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. Author manuscript; available in PMC: 2024 Jun 4.
Published in final edited form as: Am J Med Genet A. 2022 Feb 23;188(6):1868–1874. doi: 10.1002/ajmg.a.62704

PRUNE1 c.933G>A synonymous variant induces exon 7 skipping, disrupts the DHHA2 domain, and leads to an atypical NMIHBA syndrome presentation: Case report and review of the literature

Christina L Magyar 1,2,3,4, David R Murdock 5, Lindsay C Burrage 5, Hongzheng Dai 5, Seema R Lalani 5, Richard A Lewis 5,6, Yuezhen Lin 7, Marcela F Astudillo 7, Jill A Rosenfeld 5, Alyssa A Tran 5, James B Gibson 8; Undiagnosed Diseases Network, Carlos A Bacino 5, Brendan H Lee 5, Hsiao-Tuan Chao 3,4,5,7,9,10
PMCID: PMC11149102  NIHMSID: NIHMS1997271  PMID: 35194938

Abstract

Prune exopolyphosphatase-1 (PRUNE1) encodes a member of the DHH (aspartic acid-histidine-histidine) phosphodiesterase superfamily that regulates cell migration and proliferation during brain development. In 2015, biallelic PRUNE1 loss-of-function variants were identified to cause the Neurodevelopmental Disorder with Microcephaly, Hypotonia, and Variable Brain Abnormalities (NMIHBA, OMIM#617481). NMIHBA is characterized by the namesake features and structural brain anomalies including thinning of the corpus callosum, cerebral and cerebellar atrophy, and delayed myelination. To date 47 individuals have been reported in the literature, but the phenotypic spectrum of PRUNE1-related disorders and their causative variants remains to be characterized fully. Here, we report a novel homozygous PRUNE1 NM_021222.2:c.933G>A synonymous variant identified in a six-year-old male with intellectual and developmental disabilities, hypotonia, and spastic diplegia, but with the absence of microcephaly, brain anomalies, or seizures. Fibroblast RNA-sequencing revealed that the PRUNE1 NM_021222.1:c.933G>A variant resulted in an in-frame skipping of the penultimate exon 7, removing 53 amino acids from an important protein domain. This case represents the first synonymous variant and the third pathogenic variant known to date affecting the DHH-associated domain (DHHA2 domain). These findings extend the genotypic and phenotypic spectrums in PRUNE1-related disorders and highlight the importance of considering synonymous splice site variants in atypical presentations.

Keywords: neurodevelopmental disorder, hypotonia, spastic paraparesis, splice site variant, alternative splicing

Introduction.

Prune exopolyphosphatase-1 (PRUNE1) encodes a member of the DHH (aspartic acid-histidine-histidine) phosphodiesterase superfamily containing aspartic-acid-histidine-histidine (DHH) and DHH-associated (DHHA2) domains (Nistala et al., 2021). PRUNE1 is expressed in both nervous system and non-nervous system tissues and was found to be highly expressed during embryonic nervous system development in mice (Karaca et al., 2015; Nistala et al., 2021; Reymond et al., 1999; Uhlen et al., 2015). Prior studies reveal that PRUNE1 interacts with β-tubulin, nucleoside diphosphate kinase A, and glycogen synthase kinase 3 beta (GSK-3β) to promote cell proliferation, migration, and differentiation (Aravind & Koonin, 1998; Carotenuto et al., 2006; D’Angelo & Zollo, 2004; Hur & Zhou, 2010; Middelhaufe et al., 2007; Reymond et al., 1999; Zollo et al., 2017). The complete genetic ablation of Prune1 in mice results in mid-gestational lethality due to disrupted growth and vascular development (Nistala et al., 2021). However, the neurological consequences of Prune1 loss-of-function in mice remains to be elucidated.

