scholar]
  • 16. Issaeva I., Zonis Y., Rozovskaia T., Orlovsky K., Croce C.M., Nakamura T., Mazo A., Eisenbach L., Canaani E. (2007) Knockdown of ALR (MLL2) reveals ALR target genes and leads to alterations in cell adhesion and growth. Mol. Cell. Biol., 27, 1889–1903. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17. Lederer D., Grisart B., Digilio M.C., Benoit V., Crespin M., Ghariani S.C., Maystadt I., Dallapiccola B., Verellen-Dumoulin C. (2012) Deletion of KDM6A, a histone demethylase interacting with MLL2, in three patients with Kabuki syndrome. Am. J. Hum. Genet., 90, 119–124. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18. Miyake N., Mizuno S., Okamoto N., Ohashi H., Shiina M., Ogata K., Tsurusaki Y., Nakashima M., Saitsu H., Niikawa N.. et al. (2013) KDM6A point mutations cause Kabuki syndrome. Hum. Mutat., 34, 108–110. [DOI] [PubMed] [Google Scholar]
  • 19. Micale L., Augello B., Maffeo C., Selicorni A., Zucchetti F., Fusco C., De Nittis P., Pellico M.T., Mandriani B., Fischetto R.. et al. (2014) Molecular analysis, pathogenic mechanisms, and readthrough therapy on a large cohort of Kabuki syndrome patients. Hum. Mutat., 35, 841–850. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20. Hong S., Cho Y.W., Yu L.R., Yu H., Veenstra T.D., Ge K. (2007) Identification of JmjC domain-containing UTX and JMJD3 as histone H3 lysine 27 demethylases. Proc. Natl. Acad. Sci. U.S.A., 104, 18439–18444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21. Lan F., Bayliss P.E., Rinn J.L., Whetstine J.R., Wang J.K., Chen S., Iwase S., Alpatov R., Issaeva I., Canaani E.. et al. (2007) A histone H3 lysine 27 demethylase regulates animal posterior development. Nature, 449, 689–694. [DOI] [PubMed] [Google Scholar]
  • 22. Bogershausen N., Gatinois V., Riehmer V., Kayserili H., Becker J., Thoenes M., Simsek-Kiper P.O., Barat-Houari M., Elcioglu N.H., Wieczorek D.. et al. (2016) Mutation update for Kabuki syndrome genes KMT2D and KDM6A and further delineation of X-linked Kabuki syndrome subtype 2. Hum. Mutat, 37, 847–864. [DOI] [PubMed] [Google Scholar]
  • 23. Bjornsson H.T., Benjamin J.S., Zhang L., Weissman J., Gerber E.E., Chen Y.C., Vaurio R.G., Potter M.C., Hansen K.D., Dietz H.C. (2014) Histone deacetylase inhibition rescues structural and functional brain deficits in a mouse model of Kabuki syndrome. Sci. Transl. Med., 6, 256ra135.. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24. Kim J.H., Sharma A., Dhar S.S., Lee S.H., Gu B., Chan C.H., Lin H.K., Lee M.G. (2014) UTX and MLL4 coordinately regulate transcriptional programs for cell proliferation and invasiveness in breast cancer cells. Cancer Res., 74, 1705–1717. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25. Dhar S.S., Lee S.H., Kan P.Y., Voigt P., Ma L., Shi X., Reinberg D., Lee M.G. (2012) Trans-tail regulation of MLL4-catalyzed H3K4 methylation by H4R3 symmetric dimethylation is mediated by a tandem PHD of MLL4. Genes Dev., 26, 2749–2762. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26. Shinsky S.A., Hu M., Vought V.E., Ng S.B., Bamshad M.J., Shendure J., Cosgrove M.S. (2014) A non-active-site SET domain surface crucial for the interaction of MLL1 and the RbBP5/Ash2L heterodimer within MLL family core complexes. J. Mol. Biol., 426, 2283–2299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27. Au P.Y.B., You J., Caluseriu O., Schwartzentruber J., Majewski J., Bernier F.P., Ferguson M., Valle D., Parboosingh J.S., Sobreira N.. et al. (2015) GeneMatcher aids in the identification of a new malformation syndrome with intellectual disability, unique facial dysmorphisms, and skeletal and connective tissue abnormalities caused by de novo variants in HNRNPK. Hum. Mutat., 36, 1009–1014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28. Lange L., Pagnamenta A.T., Lise S., Clasper S., Stewart H., Akha E.S., Quaghebeur G., Knight S.J., Keays D.A., Taylor J.C.. et al. (2016) A de novo frameshift in HNRNPK causing a Kabuki-like syndrome with nodular heterotopia. Clin. Genet., 90, 258–262. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29. Yuan B., Pehlivan D., Karaca E., Patel N., Charng W.L., Gambin T., Gonzaga-Jauregui C., Sutton V.R., Yesil G., Bozdogan S.T.. et al. (2015) Global transcriptional disturbances underlie Cornelia de Lange syndrome and related phenotypes. J. Clin. Invest., 125, 636–651. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30. Parenti I., Teresa-Rodrigo M.E., Pozojevic J., Ruiz Gil S., Bader I., Braunholz D., Bramswig N.C., Gervasini C., Larizza L., Pfeiffer L.. et al. (2017) Mutations in chromatin regulators functionally link Cornelia de Lange syndrome and clinically overlapping phenotypes. Hum. Genet., 136, 307–320. [DOI] [PubMed] [Google Scholar]
  • 31. Sobreira N., Brucato M., Zhang L., Ladd-Acosta C., Ongaco C., Romm J., Doheny K.F., Mingroni-Netto R.C., Bertola D., Kim C.A.. et al. (2017) Patients with a Kabuki syndrome phenotype demonstrate DNA methylation abnormalities. Eur. J. Hum. Genet, 25, 1335–1344. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32. Guenther M.G., Jenner R.G., Chevalier B., Nakamura T., Croce C.M., Canaani E., Young R.A. (2005) Global and Hox-specific roles for the MLL1 methyltransferase. Proc. Natl. Acad. Sci. U.S.A., 102, 8603–8608. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33. Zhang J., Dominguez-Sola D., Hussein S., Lee J.E., Holmes A.B., Bansal M., Vlasevska S., Mo T., Tang H., Basso K.. et al. (2015) Disruption of KMT2D perturbs germinal center B cell development and promotes lymphomagenesis. Nat. Med., 21, 1190–1198. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34. Lindsley A.W., Saal H.M., Burrow T.A., Hopkin R.J., Shchelochkov O., Khandelwal P., Xie C., Bleesing J., Filipovich L., Risma K.. et al. (2016) Defects of B-cell terminal differentiation in patients with type-1 Kabuki syndrome. J. Allergy Clin. Immunol., 137, 179–187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35. Lv S., Ji L., Chen B., Liu S., Lei C., Liu X., Qi X., Wang Y., Lai-Han Leung E., Wang H.. et al. (2018) Histone methyltransferase KMT2D sustains prostate carcinogenesis and metastasis via epigenetically activating LIFR and KLF4. Oncogene, 37, 1354–1368. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36. Andreu-Vieyra C.V., Chen R., Agno J.E., Glaser S., Anastassiadis K., Stewart A.F., Matzuk M.M. (2010) MLL2 is required in oocytes for bulk histone 3 lysine 4 trimethylation and transcriptional silencing. PLoS Biol., 8, e1000453.. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37. Richards S., Aziz N., Bale S., Bick D., Das S., Gastier-Foster J., Grody W.W., Hegde M., Lyon E., Spector E.. et al. (2015) Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet. Med., 17, 405–424. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38. Butcher D.T., Cytrynbaum C., Turinsky A.L., Siu M.T., Inbar-Feigenberg M., Mendoza-Londono R., Chitayat D., Walker S., Machado J., Caluseriu O.. et al. (2017) CHARGE and Kabuki syndromes: gene-specific DNA methylation signatures identify epigenetic mechanisms linking these clinically overlapping conditions. Am. J. Hum. Genet., 100, 773–788. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39. Choufani S., Cytrynbaum C., Chung B.H., Turinsky A.L., Grafodatskaya D., Chen Y.A., Cohen A.S., Dupuis L., Butcher D.T., Siu M.T.. et al. (2015) NSD1 mutations generate a genome-wide DNA methylation signature. Nat. Commun., 6, 10207.. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40. Grafodatskaya D., Chung B.H., Butcher D.T., Turinsky A.L., Goodman S.J., Choufani S., Chen Y.A., Lou Y., Zhao C., Rajendram R.. et al. (2013) Multilocus loss of DNA methylation in individuals with mutations in the histone H3 lysine 4 demethylase KDM5C. BMC Med. Genomics, 6, 1.. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41. Kernohan K.D., Cigana Schenkel L., Huang L., Smith A., Pare G., Ainsworth P., Boycott K.M., Warman-Chardon J., Sadikovic B. (2016) Identification of a methylation profile for DNMT1-associated autosomal dominant cerebellar ataxia, deafness, and narcolepsy. Clin. Epigenetics, 8, 91.. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42. Andersen M.S., Menazzi S., Brun P., Cocah C., Merla G., Solari A. (2014) Clinical diagnosis of Kabuki syndrome: phenotype and associated abnormalities in two new cases. Arch. Argent. Pediatr., 112, 26–32. [DOI] [PubMed] [Google Scholar]
