Five new cases of syndromic intellectual disability due to KAT6A mutations: widening the molecular and clinical spectrum

The patient is a 10-year-old boy from a non-consanguineous and healthy couple. Two younger siblings, a girl aged 5½ years and a boy aged 3, are healthy. During the pregnancy of the proband, intrauterine growth retardation was detected. He was born at 37 weeks by caesarean section with Apgar scores 9/10 at 1 and 5 min, respectively. At birth, weight was 2.04 kg (2nd percentile, − 2.04 SD), height 43 cm (<3rd percentile, − 2.91 SD), and cranial circumference 30 cm (<3rd percentile, − 3.77 SD). The infant presented dysmorphic features such a triangular facies, low-set ears, and short neck, in addition to cryptorchidism. Abdominal and cranial ultrasound studies were performed with no evidence of malformations. Serology for common neonatal infections (toxoplasma, rubella, cytomegalovirus, and herpes simplex) as well as brain computerized tomography (CT) and funduscopy were normal.

At the 5th month of life, a prominent metopic suture was observed. The helical CT confirmed craniosynostosis of the metopic suture and ossification of the anterior fontanelle. At this follow-up visit developmental delay and limb hypertonia were detected. The subject began to crawl at 4 years of age and made his first unassisted steps at age 5. At 9 years of age absence seizures were clinically suspected and he was treated with valproate, showing good response. At 11 years the treatment was discontinued as the patient did not present seizures anymore and EEG was normal. Brain MRI (magnetic resonance imaging) was always normal. He was also treated with botulinum toxin because of hypersalivation due to dysphagia. The boy was also followed-up for severe myopia and constipation. He had a non-symptomatic large atrial septal defect, ASD (ostium secundum type).

At 10 years of age the dysmorphic traits continued, suggestive of Opitz C syndrome. The patient presented bulging eyes with true proptosis, mild epicanthus, hypoplastic nose, small mouth with normal philtrum and palate, and normal auricles (see detailed description in Table 1 and Fig. 1: a, g and l). From the neurological point of view he presented with spastic tetraparesis, with ulnar deviation of the hands, wide-base gait (increased distance between the tibial malleolus when walking) not in the range of a cerebellar ataxia but denoting clumsiness and a subtle gait disturbance, and flexion of hips and knees. The patient lacks expressive language, but has improved his ability to understand and to establish communication. He does not respond to simple commands and has poor attention span. He is unable to chew and is fed mashed food. He walks with increased lift base and can take some steps alone. Usually, he needs walker support to get around and has no sphincter control. The patient is currently on physiotherapy and speech therapy, and pharmacological treatment with risperidone and methylphenidate due to behavioural disturbances and ADHD (attention deficit-hyperactivity disorder) traits, respectively.

The patient is an 11-year-old boy, the first child of healthy non-consanguineous parents. He was born at 38 + 5 weeks via caesarean section due to breech presentation after an uneventful pregnancy. At birth, weight was 2.97 kg (30th percentile, − 0.45 SD), length was 49 cm (25th percentile, − 0.28 SD), and cranial circumference was 33 cm (10th percentile, − 0.61 SD). On neonatal examination, sacral and suprasternal dimples were observed on ultrasound with normal subjacent tissues and reducible bilateral inguinal hernia.

The neonatal period was significant for poor feeding, mild hypertonia of limbs, and delayed gross motor development. He achieved head control at 3 months, sitting at 15 months, and crawling and kneeling later than 24 months; currently he walks only with a walker. His speech was also delayed as he started babbling at 22 months. His fine motor skills have improved with therapy; he is able to grab objects and play with both hands. Recurrent pneumonias were diagnosed with several hospital admissions, and dysphagia to liquids was observed, requiring thickeners. At age 2 years, an echocardiogram showed a 12-mm small ASD, ostium secundum type that was occluded with vascular plug system. Testing for inborn errors of metabolism (with plasma amino acids, acylcarnitine profile, total and free carnitines, and urine organic acids) was negative.

At age 6 years, he presented 2 episodes of loss of consciousness but electroencephalogram (EEG) did not confirm electric seizures. He was treated with valproic acid, currently on withdrawal. Brain MRI showed areas of polymicrogyria on the posterior right insula, delayed myelination, and slight descent of the cerebellar tonsils through the foramen magnum (Arnold-Chiari malformation).