The disease association of PRUNE1 variants with a human condition was initially identified in 2015 when exome sequencing (ES) studies identified biallelic PRUNE1 variants in five individuals from four unrelated families with delayed development and intellectual disability (DD/ID), microcephaly, cortical atrophy, and cerebellar atrophy (Karaca et al., 2015). Two of the individuals were non-verbal and non-ambulatory with axial hypotonia, spastic quadriparesis, and hyperreflexia (Karaca et al., 2015; Zollo et al., 2017). This led to the recognition of the Neurodevelopmental Disorder with Microcephaly, Hypotonia, and Variable Brain Abnormalities (NMIHBA, OMIM#617481). To date, 47 individuals are reported in the literature, of which 40 individuals have sequence confirmation of PRUNE1 variants and phenotypic information is available for review (Table S1). Prominent features of NMIHBA include congenital or acquired microcephaly defined as occipito-frontal circumference (OFC) smaller than −2 standard deviations (SD) or 5th centile, DD/ID, hypotonia, and spastic quadriparesis. Brain anomalies are common, including cerebral and cerebellar atrophy, thinning of the corpus callosum, and white matter abnormalities. Epilepsy, ophthalmologic findings, gastrointestinal complications, and peripheral neuropathy are other features (Karaca et al., 2015; Zollo et al., 2017).

Here, we present a novel homozygous synonymous PRUNE1 NM_021222.1:c.933G>A variant identified in a six-year-old male with DD/ID, central hypotonia, and spastic paraparesis, but without microcephaly, structural brain anomalies, or seizures. These findings extend the genotype-phenotype spectrum in PRUNE1-related NMIHBA syndrome and highlight the importance of considering synonymous variants affecting important functional domains in atypical presentations.

Case Presentation.

This six-year-old male of Pakistani descent was born in the United States at 41 weeks gestational age to a 27-year-old mother (gravidity three, parity two) and 40-year-old father. Family history was significant for consanguinity, infantile demise in six siblings of the maternal grandfather, and maternal bilateral hearing loss (Fig. 1a). The pregnancy was complicated by hyperemesis gravidarum. Prenatal ultrasounds were unremarkable and delivery was uncomplicated. Birth weight was 3.31 kg (−0.1SD), length was 50 cm (+0.06SD), and OFC at birth was 35.5 cm (−0.18SD), which is at the 43rd centile. Maternal OFC was 54.5 cm (+0.18SD), and paternal OFC was 55.5 cm (+0.28SD). Neonatal course was notable for reduced vocalizations and activity.

Figure 1: Pedigree, brain MRI images, physical features, and genetic findings.

Figure 1:

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a: Proband family pedigree displaying consanguineous union and family history of early infantile demise. b: Physical characteristics demonstrating light hair color with coarse texture, hypotonic facies, mild midface hypoplasia, and widely spaced teeth. c: T1-weighted (T1W) MRI brain imaging of proband at 20 months of age i, ii) axial image showing no gross structural brain anomalies, iii) sagittal image with no structural anomalies, iv) sagittal imaging of 24-month-old neurotypical control. d: Trio exome sequencing results showing homozygous PRUNE1 c.933G>A variant. e: Fibroblast RNA-sequencing shows penultimate exon 7 skipping. The mis-spliced transcript escapes NMD and results in Prune1 p.Ala259_Thr311del that is predicted to disrupt the DHHA2 domain. f: i) position of PRUNE1 variants within the linear protein structure showing enrichment in the DHH domain (NP_067045.1), ii) position of PRUNE1 variants within the genomic sequence (NC_000001.10). The 53 amino acid region that would be lost from the exon 7 skipping is marked by the orange star.

Delayed development was identified at eight months of age with the inability to sit without support and poor head control. At 18 months of age, he was able to crawl on his belly, roll over, perform fine motor tasks including finger feeding, pincer grasp, object stacking, scribbling, and object transfer between hands. By three years of age, neurologic decline was apparent with progressive loss of acquired gross motor skills, dysphagia, and bradykinesia. At six years of age, he was unable to sit without support, lost purposeful hand use, and required gastrostomy tube placement. He remained non-verbal but was able to engage socially with good eye contact. Magnetic resonance imaging (MRI) of the brain and spinal cord obtained at two years (Fig. 1b) and 4.5 years of age revealed age-appropriate neuroanatomy. Metabolic studies including plasma amino acids, very long chain fatty acids, creatine kinase, lactate, carbohydrate deficient transferrin, and cerebrospinal fluid studies were normal. Thyroid-stimulating hormone (TSH) was elevated at 6.4 mIU/L but normalized to 2.4 mIU/L on subsequent testing.

