Bi-allelic TTC5 variants cause delayed developmental milestones and intellectual disability

Abstract

Background:

Intellectual disability syndromes with or without developmental delays affect up to 3% of the world population. We sought to clinically and genetically characterize a novel intellectual disability syndrome segregating in five unrelated consanguineous families.

Methods:

Clinical analyses were performed for eight patients with intellectual disability. Whole-exome sequencing for selected participants followed by Sanger sequencing for all available family members was completed. Identity-by-decent (IBD) mapping was carried out for patients in two Egyptian families harboring an identical variant. RNA was extracted from blood cells of Turkish participants, followed by cDNA synthesis and real-time PCR for TTC5.

Results:

Phenotype comparisons of patients revealed shared clinical features of moderate-to-severe intellectual disability, corpus callosum agenesis, mild ventriculomegaly, simplified gyral pattern, cerebral atrophy, delayed motor and verbal milestones, and hypotonia, presenting with an intellectual disability syndrome. Four novel homozygous variants in TTC5: c.629A>G;p.(Tyr210Cys), c.692C>T;p.(Ala231Val), c.787C>T;p.(Arg263Ter), and c.1883C>T;p.(Arg395Ter) were identified in the eight patients from participating families. IBD mapping revealed that c.787C>T;p.(Arg263Ter) is a founder variant in Egypt. Missense variants c.629A>G;p.(Tyr210Cys) and c.692C>T;p.(Ala231Val) disrupt highly conserved residues of TTC5 within the fifth and sixth TPR motifs which are required for p300 interaction, while the nonsense variants are predicted to decrease TTC5 expression. Functional analysis of variant c.1883C>T;p.(Arg395Ter) showed reduced TTC5 transcript levels in accordance with nonsense-mediated decay.

Conclusion:

Combining our clinical and molecular data with a recent case report, we identify the core and variable clinical features associated with TTC5 loss-of-function variants and reveal the requirement for TTC5 in human brain development and health.

INTRODUCTION

Intellectual disability syndromes (IDSs) are a widespread group of neurological disorders impacting 1–3% of the world-wide population.[1] An individual is diagnosed with intellectual disability (ID) when they exhibit significant limitations of intellectual functioning and adaptive behaviors prior to 18 years of age.[2] Hundreds of genomic variants have been associated with IDSs, yet nearly 60% of cases have unknown molecular etiology.[3] The recent use of next-generation sequencing technologies in the clinic have enabled the discovery of novel candidate genes and variants for this genetically complex and heterogeneous disorder.[4] For example, heterozygous variants in EP300, encoding p300, a co-activator of p53, are observed in Rubinstein-Taybi syndrome type 2 (RSTS [MIM: 613684]) in roughly 3% of patients. RSTS2 patients present with intellectual disability, pronounced microcephaly, broad thumbs and halluces, and characteristic dysmorphic facial features that include high arched eyebrows, mild downslanting of palpebral fissures, and retrognathia.[5] Most of the reported variants are small, frameshifting insertions or deletions within the EP300 coding sequence, suggesting the mechanism of disease may be haploinsufficiency.[6–10] Only recently, genotyping of two female patients with Menke-Hennekam syndrome 2 (MKHK2 [MIM: 618333]) uncovered de novo missense and in-frame indel heterozygous variants in EP300.[11] Both individuals had low IQ, feeding difficulties, recurrent upper airway infections, hearing impairment, hypermetropia, and facial features consisting of square flat face with upslanting, short palpebral fissures.[11, 12] Together, these genetic and clinical findings suggest the functional impact and position of variants in EP300, and likely components of the p300 co-activator complex, can lead to diverse clinical phenotypes and distinct IDSs.

p300 directly interacts with TTC5 (also known as STRAP, stress-responsive activator of p300) and JMY (junction mediating and regulatory protein, p53 cofactor) in the nucleus to form the p300 co-activator complex that mediates the p53 transcriptional response to cell stress.[13, 14] TTC5 encodes a 440 amino acid protein containing six tetratricopeptide repeat (TPR) domains and an oligonucleotide-binding (OB) fold that functions as a scaffold to stabilize p300-JMY interactions. This interaction prevents MDM2-dependent degradation of p53 to promote apoptosis.[13] In addition to this nuclear role, TTC5 has two known functions in the cytoplasm. TTC5 inhibits actin nucleation and autophagosome formation in the cytoplasm during cell stress,[15] as well as associates with the active ribosome and nascent tubulin polypeptides during protein synthesis to control tubulin autoregulation.[16] TTC5 is ubiquitously expressed in human tissues, with the highest levels found in the brain.[13]