  • 43. Cappuccio G., Rossi A., Fontana P., Acampora E., Avolio V., Merla G., Zelante L., Secinaro A., Andria G., Melis D. (2014) Bronchial isomerism in a Kabuki syndrome patient with a novel mutation in MLL2 gene. BMC Med. Genet., 15, 15, [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44. Ejarque I., Uliana V., Forzano F., Marciano C., Merla G., Zelante L., Di Maria E., Faravelli F. (2011) Is Hardikar syndrome distinct from Kabuki (Niikawa-Kuroki) syndrome? Clin. Genet., 80, 493–496. [DOI] [PubMed] [Google Scholar]
  • 45. Makrythanasis P., van Bon B.W., Steehouwer M., Rodriguez-Santiago B., Simpson M., Dias P., Anderlid B.M., Arts P., Bhat M., Augello B.. et al. (2013) MLL2 mutation detection in 86 patients with Kabuki syndrome: a genotype-phenotype study. Clin. Genet., 84, 539–545. [DOI] [PubMed] [Google Scholar]
  • 46. Micale L., Augello B., Fusco C., Selicorni A., Loviglio M.N., Silengo M.C., Reymond A., Gumiero B., Zucchetti F., D'Addetta E.V.. et al. (2011) Mutation spectrum of MLL2 in a cohort of Kabuki syndrome patients. Orphanet J. Rare Dis., 6, 38.. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47. Priolo M., Micale L., Augello B., Fusco C., Zucchetti F., Prontera P., Paduano V., Biamino E., Selicorni A., Mammi C.. et al. (2012) Absence of deletion and duplication of MLL2 and KDM6A genes in a large cohort of patients with Kabuki syndrome. Mol. Genet. Metab., 107, 627–629. [DOI] [PubMed] [Google Scholar]
  • 48. Ratbi I., Fejjal N., Micale L., Augello B., Fusco C., Lyahyai J., Merla G., Sefiani A. (2013) Report of the first clinical case of a Moroccan Kabuki patient with a novel MLL2 mutation. Mol. Syndromol., 4, 152–156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49. Adzhubei I.A., Schmidt S., Peshkin L., Ramensky V.E., Gerasimova A., Bork P., Kondrashov A.S., Sunyaev S.R. (2010) A method and server for predicting damaging missense mutations. Nat. Methods, 7, 248–249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50. Tavtigian S.V., Deffenbaugh A.M., Yin L., Judkins T., Scholl T., Samollow P.B., de Silva D., Zharkikh A., Thomas A. (2005) Comprehensive statistical study of 452 BRCA1 missense substitutions with classification of eight recurrent substitutions as neutral. J. Med. Genet., 43, 295–305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51. Choi Y., Sims G.E., Murphy S., Miller J.R., Chan A.P. (2012) Predicting the functional effect of amino acid substitutions and indels. PLoS One, 7, e46688.. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52. Kumar P., Henikoff S., Ng P.C. (2009) Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm. Nat. Protoc., 4, 1073–1081. [DOI] [PubMed] [Google Scholar]
  • 53. Frederic M.Y., Lalande M., Boileau C., Hamroun D., Claustres M., Beroud C., Collod-Beroud G. (2009) UMD-predictor, a new prediction tool for nucleotide substitution pathogenicity – application to four genes: fBN1, FBN2, TGFBR1, and TGFBR2. Hum. Mutat., 30, 952–959. [DOI] [PubMed] [Google Scholar]
  • 54. Schwarz J.M., Cooper D.N., Schuelke M., Seelow D. (2014) MutationTaster2: mutation prediction for the deep-sequencing age. Nat. Methods, 11, 361–362. [DOI] [PubMed] [Google Scholar]
  • 55. Desmet F.O., Hamroun D., Lalande M., Collod-Beroud G., Claustres M., Beroud C. (2009) Human splicing finder: an online bioinformatics tool to predict splicing signals. Nucleic Acids Res., 37, e67.. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 56. Reese M.G., Eeckman F.H., Kulp D., Haussler D. (1997) Improved splice site detection in Genie. J. Comput. Biol., 4, 311–323. [DOI] [PubMed] [Google Scholar]
