A Chinese girl like atypical Rubinstein-Taybi syndrome caused by a novel heterozygous mutation of the EP300 gene

DOI: https://doi.org/10.21203/rs.3.rs-1815122/v1

Abstract

Background

Rubinstein–Taybi syndrome (RSTS) is an extremely rare autosomal dominant inheritable disorder caused by CREBBP and EP300 mutations, while atypical RSTS harbouring variant from the same genes but not obvious resembling RSTS. There are only a few cases of Menke-Hennekam syndrome (MKHK) with variant of exon 30 or 31 of CREBBP or EP300 gene have been reported that not resembling RSTS recent years. Atypical RSTS cannot be accurately classified as MKHK, nor is it easy to identify the obvious classic characteristics of RSTS. The clinical manifestations and genetic variation of atypical RSTS are not fully understood.

Methods

We recruited a Chinese core family with a girl had recurrent respiratory tract infection and developmental delay, the patient was investigated using whole exome sequencing. The detected variation of NGS was verified by PCR-based Sanger sequencing, and the carrying status of candidate variant of her normal parents were also tested. The pathogenity of candidate mutation was predicted by software Mutation Taster, population frequency and sequence conservation of the variation were accounted for, too. Finally, it was evaluated according to standard and guidelines recommended by the American College of Medical Genetics and Genomics. We have made comprehensive analysis of the case through sequencing data and clinical examination information.

Results

The patient with language and motor mild development retardation, she has slight abnormal facial features, mild hirsutism and post-axial hexadactylia of left foot. Her cisterna magna is enlarged to connect with the fourth ventricle, and the ventricular system is enlarged. She has a malacia beside the posterior horn of the left lateral ventricle. The patient has primary low immunoglobulin G and A, but her level of immunoglobulin M content in blood is normal. The patient harbors a novel heterozygous frameshift variant of c.2499dupG in exon 14 of EP300 gene, that it is proved de novo origin. The mutation is judged to be a pathogenic mutation, and it has high-grade pathogenic evidence.

Conclusion

The clinical and genetic evaluation of this case corroborates that clinical features with c.2499dupG in exon 14 of EP300 are less marked than in RSTS2 patient although it is difficult to establish an accurate genotype-phenotype correlation. Our additional case also helps to deepen the clinical and genetic spectrum in this disorder. Our study provides a novel mutation outside exon 30 or 31 of EP300 and enriches the phenotypes related with the gene. We have contributed new variation and disease information for guardians and doctors to broaden the knowledge about EP300-RSTS genotype and phenotype, this may contribute to ameliorate the health management of patients and improve the genetic counseling to the families.

Background

Rubinstein–Taybi syndrome (RSTS; OMIM #180849, #613684) is a well-known disorder characterized by a distinctive face, broad thumbs, broad halluces and big toes, short stature, and variable degree of intellectual disability [1, 2]. It is a rare autosomal dominant genetic pluri-malformative syndrome, which well-defined facial features include: down slanting palpebral fissures, convex nasal bridge, columella below alae nasi, and a characteristic grimacing smile [3]. The other aspects of manifestation include growth retardation, microcephaly and behavioural problems [3]. Broad and angulated thumbs and halluces are usually considered as hallmarks for clinical diagnosis[4]. RSTS1 (OMIM #180849) is caused by variants in CREBBP that coding CREB-binding protein acted as transcriptional co-activators, and RSTS2 (OMIM #613684) is caused by variants in its paralog EP300 that coding E1A associated protein p300. CREBBP and EP300 mutations have been identified in majority (50 ~ 60%) and minority (3 ~ 5%) of RSTS affected individuals, respectively [46], most, if not all, of them occur de novo.

Recently Menke-Hennekam syndrome (MKHK, OMIM #618332, #618333) is put forward as an entity caused by the same gene CREBBP and EP300 mutations but not resembling Rubinstein–Taybi syndrome [7, 8]. The manifestations of MKHK include variable impairment of intellectual development and specific facial appearance, but patients with MKHK do not resemble the striking phenotype of RSTS. The other symptoms of MKHK are also consist of feeding difficulties, autistic behavior, recurrent upper airway infections, hearing impairment, and short stature, microcephaly are also frequently seen. Current evidence suggests that MKHK2 (OMIM #618333) is caused by heterozygous mutations in exon 30 or 31 of the EP300 gene, which symptom is milder than MKHK1 (OMIM #618332) caused by heterozygous mutations in exon 30 or 31 of the CREBBP gene [9, 10]. Here we report one further child with an exon 14 frameshift variant, although outside exon 30 or 31 of EP300, without the typical RSTS features.