Based on the expression pattern of TTC5 and biological association with JMY and p300, we hypothesized that variants in TTC5 might underlie an intellectual disability syndrome. A recent report identified TTC5 as a candidate gene for ID among 183 other genes profiled in 404 families by whole genome sequencing.[17] The authors reported two variants (c.51+1G>A and c.599delC;p.(Pro200LeufsTer29)) in three patients from two families with ID and discordant clinical features.[17] Here, we expand on these findings by using whole-exome sequencing to identify four novel bi-allelic TTC5 variants in eight individuals from five unrelated families with an intellectual disability syndrome.

MATERIALS AND METHODS

Patient Recruitment

This research was conducted after obtaining approval from Institutional Review Board of School of Biological Sciences, University of the Punjab, Lahore Pakistan (IRB No. 00005281), the Regional Ethics Committee of Harran University, Sanliurfa, Turkey, and the University of California, San Diego (140028). All participants or legal guardians signed written informed consent, including permission to publish photographs of the affected children. Medical histories were obtained by questioning. Collaboration was enabled by GeneMatcher.[18] For inclusion in the study, patients needed to be diagnosed with a moderate to severe intellectual disability with no known etiology.

Sampling

Peripheral blood was obtained from all available family members. DNA was extracted using a standard sucrose lysis, proteinase K digestion and salting out method (RDHM-05) or the MagNA Pure Compact Nucleic Acid Isolation Kit (CW07) (Roche, Switzerland) or as previously described.[19]

Whole-exome Sequencing

Whole-exome sequencing (WES)[20] was performed on DNA from all affected individuals in family RDHM-05, or individual CW07-A, 5543-A, 5377-A, or 6772-B at 100X resolution using Agilent V4 enrichment kit (Agilent Technologies, Santa Clara, CA, USA) or Nextera Rapid Capture Exome (Illumina, San Diego, CA USA), using the Illumina platform.[19, 21] ANNOVAR (http://wannovar.usc.edu/)[22] was used to annotate the variants. 97.41% of target regions had a coverage depth of more than 10-fold.

Variant Prioritization and amino acid conservation analyses

Rare homozygous exonic and splice variants with a population allele frequency of less than 1% in public databases (1000 genome database (http://www.internationalgenome.org/), Exome Aggregation Consortium (ExAC) (www.exac.broadinstitute.org/),[23] Exome Sequencing Project ESP (http://evs.gs.washington.edu/EVS/), genome Aggregation Database (gnomAD) (http://gnomad.broadinstitute.org/)[24] were filtered for further consideration. Missense variants were prioritized by the pathogenicity scores predicted from CADD (Combined Annotation Dependent Depletion),[25] SIFT (Scale-Invariant Feature Transform),[26] REVEL (Rare Exome Variant Ensemble Learner)[27] and ClinPred (https://omictools.com/clinpred-tool).[28] Each affected individual was analyzed independently for disease candidate variants; however, if multiple affected family members were sequenced, shared variants were prioritized as potentially pathogenic. Variants segregation was tested by PCR amplification and Sanger sequencing using Big Dye Terminator v 3.1 with Seqman (Lasergene, DNAstar) or SeqScape Software 3 (Life Technologies Corp.) analyses. In addition, the allele frequency of the TTC5 missense and nonsense variants were validated using DNA from ethnically matched controls. Protein sequence alignments from the UCSC genome browser (https://genome.ucsc.edu/) and homologene (https://www.ncbi.nlm.nih.gov/homologene) were used to assess amino acid conservation among diverse species. TTC5 protein orthologue sequences were obtained from UniProtKB (https://www.uniprot.org/help/uniprotkb) and amino acid conservation was observed following Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/) alignment.

Identity-by-descent mapping

Homozygous intervals were detected from whole -exome sequencing data of the affected individuals in family 5543 and 5377 using HomozygosityMapper.[29] Variant calls within the homozygous interval surrounding TTC5 were aligned by chromosomal position and tabulated to identify a shared haplotype.

