Deciphering rare imprinting disorders within the Beckwith-Wiedemann spectrum

Beckwith-Wiedemann syndrom (BWS) er den hyppigste og mest komplekse tilstanden blant imprintingssykdommene. Avhandlingen presenterer tre ulike BWS spektrum familier, hvor vi utvider forståelsen av arvemønsteret ved BWS, og viser hvor viktig en presis diagnose er med tanke på individuell oppfølging av f.eks kreftrisiko og svangerskapskomplikasjoner, men også gjentakelsesrisiko i familien. Vi presenterer også en kompleks genvariant i CDKN1C som vi foreslår forårsaker ikke bare en, men hele tre ulike tilstander samtidig, bl.a grunnet en ikke tidligere beskrevet isoform av proteinet som kan tenkes å være viktig for hjernens utvikling. I tillegg beskriver vi for første gang en person med et dobbelt kromosomsett (UPD) fra far for to kromosomer (kromosom 7 og 15) som har gitt innsikt i nettverket av imprintede gener. BWS karakteriseres av bl.a svangerskapskomplikasjoner, lavt blodsukker ved fødsel, høy fødselsvekt og stor morkake, stor tunge og store indre organer, midtlinjedefekter i buk og eventuelt tarmbrokk, økt risiko for barnekreft som Wilms tumour, og hos enkelte spesielle ansiktstrekk og mindre utviklingsavvik. Som hovedregel er den psykomotoriske utviklingen normal. Imprinting er en viktig genreguleringsmekanisme som ikke innebærer endringer i selve DNA-koden, men derimot en reversibel epigenetisk endring av DNA vha cytosin-metylering av hovedsaklig CpG posisjoner. For enkelte gener er ikke bare regulering av gendosen men også genkilden viktig, dvs om genet uttrykket på det kromosomet som er arvet fra mor eller fra far. Dette kalles kjønnsavhengig imprinting. Ved feil i dette kjønnsavgengige imprintingsystemet oppstår imprintingsykdommene. Ved BWS styres imprintingen av to imprintingssentre, IC1 og IC2, på den aktuelle kromosomregion på enden av kromosom 11 (11p15.5). I en normal situasjon er vektsfaktoren IGF2 i IC1 kun aktivt på genkopien nedarvet fra far, mens veksthemmeren CDKN1C i IC2 kun uttrykkes fra genkopien nedarvet fra mor. 7 Metyleringsforstyrrelser i et (eller begge) av de to imprintingssentrene medfører BWS. Den hyppigste årsaken er sporadisk tap av metylering i IC2 på 11p15.5, som bl.a medfører at veksthemmeren CDKN1C slås helt eller delvis av. BWS kan også skyldes økt metylering av det maternelle IC1 som fører til at vekstfaktoren IGF2 også uttrykkes fra mors genstreng, ikke bare fra fars. En slik økt metylering av IC1 kan være sporadisk, men kan også skyldes underliggende avvik i IC1 sin DNA sekvens, som kan nedarves og gi BWS dersom nedarving via morsledd. Artikkel I beskriver en en slik familie, hvor familiemedlemmer i tre generasjoner har en sjelden IC1-genfeil som gir opphav til dominant arvelig BWS dersom maternell nedarving. Denne formen for BWS er forbundet med risiko for nyrekreft hos barn. Vi viser også at BWS symptomene øker fra andre til tredje generasjon (antesipasjon) og samtidig korrelerer med metyleringsgraden i IC1. Dette er ikke tidligere beskrevet ved imprintingssykdommer. Vi presenterer en hypotese for å forklare denne antesipasjonen. Som forventet medfører genfeilen forstyrrelse av skifte av metyleringsmønster på fars kromosom 11 i hans datters eggstokker, dvs at demetyleringen er inkomplett her. Det nye her er at denne metyleringsfeilen forsterkes til neste generasjon, dvs hos datterens barn, selv om genfeilen nå sitter på et maternelt nedarvet kromosom. Dette antyder at metyleringen på det maternelle kromosom 11 passivt nedarves til neste generasjon gjennom kvinneledd, mens kun det paternelle kromosom 11 aktivt demetyleres, altså at mors og fars BWS-regioner på kromosom 11 behandles ulikt i eggstokkene. Artikkel II presenterer en familie med en kompleks variant i CDKN1C, som medfører både tap og økning av genfunksjon, slik at gutten har en unik kombinasjon av både BWS-lignende overvekst og en veksthemming. Sistnevnte minner om speilbildetilstanden til BWS som kalles IMAGe. Ved å benytte RNA dypsekvensering viser vi at varianten i tillegg rammer en hittil ikke beskrevet transkriptutgave (isoform) av genet. Vi spekulerer i at genfeil i denne ubeskrevne isoformen kan forklare forsinket utvikling og liten hodeomkrets. Artikkel III presenterer en gutt med en BWS-lignende trekk i tillegg til psykomotorisk utviklingsavvik. Han har normalt kromosomantall, men begge utgaver av både kromosom nr 7 og nr 15 er kun nedarvet fra far (paternelt), dvs at det 8 maternelle kromosom 7 og 15 ikke påvises. Dette kalles paternell uniparental disomi (UPD). Når UPD rammer kromosom som inneholder imprintede gener, kan imprintingsykdom oppstå. Begge disse kromosomene inneholder en rekke imprintede gener. Paternell UPD(15) er en av årsakene til Angelman syndrom, en velkjent imprintingssykdom som medfører psykomotorisk utviklingshemming, mens paternell UPD(7) ikke er assosiert med noen kjent imprintingssykdom. Vi finner imidlertid ingen annen forklaring enn paternell UPD(7) på hans BWS-lignende symptomer, som