On physical examination at age 11 years (Table 1 and Fig. 1: b, c and h), the patient was found to have a short stature (height 113 cm, <3rd percentile, − 4.75 SD), microcephaly (cranial circumference of 49.5 cm, <3rd percentile, − 3.74 SD), and dysmorphic facial features such as flat facies, hypertelorism, mildly blue sclera and proptosis, full lower lip and protruding tongue that led to an open mouth expression, and low-set dysplastic ears. Ocular abnormalities included endotropia, astigmatism, and myopia. Additionally, the patient had one right supernumerary nipple, a single palmar crease on the right hand, poor palmar sulcation, and mild fifth finger clinodactyly in both hands. Neurologically, he showed intellectual disability, poor eye contact, generalized hypertonia, the need for a stroller to walk, and absence of comprehensible language, but with babbling. He presents midline manual stereotypies and suffers sleep disturbances with frequent awakenings.

The patient is a 9-year-old female, the only child of healthy non-consanguineous parents. Intrauterine growth retardation and mild left pyelectasis were detected during pregnancy. Birth weight was 2.30 kg (1st percentile, − 2.25 SD) and cranial circumference was 30.5 cm (<3rd percentile, − 3 SD). Screening for congenital disorders of metabolism was negative. Feeding difficulties and oral candidiasis were observed during the perinatal period. The subject achieved head control at 2 months, sat up at 10–12 months, began to crawl at 23 months, and presented unstable gait at age 3. At age 2, she was diagnosed with beta-thalassaemia minor (of paternal inheritance). Mild valvular pulmonary stenosis was also observed, although this problem had disappeared by 6 years of age. EEG was normal at the age of 3 years. Brain MRI, performed at few months of age, showed unspecific delayed myelination, although repeated imaging was considered normal when the patient was 3 years old.

At 6 years of age, when she was recruited by the SpainUDP (Spanish Undiagnosed Rare Diseases Program, http://​spainudp.​isciii.​es/​), she had global developmental delay, intellectual disability, language impairment, stereotypies, astigmatism, amblyopia, and recurrent conjunctivitis. Her physical exam showed craniofacial dysmorphisms including microcephaly, midface retraction, mild hypoplasia of the ear lobe, prominent antitragus, sparse medial eyebrows, proptosis, strabismus, broad and bifid nasal tip, short philtrum, prognathism, and big mouth (Table 1 and Fig. 1: d and i). She also had wide intermammillary distance, bulging abdomen, protruding umbilicus placed on a small depression of the abdomen, mild genu valgo, pes planus, and fibular deviation of halluces.

The patient is an 8-year-old boy, the only child of non-consanguineous parents. No problems were detected during pregnancy and the neonatal period. He was born at term with a birth weight of 3.7 kg (76th percentile, + 0.70 SD) and a cranial circumference of 35 cm (34th percentile, − 0.42 SD). He achieved head control at 3 months, sat up at 8.5 months, and began to walk independently at 16 months. Microcephaly was detected at 2 months of age and he was diagnosed with atypical absences with eyelid myoclonias at the age of 16 months. He was treated with valproate from 21 months to 5 years with acceptable control of seizures. When the proband was 5 years old, it was withdrawn due to its association with adverse reactions (nauseas, vomiting, and weight loss). A few months later, valproate treatment was administered again since the number and intensity of epileptic crises had increased. In this recurrence of epilepsy, ethosuximide was needed together with valproate to achieve seizure control. Several EEGs carried out starting at age 3 years have disclosed spike-wave anomalies. When he was admitted to SpainUDP, at 6 years of age, the subject showed intellectual disability with altered fine motor coordination, unstable gait with frequent falls, language impairment, and autistic behaviour. He has always had sleep disturbances and hyporexia. Hypotonia with reduced muscle bulk has also been observed. Physical examination (Table 1 and Fig. 1: e and j) showed microcephaly, long face, mid facial asymmetry, frontal central bossing, swollen skin on the upper eyelids, epicanthus, deep set eyes, mildly everted lower eyelid in its external part, and deep horizontal groove under the lower lip. Additional features include a slender appearance, scarce body adiposity, long and narrow thorax, mild genu varo (bilateral), pes planus, short halluces, sandal gap between the first and second toes, and flexed fifth toes.

Metabolic testing (including urine organic acids and glucose in cerebrospinal fluid) was normal. Brain MRI was normal.

The patient is a 6.5-year-old girl, first child of healthy non-consanguineous parents and with a healthy younger sister. The subject was born at term with an uneventful delivery and with a birth weight of 3.14 kg (42nd percentile, − 0.2 SD), a cranial circumference of 33 cm (10th percentile, − 0.61 SD), and Apgar 9/10 at 1 and 5 min. New-born screening for inborn errors of metabolism was normal. She had feeding problems from the neonatal period. During her hospital admission for dysphagia at 2 months of age, an atrial septal defect (ASD) was detected, which was surgically repaired at 16 months. She had delayed motor skills, with head control at 6 months, sitting up at 13 months, crawling at 16 months, and autonomous although unstable gait developing at age 2.