TTC5 qRT-PCR

Peripheral blood mononuclear cells were isolated from patients and family matched controls for family CW07 that consented to blood resampling using Lymphoprep (Stemcell Technologies, Inc.) according to manufacturer recommendations. The cells were lysed and the total RNA was extracted using TriZOL LS (Thermo Fisher Scientific). The cDNA libraries were prepared with SuperScript Vilo with ezDNase (Invitrogen). qRT-PCR was performed for TTC5 and GAPDH (loading control) expression using equal amounts of cDNA input per individual, Power SYBR Green Master Mix (Thermo Fisher Scientific), and primers: (TTC5 fwd) 5ʹ– TCA TCC TGG GAA AGG TGG TA – 3ʹ, (TTC5 rev) 5ʹ – GCT TCC CAT TCA CCA CTA GC – 3ʹ, or (GAPDH fwd) 5ʹ – GAG GCA GGG ATG ATG TTC TG – 3ʹ, (GAPDH rev) 5ʹ – CTG CAC CAC CAA CTG CTT AG – 3ʹ in triplicate. Triplicate Ct values were averaged and normalized to the loading control (GAPDH) for each individual. Normalized expression of TTC5 in unaffected relatives was set to 1, and the ΔCt calculated to determine fold change of TTC5 in affected patients.

RESULTS

Clinical Analyses:

Family 1, (RDHM-05, figure 1A, table 1) with two affected siblings from a first cousin marriage was ascertained from Lahore, Pakistan. Male patient RDHM-05-A, now age 8, was born through C-section and had an undescended testicle (HP:0000028), which was repaired through surgery. He suffered from lactose intolerance (HP:0004789) during early infancy. All assessed verbal (HP:0000750) and motor milestones (HP:0002194) were developmentally delayed (table 1) and he was diagnosed with moderate intellectual disability (HP:0002342). Muscular hypotonia (HP:0001252), aggression (HP:0000718) and recurrent chest infections (HP:0002205) were observed. Brain MR imaging revealed cerebral atrophy (HP:0002059) (data not shown). Facial dysmorphisms of pointed chin (HP:0000307), thin upper lip (HP:0000219), low-set ears (HP:0000369), and prominent midface were noted. Female patient RDHM-05-B, age 3, was born pre-term (HP:0001622) at 34 weeks gestation by C-section. Similar to her brother, her developmental milestones were delayed (HP:0001263). To date, she has shown mild, but identical age-matched features to her brother but with the addition of hirsutism (HP:0001007). Facial dysmorphisms include thin upper lip (HP:0000219) and low-set ears (HP:0000369). (figure 1B, table 1).

Figure 1. Pedigree and clinical data from families RDHM-05, CW07, 5543 and 5377.

A. Family RDHM-05, CW07, 5543, 5377 and 6772. Parental consanguinity, double bar; square, male; circle, female; diamond, sex unknown; filled, affected; unfilled, unaffected; backslash, deceased.

B. Photographs of patients from family CW07 showing dry, acneiform lesions of the skin, RDHM-05, 5543, 5377 and 6772 showing thin upper lips.

Table 1.