for øvrig overlapper med det man ser hos pasienter med generell paternell UPD mosaisisme. Ved RNA dypsekvensering finner vi oppregulering av flere vekstrelaterte gener. Mest interessant er en betydelig oppregulering av et imprintet gen på kromosom 7 kalt PEG10, som også kan tenkes å ha betydning for vekst. En slik dobbel UPD er ikke rapportert tidligere, og vi presenterer også en hypotese på hvordan dette har oppstått.Background: Beckwith-Wiedemann syndrome spectrum (BWSp) is the most common and probably the most complex imprinting disorder in humans. BWSp is caused by different molecular and epigenetic mechanisms involving the chromosome 11p15.5 subdomain and is associated with overgrowth, endocrine disturbances, congenital malformation, and an increased childhood cancer risk. We see many BWSp families in our clinical practice, but the three families described in this dissertation have been selected because of their unique features, atypical inheritance patterns, or novel genetic causes. Objective: We aimed to explore each family's clinical characteristics and the underlying molecular defects and further explain the genotype-phenotype correlation to improve clinical diagnostics and follow-up of these patients. Materials and Methods: Three families were ascertained in our outpatient clinic, and we performed clinical and molecular investigations and gave genetic counseling. We used Sanger sequencing to identify the causal variants in the families described in articles I and II. In article III, SNP array-based copy number analysis in the affected boy revealed double isodisomy. In the family in article I, we determined the methylation status of Imprinting Centre 1 (IC1) in the BWS locus by MS-MLPA (Methylation Specific-Multiplex Ligation-dependent Probe Amplification) and bisulphite conversion followed by both subclone Sanger sequencing and NGS (Next-Generation Sequencing) of CTCF (CCCTC binding factor) binding sites in IC1. We compared the degree of methylation between the generations and between individual CTCF binding sites. In articles II and III, we performed NGS-based RNA deep sequencing to decipher the different CDKN1C transcripts and study the expression of individual genes or gene sets. Trio-based whole-exome sequencing (WES) was performed in articles II and III to exclude other molecular causes of the phenotype. Results: In the three-generations BWS-family described in article I, we found evidence for anticipation through the generations, with increased methylation of IC1 correlating with increased severity of the developmental disturbance. This epimutation was secondary to a previously described heterozygous variant in the 10 OCT binding site NCBI36:11:g.1979595T4C in IC1. In the family presented in article II, an inherited and complex delins-variant in CDKN1C ((NM_000076.2) c.822_826delinsGAGCTG) was detected. This variant probably induced concurrent gain-of-function and loss-of-function effects, which correlated with the mirror phenotypes of both growth restriction and IMAGe features, and overgrowth and BWSp features, both present in the same individual. In addition, this variant affected a hitherto not reported CDKN1C transcript with a different C-terminal reading frame that might explain his developmental delay. In article III, we studied a baby boy with a clinical presentation of BWS, but without a molecular correlate. Here we described for the first time a double uniparental disomy (UPD) affecting two imprinted chromosomes: 7 and 15. This caused paternal isoUPD(7) and isoUPD(15), the latter explaining Angelman syndrome. We also present a hypothesis for how the double UPD arose. However, we did not identify a known molecular explanation for the boy's BSWp, a phenotype reminiscent of general paternal UPD mosaicism. We explored if the UPDs could induce a global imprinting defect, but this was not detected. However, RNA-sequencing detected upregulation beyond expectation (>2 fold) of the maternally imprinted gene PEG10 located to 7q21.3. Possibly, PEG10 overexpression could cause an overgrowth phenotype, but this remains to be proven. Conclusions: This work broadens the phenotypic and molecular spectrum of BWS and highlights the importance of a precise molecular diagnosis for clinical follow-up and recurrence risk. We show that anticipation can also occur in imprinting disorders and hypothesize that the paternal and maternal IC1 alleles are treated differently in the gonads. In addition, we present a hitherto not described CDKN1C transcript which might have importance for brain function. The study also gives insight into the so-called imprinted gene network. We investigated how this specific double paternal UPD(7) and UPD(15) could cause BWSp and ended up with PEG10 overexpression as the most likely mechanism. Intriguingly, the two small, imprinted genes that we have studied, CDKN1C and PEG10, both have multiple reading frames, which is rare in human genes.Doktorgradsavhandlin