Methods

Subject recruitment

A Chinese family from central China was recruited for this study. The patient was a girl at 4 years of age, she was hospitalized for recurrent respiratory infections and pneumonia in the past half one year. The sufferer's parents were 33 and 33 years old and in good health. The patient was examined and treated in the Pediatrics Department. The specific clinical manifestation of the patient was recorded. Samples were obtained with written informed consent. The patient sought medical and genetic consultations in the hospital from September 2020 to February 2021, and 4 mL peripheral blood was from individuals in her core family. The clinical data for the patient was collected at the Genetic Counseling Clinic.

In this research, we chose a family-based strategy to determine the exact inheritance pattern and candidate disease causing gene mutation. Using such a family-based strategy, we can also determine whether phenotype and genotype co-segregate in the family, which helps to estimate the pathogenicity of candidate mutation and explore the relationship between phenotype and genotype. The proband was screened by NGS, then, the parents of the proband were tested by Sanger sequencing to detect and verify the carrying status of candidate mutation screened through NGS.

Whole exome sequencing and Sanger sequencing

Genomic DNA was extracted from EDTA-treated blood samples using a Blood DNA Midi Kit D3494 (Omega Bio-tek, USA) with nucleic acid automatic extraction equipment (Eppendorf epMotion 5075m, Germany). NextEra flex for enrichment (Illumina Inc., USA) was used to capture the whole exome and NovaSeq 6000 sequencer (Illumina Inc., USA) was used for high-throughput sequencing. The average sequencing depth of the targeted region was more than 100×, and the coverage was 99%. Version GRCh37 is the human reference genome used for short-read mapping (https://www.gencodegenes.org/human/release_37lift37.html). The transcript reference sequence number was obtained from the Ensembl database (http://asia.ensembl.org)[11]. GoldeneyeTM20A kit (Bejing peoplespot Ltd, China) was used for paternity test of the patient and her parents. PCR-based Sanger sequencing was used to validate disease-causing mutation of the proband based on NGS and to cheek carrying status of the mutation in the proband’s parents. The primers used for PCR were designed by GeneTool software. A capillary electrophoresis apparatus (ABI 3130xl, USA) and dGTP BigDye® Terminator sequencing kit (ABI, USA) were used for Sanger sequencing.

Pathogenicity analysis

The frequency of the detected mutation in the population was retrieved from Genome Aggregation Database (gnomAD, http://gnomad-old.broadinstitute.org/) because of its wide large-scale sequencing data. We referred to the frequency of mutation site in all populations and in the East Asian population. Candidate sites in HGMD (The Human Gene Mutation Database at the Institute of Medical Genetics in Cardiff, http://www.hgmd.cf.ac.uk/ac/index.php), professional version, were also searched in to determine whether pathogenicity has been reported in the literature. The effect of variant was predicted using in silico prediction program MutationTaster[12]. The pathogenicity of candidate mutation was graded and judged according to the 2015 edition of the ACMG standard and guidelines[13].

Results

Phenotypes and clinical manifestation

The patient is the first child of nonconsanguineous Chinese parents born at 41 weeks gestation, following an unremarkable pregnancy with a birth weight of 3.1 kg. At birth, her left foot was noted to be hexadactylia. She was low neonatal response. Her parents and family feel that she has a peculiar appearance, neither like father nor mother. There are no concerns about her hearing and vision, but eye-tracking and listen -tracking was poor during infancy. From 3 months to 8 months she underwent rehabilitation training for half for year for movement development retardation. She rolled over at 7 months, sat unsupported at 9 months, crawled at 12 months and walked independently at 16 months of age. At 2 years of age she received rehabilitation training for language delay. She is mild growth retardation. She had no self-injurious behavior. She often pushes other children, but is sociable and interactive. At present, she is 101 cm tall and weighs 15.5 kg, which are at low level among the normal range of girls aged 4 years. She can ride a tricycle independently and freely (Short Video S1). She also builds blocks and draws with no problem (Short Video S2 and S3). Her language comprehension is somewhat poor. She can now express long sentences or recite simple enlightening poems of the Tang dynasty such as singing goose, spring dawn and so on. But when she said long sentences, she sometimes would miss one word. She is now in kindergarten middle shift.