Clinical features of individuals with bi-allelic TTC5 variants

Family	RDHM-05		CW07		5543	5377	Summary of features
Subject	A	B	A	B	A	A
TTC5 Variant	c.629A>G p.Tyr210Cys	c.629A>G p.Tyr210Cys	c.1183 C>T p.Arg395Ter	c.1183 C>T p.Arg395Ter	c.787C>T p.Arg263Ter	c.787C>T p.Arg263Ter
Sex	Male	Female	Male	Male	Male	Female	2f / 4m
Gestational age at delivery (weeks)	37	34	35	34	39	40	34–40 weeks
Head Size (cm)	BPD: 8.8	BPD: 8.5	BPD: 8.7	NA	HC: 32	HC: 31	0.1–50.8 percentile
Onset of disorder	At birth	At birth	At birth	At birth	At birth	At birth	At birth
Birth weight (kg)	2.5	1.8	2.0	2.4	2.75	3	3.2–56.9 percentile
Present weight (kg)	21	10	18.5	16.1	6.8	10	0.1–12.71 percentile
Current age (years)	8	3	7	5	5	6	3–8 years
Present height (cm.)	106.68	86.36	115	110	92	82	0.1–57.1 percentile
Age at unassisted sitting (months)	12	NA	After 1 year	After 1 year	Not Yet	Not Yet	3/6 1–6+ years
Age at unassisted standing (months)	36	12	After 3 years	After 2 years	Not Yet	Not Yet	4/6 1–3+ years
Age at first word (months)	24	None	None	None	None	None	6/6 24+ months
Present speech ability	Equals 2 years and 3 months	No speech	No speech	No speech	No speech	No speech	5/6 no speech
Intellectual disability	Moderate	Mild	Severe	Moderate	Severe	Severe	6/6
Abnormal brain MRI	+	NA	+	+	+	+	5/5
Cerebral atrophy	+	NA	+	+	+	+	5/5
Enlarged lateral ventricles	−	NA	+	+	+	+	4/5
Corpus callosum atrophy	−	NA	+	+	+	+	4/5
Simplified gyral pattern	−	NA	+	+	+	+	4/5
Other brain abnormality	−	NA	+ distended straight sinus, gliosis	+ distended straight sinus, enlarged third ventricle, bilateral silvian fissures observed	+	+ central atrophy	4/5
Hypotonia	−	−	+	+	+	+	4/6
Facial Dysmorphism	−	−	+ abnormal outer ear, wide forehead, flared eyebrows and thin upper lip (v-shaped)	+ abnormal outer ear, wide forehead, flared eyebrows, v-shaped upper lip	+ V-shaped upper lip, high arched palate, long philtrum, prominent eyes	+ Thin upper lip, high arched palate	4/6 Thin v-shaped upper lip
Microcephaly	−	−	−	−	−	+	1/6
Skin Anomaly	−	+ Hirsutism	+ Acneiform lesions, dry skin	+ Acneiform lesions, dry skin	−	−	3/6
Paranasal sinus volume increased	−	NA	+	+	−	−	2/5
Autistic behavior	−	−	+	+	−	−	2/6
Aggression	+	−	−	+	−	−	2/6
Genitourinary Abnormalities	+ Undescended testes	−	−	−	+ Bilateral undescended testes	−	2/6
Sleep Disturbance	−	−	−	−	+	+	2/6
Scoliosis or kyphosis	−	−	−	−	−	+ Mild kyphosis	1/6
Seizures	−	−	−	−	−	+ Left centro- parieto- temporal epileptogenic discharge	1/6
Internipple distance	NA	NA	−	+ wide	−	−	1/6

NA, not available; HC, head circumference; BPD, biparietal diameter; +, present; −, absent.

Family 2, (CW07, figure 1A, table 1) with two affected brothers, aged 7 and 5, from a first cousin marriage was ascertained from Sanliurfa, Turkey after approval by the Regional Ethics Committee of Harran University, Sanliurfa, Turkey. Patient CW07-A was born pre-term at 35 weeks gestation (HP:0001622). He presented with autistic behaviors (HP:0000729), severe intellectual disability (HP:0010864), delayed motor milestones (HP:0002194), lack of speech (HP:0001344), and muscular hypotonia (HP:0001252) (table 1). MRI revealed several brain abnormalities, including prominent anterior ventricle horns (HP:0002119), dilated paranasal sinus (HP:0000245), distended straight sinus, atrophy of the corpus callosum (HP:0007371), simplified gyral pattern (HP:0009879), and gliosis (HP:0002171) (figure 2). Moreover, he had dermatological findings of acneiform lesions with dry skin (HP:0000958) (figure 1B) together with minor dysmorphic features of abnormal outer ear (HP:0000356), wide forehead (HP:0000337), flared eyebrows (HP:0011229), and v-shaped, thin upper lip (HP:0000219). CW07-B presented with autistic (HP:0000729) and aggressive behavioral pattern (HP:0000718), moderate intellectual disability (HP:0002342), delayed motor milestones (HP:0001270), lack of speech (HP:0001344), muscular hypotonia (HP:0001252), and severe brain abnormalities, including enlarged paranasal (HP:00000245) and distended straight sinus, cerebral atrophy (HP:0002059) with simplified gyral pattern (HP:0009879), ventriculomegaly of the third and lateral ventricles (HP:0002119), atrophy of the corpus callosum (HP:0007371) and prominent sylvian fissures (HP:0100952) (figure 2). He shared the dermatological and dysmorphic findings observed in his brother, as well as increased internipple distance (HP:0006610) (table 1, figure 1B).