Deep exploration of a CDKN1C mutation causing a mixture of Beckwith-Wiedemann and IMAGe syndromes revealed a novel transcript associated with developmental delay

Background: Loss-of-function mutations in CDKN1C cause overgrowth, that is, Beckwith-Wiedemann syndrome (BWS), while gain-of-function variants in the gene’s PCNA binding motif cause a growth-restricted condition called IMAGe syndrome. We report on a boy with a remarkable mixture of both syndromes, with developmental delay and microcephaly as additional features. Methods: Whole-exome DNA sequencing and ultra-deep RNA sequencing of leucocyte-derived and fibroblast-derived mRNA were performed in the family. Results: We found a maternally inherited variant in the IMAGe hotspot region: NM_000076.2(CDKN1C) c.822_826delinsGAGCTG. The asymptomatic mother had inherited this variant from her mosaic father with mild BWS features. This delins caused tissue-specific frameshifting resulting in at least three novel mRNA transcripts in the boy. First, a splice product causing CDKN1C truncation was the likely cause of BWS. Second, an alternative splice product in fibroblasts encoded IMAGe-associated amino acid substitutions. Third, we speculate that developmental delay is caused by a change in the alternative CDKN1C-201 (ENST00000380725.1) transcript, encoding a novel isoform we call D (UniProtKB: A6NK88). Isoform D is distinguished from isoforms A and B by alternative splicing within exon 1 that changes the reading frame of the last coding exon. Remarkably, this delins changed the reading frame back to the isoform A/B type, resulting in a hybrid D–A/B isoform. Conclusion: Three different cell-type-dependent RNA products can explain the co-occurrence of both BWS and IMAGe features in the boy. Possibly, brain expression of hybrid isoform D–A/B is the cause of developmental delay and microcephaly, a phenotypic feature not previously reported in CDKN1C patients.publishedVersio

Double paternal uniparental isodisomy 7 and 15 presenting with Beckwith–Wiedemann spectrum features