The subject has received physical, speech, and behavioral therapies and has attended a special school since 6 years of age. She is not able to talk, shows difficulties in adapting to new environments, shows significant behavioral problems with particular impact on the social domain, and has poor eye contact. Physical examination (Table 1 and Fig. 1: f, k, m and n) showed dysmorphic features characterized by midface hypoplasia, almond-shaped eyes, and slightly upslanted palpebral fissures, and absent Cupid’s bow. Ears have underdeveloped antihelix, and are slightly low set, with increased posterior angulation. The hands show a complex palmar dermatoglyphic pattern with abnormal square radial border morphology. Tone, strength, and deep tendon reflexes are normal.

Remarkably, cranial MRI at age 6 revealed prominent cerebellar interfolia space compared with a previous MRI at 2 years of age; this is compatible with progressive cerebellar atrophy. No other significant alterations were detected on MRI. The subjected was recruited by the Undiagnosed Rare Disease Program of Catalonia (www.​urdcat.​cat).

A normal karyotype (46, XY) was determined. A trio-based whole exome study (WES) revealed a de novo heterozygous variant NM_006766:c.3385C > T (p.Arg1129*) in the KAT6A gene, which was confirmed by Sanger sequencing. This change was previously identified in a child with global developmental delay [5].

Other variants of unknown clinical relevance (VUS) are summarized in Additional file 1: Table S1.

Karyotype, subtelomeric fluorescent in situ hybridization (FISH), kit MLPA panel studying recurrent genomic disorders (SALSA® P245-B1), chromosomal microarray, and testing for fragile X, MECP2/FOXG1 genes, Angelman syndrome, Pitt-Hopkins syndrome, mucopolysaccharidoses, and congenital disorders of glycosylation, yielded normal results. Clinical exome of the index case revealed a heterozygous de novo variant in KAT6A in the proband (NM_006766: c.3640 A > T (p.Lys1214*)]. Other VUS detected in the patient were: heterozygous c.852G > A (p.Leu284Leu) variant and heterozygous c.1467 + 16A > C variant in the FOXG1 gene (Sup. Table 2).

Before WES, several genetic tests were carried out, with negative results: karyotype, array-CGH (60 K), Fragile X, Angelman, and diagnostic exome sequencing (DES) for epilepsy (543 genes). Trio-based WES was performed and a de novo KAT6A variant was identified in the patient (NM_006766:c.1075G > A), which was confirmed through Sanger sequencing. This variant predicts a replacement of guanine by adenine at codon 359 in exon 7 [p.(Gly359Ser)]. It has been found in only one carrier out of 250,564 alleles in gnomAD. It is predicted to be ‘deleterious’ or ‘putatively pathogenic’ by a variety of bioinformatic tools (PolyPhen2, Mutation Taster) and to be ‘tolerated’ by SIFT (score 0.28); CADD score is 16.9. Altogether, this variant is classified as ‘pathogenic’ according to the AMCG/AMP 2015 guidelines. In addition, the Human Splicing Finder software (3.1 version; January 10, 2018) predicts a potential alteration of splicing consisting of the generation of a cryptic acceptor site (AGTTCGAACTAGCC) at exon 7 (which was not experimentally observed, see below), and the loss of a predicted ESE (GGCCCTGG) for splicing factor SC35 (starting at c.1075G, which is not present in the mutant allele).

The subject has a normal karyotype (46,XX) and molecular karyotype by array-CGH (60 K). Singleton WES was performed followed by trio-based segregation with Sanger sequencing. The analysis showed a heterozygous de novo frameshift deletion of four nucleotides (NM_006766: c.4254_4257delTGAG) in exon 17 of the KAT6A gene, already defined as ‘pathogenic’ in ClinVar and reported by Kennedy et al. [13]. The mutant mRNA is predicted to be translated as a truncated protein with a premature termination 12 amino acids after the frameshift (p. Glu1419Trpfs*12). The variant has not been described in healthy population in the genomic databases.

Whole exome sequencing of the proband and his parents was performed in the National Centre of Genomic Analysis (CNAG; Barcelona, Spain) using the Illumina HiSeq-2000 platform. Exome capture was performed with Agilent SureSelect v5 (Agilent, CA, USA). The samples were sequenced at a coverage of 140x. The data were analyzed as described elsewhere [18]. Annotation, filtering, and prioritization of variants were carried out using VarAFT software [19]. The results were then filtered under de novo dominance and recessive hypotheses. Variants with a minimum allele frequency (MAF) above 0.001 for AD filtering and above 0.01 for AR filtering in the common population (according to GnomAD) were excluded. Variants in genes included in DDG2P (The Development Disorder Genotype-Phenotype Database [15, 20]), and covered by at least 10 reads, were prioritized for validation (it should be noted that those who carried out the original DECIPHER analysis and collection of the data bear no responsibility for the further analysis or interpretation of it).