Figure 2. Magnetic resonance images (MRI) of patients from families CW07, 5543, 5377, and 6772.

MRIs from a healthy control individual (left column) and affected individuals. Midline sagittal images (top row) show corpus callosum atrophy (red arrow), increased paranasal sinus area (green arrow), and distended straight sinus (pink arrow). Axial (middle row) images depict dilated ventricles (yellow arrow), white matter changes at the occipital horn (blue arrow), and abnormal gyri (orange arrow). Coronal (bottom row) images show prominent sylvian fissures in patient CWO7-B (purple arrow) and abnormal gyri in patient 5543-A and 6772-B (orange arrow). Control cases courtesy of Dr. Derek Smith, Radiopaedia.org, rID:53692

Family 3 (5543, figure 1, table 1), with an affected male from a first cousin marriage, was ascertained from Cairo, Egypt. Patient 5543-A, now 5 years of age, was born full term at 39 weeks gestation with bilateral undescended testes (HP:0000028). He presented with severe intellectual disability (HP:0010864), delayed motor milestones (HP:0001270), lack of speech (HP:0001344), muscular hypotonia (HP:0001252), and facial dysmorphisms of v-shaped upper lip (HP:0010804), high arched palate (HP:0002705), long philtrum (HP:0000343), and prominent eyes (HP:0000520) (table 1, figure 1B). MR imaging revealed cerebral atrophy (HP:0002059) with simplified gyral pattern (HP:0009879), hypoplastic corpus callosum (HP:0002079), white matter abnormalities around the occipital horn (HP:0007052), and dilated lateral ventricles (HP:0002119) (figure 2).

Family 4, (5377, figure 1, table 1), with an affected female of noted parental consanguinity, was ascertained from Cairo, Egypt. Patient 5377-A, now 6 years of age, was born by vaginal delivery at 40 weeks gestation. She presented with severe intellectual disability (HP:0010864), microcephaly (HP:0011451), hypotonia (HP:0001290), delayed motor milestones (HP:0001270), no speech (HP:0001344), mild kyphosis (HP:0002808), and minor facial dysmorphisms of thin upper lip (HP:0000219), curly hair (HP:000212) and high arched palate (HP:0002705) (table 1, figure 1B). At the age of 2, she developed severe epilepsy resulting in roughly four tonic (HP:0010818) and two myoclonic (HP:0002123) seizures per day with cyanosis (HP:0000961) that is now controlled with antiepileptic medications (table 1). She has severe developmental brain abnormalities including abnormal gyri (HP:0009879), dilated lateral ventricles (HP:0002119), a thin corpus callosum (HP:0002079), and central (HP:0002506) and cortical atrophy (HP:0002120) (data not shown).

Family 5, (6772, figure 1, table 1), with two affected siblings from a first cousin marriage, was ascertained from Cairo, Egypt. Female patient 6772-A, now age 12, was born by vaginal delivery at 40 weeks gestation. She presented with moderate intellectual disability (HP:0010864), microcephaly (HP:0011451), hypotonia (HP:0001290), delayed verbal (HP:0000750) and motor (HP:0001270) milestones, increased internipple distance (HP:0006610), and minor facial dysmorphisms of long face (HP:0000276), high-arched eyebrows (HP:0002553), prominent nose (HP:0000448), short philtrum (HP:0000322), and large low-set ears (HP:0000369). MR imaging revealed severe structural brain defects including abnormal gyri (HP:0009879), dilated lateral ventricles (HP:0002119), a thin corpus callosum (HP:0002079), and cortical atrophy (HP:0002120) (data not shown). Male patient 6772-B, now age 5, was born by vaginal delivery at 38 weeks gestation. He was diagnosed with severe intellectual disability and presented with delayed motor milestones (HP:0001270), lack of speech (HP:0001344), muscular hypotonia (HP:0001252), increased internipple distance (HP:0006610), and facial dysmorphisms of long face (HP:0000276), brachycephaly (HP:0000248), low anterior hairline (HP:0000294), woolly scalp hair (HP:0040149), mild sagging eyelid (HP:0001488), prominent nose (HP:0000448) and nasal tip (HP:0005274), v-shaped upper lip (HP:0010804), short philtrum (HP:0000322), and low-set protruding ears (HP:0000369) (table 1, figure 1B). MR imaging revealed cerebral atrophy (HP:0002059) with simplified gyral pattern (HP:0009879), hypoplastic corpus callosum (HP:0002079), and dilated lateral and third ventricles (HP:0002119) (figure 2).