  • 57. Hebsgaard S.M., Korning P.G., Tolstrup N., Engelbrecht J., Rouze P., Brunak S. (1996) Splice site prediction in Arabidopsis thaliana pre-mRNA by combining local and global sequence information. Nucleic Acids Res., 24, 3439–3452. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 58. Zhang Y. (2008) I-TASSER server for protein 3D structure prediction. BMC Bioinformatics, 9, 40.. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 59. Kelley L.A., Mezulis S., Yates C.M., Wass M.N., Sternberg M.J. (2015) The Phyre2 web portal for protein modeling, prediction and analysis. Nat. Protoc., 10, 845–858. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 60. Li J., Deng X., Eickholt J., Cheng J. (2013) Designing and benchmarking the MULTICOM protein structure prediction system. BMC Struct. Biol., 13, 2.. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 61. Kallberg M., Wang H., Wang S., Peng J., Wang Z., Lu H., Xu J. (2012) Template-based protein structure modeling using the RaptorX web server. Nat. Protoc., 7, 1511–1522. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 62. Sali A., Blundell T.L. (1993) Comparative protein modelling by satisfaction of spatial restraints. J. Mol. Biol., 234, 779–815. [DOI] [PubMed] [Google Scholar]
  • 63. Benkert P., Kunzli M., Schwede T. (2009) QMEAN server for protein model quality estimation. Nucleic Acids Res., 37, W510–W514. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 64. Rodrigues J.P., Levitt M., Chopra G. (2012) KoBaMIN: a knowledge-based minimization web server for protein structure refinement. Nucleic Acids Res., 40, W323–W328. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 65. Zhang P., Lee H., Brunzelle J.S., Couture J.F. (2012) The plasticity of WDR5 peptide-binding cleft enables the binding of the SET1 family of histone methyltransferases. Nucleic Acids Res., 40, 4237–4246. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 66. Lauck F., Smith C.A., Friedland G.F., Humphris E.L., Kortemme T. (2010) RosettaBackrub—a web server for flexible backbone protein structure modeling and design. Nucleic Acids Res., 38, W569–W575. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 67. Guerois R., Nielsen J.E., Serrano L. (2002) Predicting changes in the stability of proteins and protein complexes: a study of more than 1000 mutations. J. Mol. Biol., 320, 369–387. [DOI] [PubMed] [Google Scholar]
  • 68. Rostkowski M., Olsson M.H., Sondergaard C.R., Jensen J.H. (2011) Graphical analysis of pH-dependent properties of proteins predicted using PROPKA. BMC Struct. Biol., 11, 6.. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 69. Baker N.A., Sept D., Joseph S., Holst M.J., McCammon J.A. (2001) Electrostatics of nanosystems: application to microtubules and the ribosome. Proc. Natl. Acad. Sci. U.S.A., 98, 10037–10041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 70. Pettersen E.F., Goddard T.D., Huang C.C., Couch G.S., Greenblatt D.M., Meng E.C., Ferrin T.E. (2004) UCSF Chimera–a visualization system for exploratory research and analysis. J. Comput. Chem., 25, 1605–1612. [DOI] [PubMed] [Google Scholar]
  • 71. Reymond A., Meroni G., Fantozzi A., Merla G., Cairo S., Luzi L., Riganelli D., Zanaria E., Messali S., Cainarca S.. et al. (2001) The tripartite motif family identifies cell compartments. EMBO J., 20, 2140–2151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 72. Thomas M., Lu J.J., Ge Q., Zhang C., Chen J., Klibanov A.M. (2005) Full deacylation of polyethylenimine dramatically boosts its gene delivery efficiency and specificity to mouse lung. Proc. Natl. Acad. Sci. U.S.A., 102, 5679–5684. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 73. Pasqualucci L., Trifonov V., Fabbri G., Ma J., Rossi D., Chiarenza A., Wells V.A., Grunn A., Messina M., Elliot O.. et al. (2011) Analysis of the coding genome of diffuse large B-cell lymphoma. Nat. Genet., 43, 830–837. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 74. Dentici M.L., Di Pede A., Lepri F.R., Gnazzo M., Lombardi M.H., Auriti C., Petrocchi S., Pisaneschi E., Bellacchio E., Capolino R.. et al. (2015) Kabuki syndrome: clinical and molecular diagnosis in the first year of life. Arch. Dis. Child, 100, 158–164. [DOI] [PubMed] [Google Scholar]