The mean coverage was of 153.43, 174.29, and 160.829 reads for the patient, father and mother respectively, and 97.2–97.9% of the target region was covered with at least 10 reads (C10). A total of 5 variants in 4 genes were selected for validation by Sanger sequencing. Primer sequences and PCR conditions are available on request. PCR reaction, purification, and sequencing were performed as described previously [20].

Whole exome sequencing of the index case was carried out with the platform Illumina NextSeq500, using the Agilent SureSelect v6 QXT capture kit. For the analysis, several bioinformatics tools were used. The exome design covered approximately 95% of the coding regions of the analysed genes. The sequences used as reference can be found at the RefSeq database. The estimated frequencies were calculated from the 1000 genomes, Complete Genomics and NHLBI Exome Sequencing Project databases. The nomenclature of mutations was based on the recommendations of the Human Genome Variation Society. The analysis was carried out according to the recommendations of the American College of Medical Genetics. The results were then filtered under de novo dominance and recessive hypotheses. Variants with a minimum allele frequency (MAF) above 0.001 for AD filtering and above 0.01 for AR filtering in the common population (according to GnomAD) were excluded. Variants in genes included in DDG2P (The Development Disorder Genotype-Phenotype Database [21, 22]), and covered by at least 10 reads, were prioritized for validation (it should be noted that those who carried out the original DECIPHER analysis and collection of the data bear no responsibility for the further analysis or interpretation of it).

After exhaustive revision of clinical information of patient 3, she was admitted to the Spanish Undiagnosed Rare Diseases Program (SpainUDP) [23]. Also, this patient and her unaffected biological parents were enrolled in the FP7-funded ‘2016 BBMRI-LPC WES Call’ (Eurobiobank website, accessed on 19th July 2018) to carry out the research shown in this article.

Trio-based whole exome sequencing (WES) for this project was conducted at the Centro Nacional de Análisis Genómico (CNAG-CRG, Spain). SureSelect Human All Exon V5 (Agilent Technologies) was used to perform whole exome enrichment following the manufacturer’s instructions. The captured libraries were sequenced using TruSeq SBS Kit v3-HS (Illumina, Inc), in paired-end mode with a read length of 2x100bp. On average, 92x median coverage for each sample was generated in a fraction of a sequencing lane on HiSeq2000 following the manufacturer’s protocol. Images analysis, base calling, and quality scoring of the run were processed using the manufacturer’s software Real Time Analysis (RTA 1.13.48, HCS 1.5.15.1) and followed by generation of FASTQ sequence files by CASAVA. High-quality reads were aligned to the decoy version of the GRCh37 reference genome used by the 1000 Genomes Project (hs37d5) using BWA-MEM (version 0.7.8), and variants were identified following GATK Best Practices [24] using HaplotypeCaller (version 3.6). All variants with a minimum coverage of 8 reads and minimum genotype quality (GQ) of 20 were uploaded to the Genome-Phenome Analysis Platform (RD-Connect GPAP) [25, 26] for variant filtration and prioritization. Additionally, phenotypic terms were extracted from clinical documents stored in the patient registry by a member of SpainUDP, mapped to HPO (Human Phenotype Ontology) [27] terms, and uploaded to PhenoTips [28], a software tool available in the RD-Connect GPAP. This platform allows filtering and refining of the results by mode of inheritance, population frequencies, in silico pathogenicity prediction tools, and HPO codes [25, 26]. This filtering process was carried out by two independent researchers of SpainUDP with common criteria, and the results were compared in order to reach a consensus on selection of the candidate variants, which were confirmed by Sanger sequencing in all family members. Finally, various sources of information were consulted to build a report with a detailed review of the scientific evidence supporting the correlation between the detected causative variant and the proband’s phenotype.

Patient 4 was admitted to the SpainUDP and underwent phenotypic analysis following the standard criteria established by this program [23]. After peripheral blood genomic DNA isolation of patient 4 and his biological parents (see case 3 for details), trio-based WES and selection of candidate variants were performed as described by López et al. [23]. Variants assessed as pathogenic and possibly contributing to the proband’s phenotype were validated by Sanger sequencing in the full trio.

Patient 5 was admitted to the URDCat Program after deep phenotyping. Patient’s whole exome sequencing from extracted DNA from peripheral blood was carried out at the National Centre of Genomic Analysis (CNAG; Barcelona, Spain) using the Illumina HiSeq-2000 platform. Exome capture was performed with Nimblegen SeqCap EZ MedExome + mtDNA 47 Mb and the samples were sequenced at coverage of 90x. Sequencing data were analysed according to the project’s established pipeline, and afterwards SNV, indel, CNV, and mosaicism analysis was performed using the RDCat Genomic Analysis Platform. Variants assessed as pathogenic and possibly contributing to the proband’s phenotype were validated by Sanger sequencing in the trio.