Molecular Analyses

Affected individuals from RDHM-05 had multiple homozygous variants present (Supplementary table 1), however, only a single variant in TTC5: NM_13837:g.20298807T>C; c.629A>G; p.(Tyr210Cys), passed segregation analysis according to a strict recessive mode of inheritance (figure 3A, B, C). The variant was not previously described in public databases, including Exome Aggregation Consortium (ExAC), HGMD Professional 2018.3, 1000 genomes, gnomAD, the NHLBI GO Exome Sequencing Project (ESP), or observed in 200 chromosomes from ethnically matched controls. Likewise, exome sequencing of affected individual 6772-A in family 6772 detected a single homozygous missense variant in TTC5: NM_13837:g.20296394G>A; c.692C>T; p.(Ala231Val) which segregated with the phenotype (figure 3A, B, C). It was observed only once as heterozygous in ExAC (1/251420).

Figure 3. Molecular analysis of variants in TTC5.

A. Partial Sanger sequence traces for TTC5 exons 5, 6, 7, and 9, encompassing c.629A>G;p.(Tyr210Cys), c.1883C>T;p.(Arg395Ter), c.787C>T;p.(Arg263Ter), and c.692C>T;p.(Ala231Val) variants, respectively. Affected individuals are homozygous for the variant, while unaffected carriers are heterozygous.

B. TTC5, located on chromosome 14 on the minus strand has ten exons (shown by rectangles). Novel TTC5 variants occur at hg38 position 20298807 in exon 5 (family RDHM-05), 20296394 in exon 6 (family 6772), 20292003 in exon 7 (families 5543 and 5377), and 20292003 in exon 9 (family CW07).

C. TTC5 protein domain structure. TPR motif domains are shown in grey. Previously reported variants are annotated with black triangles and novel variants with red triangles. The cDNA and protein positions are shown. TPR; Tetratricopeptide repeat.

D. Clustal-Omega alignment of TTC5 protein orthologues in vertebrate species showing complete evolutionary conservation of p.Tyr210 and p.Ala231.

E. Quantitative RT-PCR for TTC5 from peripheral blood mononuclear cells isolated from an unaffected carrier or affected individual in family CW07 showing variant p.(Arg395Ter) decreases TTC5 expression.

The TTC5 missense variants c.629A>G; p.(Tyr210Cys) and c.692C>T; p.(Ala231Val) had high CADD (p.(Tyr210Cys), 19.99; p.(Ala231Val), 32.00),[25] Grantham (p.(Tyr210Cys), 194; p.(Ala231Val),64),[30] and GERP (p.(Tyr210Cys), 4.65; p.(Ala231Val), 5.10)[31] scores indicating high nucleotide and amino acid residue conservation at both positions. Indeed, Clustal-Omega alignment of TTC5 orthologues revealed the p.Tyr210 and p.Ala231 residues are evolutionarily conserved among all vertebrate species examined (figure 3D) as well as in ciliates and plants.[16] In addition, in-silico tools predict the variants to be damaging or disease causing: PolyPhen,[32] probably damaging; SIFT,[26] deleterious; PROVEAN,[33] deleterious; and MutationTaster,[34] disease causing.

Exome sequencing of affected individuals CW07-A, 5477-A and 5543-A revealed two novel homozygous nonsense variants in TTC5: NM_138376:g.20292003G>A; c.1883C>T; p.(Arg395Ter) in family CW07, and NM_138376:g.20295764G>A; c.787C>T; p.(Arg263Ter) in families 5477 and 5543, respectively, that segregated with the phenotypes in the respective families (figure 3A, B, C). Nonsense variant p.(Arg395Ter) was not present in 120 chromosomes from ethnically matched control individuals but was observed twice in ExAC and four times in gnomAD (4/235016), while, nonsense variant p.(Arg263Ter) was reported once in ExAC (1/120536) and twice in gnomAD (1/246074); all as heterozygous. Both nonsense variants had not been described in 1000 genomes or in the NHLBI GO Exome Sequencing Project (ESP), and are predicted to be disease causing by in-silico prediction tools.