Patient’s facial and physical features are shown in Fig. 1 and supplementary material (Figure S1a to S1g). She at 4 years of age, has slightly arched eyebrows and synophridia, long eyelashes, a square tip to his nose, normal columella, prominent two front teeth, normal tooth number. And she is absence of microcephaly, beaked nose and characteristic grimacing smile like RSTS. The fine hairs on the front of the ear and on the cheek form a hair whorl. The child has hirsute back and opisthenar. She has no broad or angulated thumbs, nor broad distal phalanges of the fingers, as seen in patients with RTS. Her brain plain scan of MRI shows as follows. Punctate long T1 and long T2 signal shadow was seen beside the posterior horn of left lateral ventricle; FLAIR showed high signal outside and low signal inside; DWI showed low signal with high b value, and the lesion had no space occupying effect. It indicated that softening lesion of the posterior horn of left lateral ventricle. The enlarged cisterna magna was connected with the fourth ventricle, the ventricular system was enlarged, and there was no widening and deepening phenomenon in the cerebral sulcus, cisterna and cerebral fissure. The morphology, structure and signal intensity of cerebellum and brainstem were normal. The midline structure did not deviate.

The patient has been hospitalized for 5 times due to recurrent respiratory infections with or without pneumonia in the last half year. The patient was found to have primary low immunity, transient EB virus infection and splenomegaly. Her examination results of content of immune globulins in peripheral blood or urine are presented in Table 1. It indicates that she with primary low immunoglobulin G/A. She has normal level of immunoglobulin M.

Table 1

Content of immune globulins in peripheral blood or urine of the Patient. IG, immune globulin; RRB, reference range in blood; RRU, reference range in urine; * denotes after intravenous immunoglobulin G therapy. T1, 2020.08.28; T2, 2020.10.24; T3, 2020.11.22; T4, 2020.12.10; T5, 2020.12.27; T6, 2021.01.25; T7, 2021.02.28.

IG Type

T1

T3

T4

T5*

T6*

T7*

RRB

T2

RRU

IgG

4.77↓

3.71↓

3.62↓

21.68

8.22

7.42

5.4–13.4 mg/L

4.67↓

5.9–14.3 mg/L

IgA

0.14↓

0.12↓

0.09↓

0.31

0.15↓

0.10↓

0.3–1.88 g/L

0.17↓

0.38–2.22 mg/L

IgM

1.03

1.39

1.37

1.42

1.54

1.43

0.43–1.93 g/L

1.42

0.45–2.08 mg/L

Genotype of EP300 gene mutation

Candidate disease causing mutation detected in the patient through whole exome sequencing, and it was verified by Sanger sequencing. The parents were also tested by Sanger sequencing to detect the carrying status of candidate mutation screened through NGS (Fig. 2). Patient harbors a heterozygous mutation of c.2499dupG in exon 14 of EP300 gene, which encodes the adenovirus E1A-associated cellular p300 transcriptional co-activator protein. NM_001429 was chosen as transcript reference sequence of EP300 gene during NGS data analysis. The genetic relationship between parents and daughter was confirmed by paternity test. Subsequent parental PCR based Sanger sequencing tests displayed that none of the parents carried the EP300 c.2499dupG mutation, and it proved de novo origin of the variant.

EP300 c.2499dupG is a frameshift mutation from exon 14, which is presumed to cause amino acid sequence changes of p.Pro834Alafs*4. It results in substitution of amino acid 834th and termination of translation after four codons shift, which is sited in a region, not domain, of the protein with still uncharacterized function (Fig. 3). The c.2499dupG p.(Pro834Alafs*4) variant in our patient is a novel variant that not been related to disease recorded in HGMD database. The variant is not found in all populations and in the East Asian population in gnomAD database, so minor allele frequency of the rare mutation is unknown (extremely low). The function prediction tool Mutation Taster indicates that the variant is disease causing and amino acid sequence in this location is highly conserved among species. According to ACMG guidelines, the novel frameshift mutation of EP300 c.2499dupG p.(Pro834Alafs*4) should be considered pathogenic, and its evidence grade is high (PVS1 + PS2 + PM2).