  • 75. Hannibal M.C., Buckingham K.J., Ng S.B., Ming J.E., Beck A.E., McMillin M.J., Gildersleeve H.I., Bigham A.W., Tabor H.K., Mefford H.C.. et al. (2011) Spectrum of MLL2 (ALR) mutations in 110 cases of Kabuki syndrome. Am. J. Med. Genet. A, 155A, 1511–1516. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 76. Banka S., Veeramachaneni R., Reardon W., Howard E., Bunstone S., Ragge N., Parker M.J., Crow Y.J., Kerr B., Kingston H.. et al. (2012) How genetically heterogeneous is Kabuki syndrome? MLL2 testing in 116 patients, review and analyses of mutation and phenotypic spectrum. Eur. J. Hum. Genet., 20, 381–388. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 77. Paulussen A.D., Stegmann A.P., Blok M.J., Tserpelis D., Posma-Velter C., Detisch Y., Smeets E.E., Wagemans A., Schrander J.J., van den Boogaard M.J.. et al. (2011) MLL2 mutation spectrum in 45 patients with Kabuki syndrome. Hum Mutat., 32, E2018–E2025. [DOI] [PubMed] [Google Scholar]
  • 78. Lin J.L., Lee W.I., Huang J.L., Chen P.K., Chan K.C., Lo L.J., You Y.J., Shih Y.F., Tseng T.Y., Wu M.C. (2015) Immunologic assessment and KMT2D mutation detection in Kabuki syndrome. Clin. Genet., 88, 255–260. [DOI] [PubMed] [Google Scholar]
  • 79. Van Laarhoven P.M., Neitzel L.R., Quintana A.M., Geiger E.A., Zackai E.H., Clouthier D.E., Artinger K.B., Ming J.E., Shaikh T.H. (2015) Kabuki syndrome genes KMT2D and KDM6A: functional analyses demonstrate critical roles in craniofacial, heart and brain development. Hum. Mol. Genet., 24, 4443–4453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 80. Cheon C.K., Sohn Y.B., Ko J.M., Lee Y.J., Song J.S., Moon J.W., Yang B.K., Ha I.S., Bae E.J., Jin H.S.. et al. (2014) Identification of KMT2D and KDM6A mutations by exome sequencing in Korean patients with Kabuki syndrome. J. Hum. Genet., 59, 321–325. [DOI] [PubMed] [Google Scholar]
  • 81. Morgan A.T., Mei C., Da Costa A., Fifer J., Lederer D., Benoit V., McMillin M.J., Buckingham K.J., Bamshad M.J., Pope K.. et al. (2015) Speech and language in a genotyped cohort of individuals with Kabuki syndrome. Am. J. Med. Genet. A, 167, 1483–1492. [DOI] [PubMed] [Google Scholar]
  • 82. Kokitsu-Nakata N.M., Petrin A.L., Heard J.P., Vendramini-Pittoli S., Henkle L.E., dos Santos D.V., Murray J.C., Richieri-Costa A. (2012) Analysis of MLL2 gene in the first Brazilian family with Kabuki syndrome. Am. J. Med. Genet. A, 158A, 2003–2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 83. Liu S., Hong X., Shen C., Shi Q., Wang J., Xiong F., Qiu Z. (2015) Kabuki syndrome: a Chinese case series and systematic review of the spectrum of mutations. BMC Med. Genet., 16, 26.. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 84. Banka S., Lederer D., Benoit V., Jenkins E., Howard E., Bunstone S., Kerr B., McKee S., Chris Lloyd I., Shears D.. et al. (2015) Novel KDM6A (UTX) mutations and a clinical and molecular review of the X-linked Kabuki syndrome (KS2). Clin. Genet, 87, 252–258. [DOI] [PubMed] [Google Scholar]
  • 85. Lederer D., Shears D., Benoit V., Verellen-Dumoulin C., Maystadt I. (2014) A three generation X-linked family with Kabuki syndrome phenotype and a frameshift mutation in KDM6A. Am. J. Med. Genet. A, 164, 1289–1292. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Data
Click here for additional data file. (8.9MB, zip)

Articles from Human Molecular Genetics are provided here courtesy of Oxford University Press

RESOURCES