To functionally determine whether the nonsense variant p.(Arg395Ter) in TTC5 would elicit nonsense-mediated decay and thereby reduce transcript levels, we investigated mRNA extracted from peripheral blood mononuclear cells from healthy and affected individuals in family CW07 that consented to resampling. Quantitative RT-PCR measurement of TTC5 transcript levels in cells isolated from the father of family CW07 (monoallelic for p.(Arg395Ter)) or CW07-A (bi-allelic for p.(Arg395Ter)) found a 4-fold decrease in expression in the affected individual (figure 3E). Taken together, the high nucleotide and amino acid conservation scores of the reference TTC5 alleles, the in-silico variant predictions, and real-time PCR results suggest that the novel TTC5 variants are deleterious and may be pathogenic.

Since patients 5377-A and 5543-A share an identical homozygous nonsense variant p.(Arg263Ter) in TTC5, and both families originated from Egypt, we sought to determine if families 5377 and 5543 share a homozygous haplotype block. Identity-by-descent mapping[29] of exome sequencing data from both affected individuals uncovered a 6,301,093 base pair region spanning the TTC5 locus, encompassing 59 single nucleotide polymorphisms (SNPs) rs199831317-rs4983176 (Supplementary table 2), indicating p.(Arg263Ter) is likely a founder mutation.

DISCUSSION

TTC5 is a member of a large family of paralogous proteins in humans, which contain a TPR motif (https://www.genenames.org/data/genegroup/#!/group/769). Variants in TTC family proteins have been reported to be causative for various neurological disorders. TTC3, TTC8 or TTC15 variants are linked to familial Alzheimer’s disease,[35] Bardet-Biedl Syndrome,[36] or encephalopathy,[37] respectively. TTC19 mutations are reported in patients with mitochondrial complex III deficiency, nuclear type 2 [MIM: 615157],[38] in which individuals have developmental delay, hypotonia, ataxia, dysarthria, dystonia, spasticity and cognitive impairment.

Protein domain interaction studies for TTC5, p300, JMY, and p53 have demonstrated that the six TPR protein-binding motifs and OB fold of TTC5 are critical for JMY and p300 associations, respectively. Loss of TPR protein-binding motifs 1–3 or 3–5 abolishes association with JMY, whereas TPR protein-binding motifs 5, 6, and the OB fold are required for p300 interaction.[13, 39] The p.(Tyr210Cys) and p.(Ala231Val) missense variants identified in this study occur within the tetratricopeptide-like helical structure of the TPR protein-binding motif 5 and 6, respectively, which are important for protein-protein interaction via the formation of anti–parallel α–helices.[40] These helices together form a super helix structure that constitutes a ligand binding pocket.[41] TPR protein-binding domain 5 is comprised of two alpha-helices of 17 residues each and harbors two phosphorylation sites that are essential for intramolecular interaction of TRP protein-binding motifs 5 and 6.[39] Phosphorylation of serine 203 by ataxia telangiectasia (ATM) and serine 221 by checkpoint kinase 2 (CHK2) stabilizes TTC5 in the nucleus to increase levels and histone acetylation activity of the p300 co-activator complex.[42, 43] Since amino acids Tyr210 and Ala231 are highly conserved and adjacent to these phosphorylation sites, we hypothesize that these residues may play a critical role in TTC5 function (figure 3D). Perhaps, p.(Tyr210Cys) or p.(Ala231Val) impairs TTC5-JMY/p300 interactions or TTC5 protein folding and stability. Western blot and co-immunoprecipitation analyses from patient derived cell lines, if available, could distinguish between these possibilities.

Premature termination codons resulting from nonsense mutations typically lead to transcript degradation by the cellular process of nonsense-mediated decay (NMD), thereby reducing overall abundance of the target mRNA.[44] However, various factors influence the efficiency of NMD, including the location of the premature termination codon within long or ultimate exons and the proximity of the variant to an intron.[45, 46] Since the homozygous nonsense variant p.(Arg263Ter) is located in TTC5 exon 7 of 10, we predict the variant will likely elicit nonsense-mediated decay. In contrast, nonsense variant p.(Arg395Ter) is located in TTC5 exon 9 of 10, 18 base pairs from the terminal intron (figure 3B). Experimental evidence has shown premature termination codons that occur less than 50–55 base pairs from the penultimate exon splice donor site might not cause NMD,[46, 47] therefore, p.(Arg395Ter) could result in a truncated TTC5 protein lacking the complete OB fold domain. However, we found a marked reduction in TTC5 expression in patients with p.(Arg395Ter) suggesting this variant diminishes levels of TTC5, consistent with NMD.