Discussion

CREBBP and EP300 mutations are well-known causes of RSTS, but recently publications reported none of the patients with missense variant in exon 30/31 of CREBBP and EP300 had typical characteristics of RSTS [7, 8, 10]. CREBBP is ubiquitously expressed and involved in the transcriptional co-activation of many different transcription factors, which is first isolated as a nuclear protein that binds to cAMP-response element binding protein (CREB) [14]. EP300 encodes the adenovirus E1A-associated cellular p300 transcriptional co-activator protein, which functions as histone acetyltransferase that regulates transcription via chromatin remodeling and is important in the processes of cell proliferation and differentiation [15]. EP300 mediates cAMP-gene regulation by binding specifically to phosphorylated CREB protein. Besides intrinsic histone or non-histone acetyltransferase activity, the protein of CREBBP acts as a scaffold to stabilize additional protein interactions with the transcription complex [15]. The proteins of EP300 and CREBBP share regions of very high sequence similarity in bromodomain, cysteine-histidine-rich regions, and histone acetyltransferase domain. They are known to play critical roles in embryonic development, growth control, and homeostasis by coupling chromatin remodeling to transcription factor recognition.

There are 25 patients reported in the medical literature without the typical RSTS features and with exon 30/31 missense or in-frame deletion CREBBP variants so far [7, 8, 10]. The main features of MKHK1 caused by CREBBP mutations include developmental delay, distinctive facial features, autistic behavior, feeding difficulties, recurrent upper airway infections, and brain abnormalities revealed by MRI. An additional Chinese case with one CREBBP in-frame deletion variant in the beginning of exon 30 has atypical RSTS phenotypes[16]. Now it seems that she is consistent with the diagnosis of MKHK1, although the mutation in the HAT domain where no pathogenic variants have been previously reported to be responsible for MKHK. The 2-year-7-month-old Chinese girl presented facial dysmorphism of MKHK included telecanthus, a depressed nasal ridge, short nose, anteverted nares, short columella, and long philtrum. Her other symptoms were mild cognitive impairments, developmental delay, short stature, recurrent upper airway infections, and mild thinning of corpus callosum revealed by MRI.

Some scholars call the atypical RSTS phenotypes with EP300 variant Menke–Hennekam syndrome 2 [7]. The main typical characteristics of previous patient E1 and E2 regarded as MKHK2 include variable impairment of intellectual development and facial dysmorphisms, and feeding difficulties, autistic behavior, recurrent infections are also frequently seen. Combined with the analysis of clinical manifestations of previous MKHK1 cases caused by CREBBP exon 30/31 mutations, Menke-Hennekam syndrome is described as a congenital disorder characterized by variable impairment of intellectual development and facial dysmorphisms that do not resemble the striking phenotype of RSTS [7, 8], and MKHK2 is milder than MKHK1. Patient E1 with EP300 c.5471A > C p.(Gln1824Pro) had recurrent otitis, airway infections and urinary tract infections owing to low immunoglobulins, and was diagnosed with autism spectrum disorder. She necessitated a percutaneous gastrostomy (PEG) when as a neonate. Patient E2 with EP300 c.5492_5494del p.(Arg1831del) continued to have recurrent otitis, and autism spectrum disorder was also diagnosed (Fig. 3). Her language and motor delayed. Brain MRI was not performed in these patients.

The number of MKHK2 cases caused by EP300 gene mutation is still small. The previously reported patients [17] with an EP300 variant in exon 31 could reconsider the name of the disease according to the new concept. Individual 8 with EP300 c.6915_6918del p.(Asn2305Lysfs*47) was male, his striking phenotypes included mild specific facial appearance, varying degrees of speech and motor retardation, reactive airway disease, undescended testicle and severely restricted growth, without broad thumb and radial deviation of the hand. Individual 8 was mild RSTS2, but cannot be attributed to MKHK2. His demise caused by severe cardiac involvement, which may be a few and isolated phenotype. While Individual 12 with EP300 frameshift variant of c.5720delC p.(Pro1907Leufs*53) had classical characteristics of RSTS. These two patients also did not do brain MRI. There were five persons with EP300 exon 30/31 variants previously reported as RSTS2 patients (Fig. 3) [18]. The Patient #42 was a 9 years old boy with an EP300 in-frame deletion variant of c.6627_6638del p.(Asn2209_Gln2213delinsLys) in exon 31, but he had the typical manifestations of RSTS. The Patient #11 was a 18 years old male youth with an EP300 frameshift variant of c.4954_4957dup p.(Cys1653Tyrfs*21) in exon 30, and both of the Patients #45 of mother and daughter had EP300 frameshift variant of c.7222_7223del p.(Gln2408Glufs*39) in exon 31. They were all RSTS2 patients with variable disease symptoms, respectively. Patients #45 have milder clinical manifestation than Patient #11, and even Patients #45 with the same mutation in the same family also realized heterogeneity of clinical features of the disease.