Here we describe for the first time double paternal uniparental isodisomy (iUPD) 7 and 15 in a baby boy with features in the Beckwith–Wiedemann syndrome spectrum (BWSp) (placentomegaly, hyperinsulinism, enlarged viscera, hemangiomas, and earlobe creases) in addition to conjugated hyperbilirubinemia. His phenotype was also reminiscent of genome-wide paternal uniparental isodisomy. We discuss the most likely origin of the UPDs: a maternal double monosomy 7 and 15 rescued by duplication of the paternal chromosomes after fertilization. So far, paternal UPD7 is not associated with an abnormal phenotype, whereas paternal UPD15 causes Angelman syndrome. Methylation analysis for other clinically relevant imprinting disorders, including BWSp, was normal. Therefore, we hypothesized that the double UPD affected other imprinted genes. To look for such effects, patient fibroblast RNA was isolated and analyzed for differential expression compared to six controls. We did not find apparent transcription differences in imprinted genes outside Chromosomes 7 and 15 in patient fibroblast. PEG10 (7q21.3) was the only paternally imprinted gene on these chromosomes up-regulated beyond double-dose expectation (sixfold). We speculate that a high PEG10 level could have a growth-promoting effect as his phenotype was not related to aberrations in BWS locus on 11p15.5 after DNA, RNA, and methylation testing. However, many genes in gene sets associated with growth were up-regulated. This case broadens the phenotypic spectrum of UPDs but does not show evidence of involvement of an imprinted gene network.publishedVersio

LRFN5 locus structure is associated with autism and influenced by the sex of the individual and locus conversions

LRFN5 is a regulator of synaptic development and the only gene in a 5.4 Mb mammalian-specific conserved topologically associating domain (TAD); the LRFN5 locus. An association between locus structural changes and developmental delay (DD) and/or autism was suggested by several cases in DECIPHER and own records. More significantly, we found that maternal inheritance of a specific LRFN5 locus haplotype segregated with an identical type of autism in distantly related males. This autism-susceptibility haplotype had a specific TAD pattern. We also found a male/female quantitative difference in the amount histone-3-lysine-9-associated chromatin around the LRFN5 gene itself (p < 0.01), possibly related to the male-restricted autism susceptibility. To better understand locus behavior, the prevalence of a 60 kb deletion polymorphism was investigated. Surprisingly, in three cohorts of individuals with DD (n = 8757), the number of deletion heterozygotes was 20%–26% lower than expected from Hardy–Weinberg equilibrium. This suggests allelic interaction, also because the conversions from heterozygosity to wild-type or deletion homozygosity were of equal magnitudes. Remarkably, in a control group of medical students (n = 1416), such conversions were three times more common (p = 0.00001), suggesting a regulatory role of this allelic interaction. Taken together, LRFN5 regulation appears unusually complex, and LRFN5 dysregulation could be an epigenetic cause of autism.publishedVersio

Further delineation of the clinical spectrum of White-Sutton syndrome: 12 new individuals and a review of the literature

White–Sutton syndrome (WHSUS) is a neurodevelopmental disorder caused by heterozygous loss-of-function variants in POGZ. Through the Deciphering Developmental Disorders study and clinical testing, we identified 12 individuals from 10 families with pathogenic or likely pathogenic variants in POGZ (eight de novo and two inherited). Most individuals had delayed development and/or intellectual disability. We analyzed the clinical findings in our series and combined it with data from 89 previously reported individuals. The results demonstrate WHSUS is associated with variable developmental delay or intellectual disability, increased risk of obesity, visual defects, craniofacial dysmorphism, sensorineural hearing loss, feeding problems, seizures, and structural brain malformations. Our series includes further individuals with rod-cone dystrophy, cleft lip and palate, congenital diaphragmatic hernia, and duplicated renal drainage system, suggesting these are rare complications of WHSUS. In addition, we describe an individual with a novel, de novo missense variant in POGZ and features of WHSUS. Our work further delineates the phenotypic spectrum of WHSUS highlighting the variable severity of this disorder and the observation of familial pathogenic POGZ variants

EPHB4 kinase-inactivating mutations cause autosomal dominant lymphatic-related hydrops fetalis.