Thus, following genetic and molecular analysis, all affected patients from the families described herein were diagnosed with TTC5-associated intellectual disability syndrome, an autosomal recessive syndrome caused by loss-of-function variants in TTC5. The four novel homozygous variants in TTC5 lead to delayed developmental milestones and intellectual disability with or without microcephaly. It is challenging to distinguish intellectual disability syndromes from one another, since many share overlapping and non-specific symptoms. Our work, together with a prior study,[17] enables us to define to-date, the cornerstone clinical features of TTC5-associated intellectual disability syndrome as severe speech impairment, cerebral atrophy and hypotonia that can be used to prioritize this syndrome for diagnosis. We find patients with bi-allelic variants present with moderate to severe intellectual disability, delayed motor and verbal milestones, hypotonia, and a thin, often v-shaped, upper lip. Shared radiological findings observed in more than one family include corpus callosum agenesis, mild ventriculomegaly, abnormal gyri, and cerebral atrophy. Variable clinical features include autistic and aggressive behavior, microcephaly, wide internipple distance, and dysmorphic facial features including flared or arched eyebrows, and low-set ears with abnormalities. Variable radiological findings of gliosis, white matter changes, and mild leukodystrophy may be present. While core clinical features of the disorder are highly consistent, phenotypes unique to families or individual patients, such as autistic behavior and dry acneiform lesions (CW07-A, B), genitourinary abnormalities (RDHM-05A and 5543-A), epilepsy (5377-A), kyphosis (5377-A), and dilated paranasal and straight sinus areas (CW07-A, B), are likely attributed to pathogenic variants in other genes. However, we cannot rule out the possibility that TTC5 variants may contribute to these phenotypes.

Patients with TTC5-associated IDS share clinical features with Rubinstein-Taybi syndrome type 2 affected individuals harboring EP300 variants, indicating the same molecular mechanism may be driving these similarities, namely reduced p300 co-activator complex activity. In our cohort of TTC5-associated IDS patients, microcephaly is present 50% of the time, while in the EP300-associated Rubinstein-Taybi syndrome type 2, this rate varies between 87–100%.[48–51] In addition to this feature, low hanging columella (HP:0009765) (92–100%), broad thumbs (69–87.5%) long eyelashes (HP:0000527) (90%), a large beaked nose (HP:0003683) (81%), and distinct facial grimacing’ (HP:0000273) (47%) are observed in patients with Rubinstein-Taybi syndrome type 2. %.[48–51] We did not find these features in our TTC5-associated IDS cohort. Furthermore, while normal IQ levels can be observed in Rubinstein-Taybi syndrome type 2, all patients with TTC5-associated IDS have moderate to severe ID. [5] Lastly, TTC5-associated IDS is inherited in an autosomal recessive pattern, with parents as carriers. Rubinstein-Taybi syndrome type 2 shows an autosomal dominant inheritance pattern and all patients identified to date have de novo variants, except one individual. [48–51]

The phenotypic differences between patients with Rubinstein-Taybi syndrome type 2 and TTC5-associated IDS may be due to a limited requirement for TTC5 during developmental periods of cell stress, while EP300 appears less restricted.[13, 14, 52, 53] Alternatively, the clinical variation observed among these different IDSs could be attributed to the additional functions of TTC5 in autophagy regulation and tubulin autoregulation in the cytoplasm, a cellular process unassociated with p300.[15],[16] Taken together, the diverse clinical features observed in TTC5-associated intellectual disability syndrome patients involve multiple organ systems and suggest a critical role of TTC5 for disparate cellular processes. Our findings establish TTC5 as a disease associated gene and delineate recurrent and variable features of this novel intellectual disability syndrome.

DATA AVAILABILITY STATEMENT

All data is available under reasonable request from the corresponding authors. The accession numbers for the TTC5 variants reported in this paper are: LOVD:0000602976 LOVD:0000604184 and dbGap:phs000288.v1.p1