These above investigations have expanded our knowledge about the spectrum of diverse phenotypes driven by dissimilar molecular and cellular consequences resulting from different class of variants in the same gene [10]. However, by comparison, we can see that it is not exactly the same as Menke and Hennekam proposed that ‘Menke-Hennekam syndrome is caused by missense mutations in the last part of exon 30 and beginning of 31 of the CREBBP/EP300 gene resulted in a gain of function. Mutations elsewhere in the gene causing Rubinstein-Taybi syndrome, result in haplo-insufficiency or perturb the function of a domain, specifically the HAT’. The previous Chinese case with, paralog of EP300, one CREBBP in-frame deletion variant in the 5’ end of HAT domain of beginning exon 30 has phenotypes more similar to typical MKHK rather than RSTS, and while EP300 in-frame deletion variant in exon 31 not always cause MKHK2 (previous Patient #42). Disease caused by mutations outside the exon 30/31 of EP300 sometimes is not always typical Rubinstein-Taybi syndrome [3, 17, 18].

Our patient has slightly arched eyebrows and synophridia, mild hirsutism, post-axial hexadactylia of left foot, and with no beaked nose or other striking features of RSTS. She has mild development retardation of language and motor, abnormality of brain structure was seen by MRI. The patient had a history of multiple respiratory tract infections and primary low immunity. All these clinical manifestations suggest that our patient is consistent with the phenotype of atypical RSTS, and she harbours a de novo frameshift EP300 variant of c.2499dupG in exon 14 that not involved HAT domain. EP300 related RSTS phenotype is often less classical than that related to pathogenic CREBBP variants, so it would be more difficult to differentiate between RSTS and MKHK. On the other hand, taking into account that only two patients with EP300 variants and MKHK have been officially described, it is appropriate for this Chinese girl to be diagnosed as atypical RSTS. Looking back at these patients with EP300 mutations, we found that a history of recurrent infection or even primary low immunity is a notable feature of the disorder. Some scholars believe that variants causing RSTS in other parts of CREBBP/EP300 would result in haplo-insufficiency of functions or perturb the function of the HAT domain, typically [7]. But not all functions of CREBBP/EP300 are dose-dependent. The disease caused by EP300 mutation shows phenotype heterogeneity and likely constitutes at least two different entities, such as RSTS and MKHK. Due to the relatively small number of patients with EP300 mutations reported, there are still a lot of details about these two entities that need to be constructed. This case contributes one novel de novo EP300 variant and novel phenotypes to the atypical RSTS. It furtherly extends the borders of the EP300 variant resulting in RSTS-like disease and expands its clinical spectrum.

Conclusion

Our clinical and genetic study about this case demonstrates that clinical features with c.2499dupG in exon 14 of EP300 are less marked than in RSTS2 patient, although previous studies have suggested that mutations beyond the exons 30 and 31 of EP300 gene can lead to RSTS. Our additional case could help to deepen the clinical and genetic spectrum in this disorder, so as to establish the correlationship between phenotypes and genotypes as much as possible. Our study provides a novel EP300 mutation for atypical RSTS and enriches the phenotypes. We have contributed new variation and disease information from this Chinese case to broaden the range of understanding about EP300-RSTS genotype and phenotype for sufferers and professionals. This could contribute to improve the health management and genetic counselling for patients, and facilitate clinical research in a certain extent.

Abbreviations

NGS

Next-generation sequencing

gnomAD

Genome Aggregation Database

HGMD

The Human Gene Mutation Database at the Institute of Medical Genetics in Cardiff

ACMG

The American College of Medical Genetics and Genomics

MRI

Magnetic Resonance Imaging

FLAIR

fluid attenuated inversion recovery

DWI

diffusion weighted imaging

Declarations

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