Hydrops fetalis describes fluid accumulation in at least 2 fetal compartments, including abdominal cavities, pleura, and pericardium, or in body tissue. The majority of hydrops fetalis cases are nonimmune conditions that present with generalized edema of the fetus, and approximately 15% of these nonimmune cases result from a lymphatic abnormality. Here, we have identified an autosomal dominant, inherited form of lymphatic-related (nonimmune) hydrops fetalis (LRHF). Independent exome sequencing projects on 2 families with a history of in utero and neonatal deaths associated with nonimmune hydrops fetalis uncovered 2 heterozygous missense variants in the gene encoding Eph receptor B4 (EPHB4). Biochemical analysis determined that the mutant EPHB4 proteins are devoid of tyrosine kinase activity, indicating that loss of EPHB4 signaling contributes to LRHF pathogenesis. Further, inactivation of Ephb4 in lymphatic endothelial cells of developing mouse embryos led to defective lymphovenous valve formation and consequent subcutaneous edema. Together, these findings identify EPHB4 as a critical regulator of early lymphatic vascular development and demonstrate that mutations in the gene can cause an autosomal dominant form of LRHF that is associated with a high mortality rate

Börjeson–Forssman–Lehmann syndrome: Delineating the clinical and allelic spectrum in 14 new families

Börjeson-Forssman-Lehmann syndrome (BFLS) is an X-linked intellectual disability syndrome caused by variants in the PHF6 gene. We ascertained 19 individuals from 15 families with likely pathogenic or pathogenic PHF6 variants (11 males and 8 females). One family had previously been reported. Six variants were novel. We analysed the clinical and genetic findings in our series and compared them with reported BFLS patients. Affected males had classic features of BFLS including intellectual disability, distinctive facies, large ears, gynaecomastia, hypogonadism and truncal obesity. Carrier female relatives of affected males were unaffected or had only mild symptoms. The phenotype of affected females with de novo variants overlapped with the males but included linear skin hyperpigmentation and a higher frequency of dental, retinal and cortical brain anomalies. Complications observed in our series included keloid scarring, digital fibromas, absent vaginal orifice, neuropathy, umbilical hernias, and talipes. Our analysis highlighted sex-specific differences in PHF6 variant types and locations. Affected males often have missense variants or small in-frame deletions while affected females tend to have truncating variants or large deletions/duplications. Missense variants were found in a minority of affected females and clustered in the highly constrained PHD2 domain of PHF6. We propose recommendations for the evaluation and management of BFLS patients. These results further delineate and extend the genetic and phenotypic spectrum of BFLS

CTCF variants in 39 individuals with a variable neurodevelopmental disorder broaden the mutational and clinical spectrum

Purpose: Pathogenic variants in the chromatin organizer CTCF were previously reported in seven individuals with a neurodevelopmental disorder (NDD). Methods: Through international collaboration we collected data from 39 subjects with variants in CTCF. We performed transcriptome analysis on RNA from blood samples and utilized Drosophila melanogaster to investigate the impact of Ctcf dosage alteration on nervous system development and function. Results: The individuals in our cohort carried 2 deletions, 8 likely gene-disruptive, 2 splice-site, and 20 different missense variants, most of them de novo. Two cases were familial. The associated phenotype was of variable severity extending from mild developmental delay or normal IQ to severe intellectual disability. Feeding difficulties and behavioral abnormalities were common, and variable other findings including growth restriction and cardiac defects were observed. RNA-sequencing in five individuals identified 3828 deregulated genes enriched for known NDD genes and biological processes such as transcriptional regulation. Ctcf dosage alteration in Drosophila resulted in impaired gross neurological functioning and learning and memory deficits. Conclusion: We significantly broaden the mutational and clinical spectrum of CTCF-associated NDDs. Our data shed light onto the functional role of CTCF by identifying deregulated genes and show that Ctcf alterations result in nervous system defects in Drosophila.Peer reviewe