He was enrolled in the Undiagnosed Diseases Network (UDN) at six-years old (Ramoni et al., 2017). Clinical data were obtained after written informed consent, and the study protocol was approved by the institutional review board at the National Institutes of Health. His weight was 15 kg (−2.51SD), height was 101.8 cm (−2.68SD), and OFC was 49.5 cm (−1.50SD, 7th centile). Examination was notable for coarse hair, hypotonic facies, mild midface hypoplasia, widely spaced teeth (Fig. 1c), hypotonia, spastic paraparesis, and hyperreflexia. He was non-verbal and non-ambulatory. Serum studies including lactate, copper, ceruloplasmin, free T4, lipids, insulin growth factor (IGF)-1, IGF-binding protein-3, and vitamin D-25-hydroxy were normal.

Genetic analysis.

Fragile X, SLC16A2 sequencing and karyotype analysis were unremarkable. High resolution chromosomal microarray (CMA) including single nucleotide polymorphism (SNP) analysis from peripheral blood did not reveal any copy number variants associated with known syndromes. However, the CMA identified regions of copy-neutral absence of heterozygosity (AOH) greater than 7 Mb on multiple chromosomes with a total AOH estimate of 84 Mb, which is consistent with consanguinity. A clinical trio ES had been performed on peripheral-blood derived DNA in which the DNA regions were sequenced using the Illumina HiSeq 2000 system. DNA was mapped to and analyzed in comparison to the reference human genome (GRCh37). The analysis identified a heterozygous maternally inherited EXOSC3 c.395A>C (p.Asp132Ala) variant, which is a known pathogenic variant associated with autosomal recessive pontocerebellar hypoplasia type 1B (OMIM#614678). However, a second EXOSC3 variant was not identified and the features were discordant.

The UDN performed a trio ES analysis and identified a homozygous PRUNE1 NM_021222.2: c.933G>A variant predicted to result in a synonymous p.Thr311= (Fig. 1d). This variant is located within his previously identified 19 Mb AOH region on chr1q21.1q23.3. Sanger sequencing identified that both parents were heterozygous for the c.933G>A variant. This PRUNE1 c.933G>A variant is present in the Genome Aggregation Database (gnomAD) (Karczewski et al., 2020) with four alleles observed out of 251,078 and minor allele frequency (MAF) of 1.593 × 10−5. There are no homozygotes present in gnomAD for this variant. Variant effect predictions were determined by the Combined Annotation Dependent Depletion (CADD) score for single nucleotide variants and insertions/deletions variants, and the Genomic Evolutionarily Rate Profiling (GERP) score for determining sequence conservation across multiple species (Davydov et al., 2010; Kircher et al., 2014; Rentzsch, Schubach, Shendure, & Kircher, 2021). The c.933G>A variant has a CADD score of 18.10 and a GERP score of 0.168, which suggests low deleterious potential. Fibroblast RNA-sequencing was performed as previously described (Murdock et al., 2021) and revealed that the PRUNE1 c.933G>A variant generated an in-frame skipping of exon 7 without evidence of nonsense-mediated decay (NMD) (Fig. 1e). Eight alternative mRNA splice isoform transcripts have been identified for PRUNE1, but the predominant isoforms contain exon 7 (Fig. 1e). Although PRUNE1’s reading frame was preserved, the exon 7 skipping is predicted to remove 53 amino acids (p.Ala259_Thr311del) from the DHHA2 domain (Fig. 1f).

Discussion.

NMIHBA syndrome has been reported to be caused by bi-allelic or homozygous PRUNE1 missense variants in the DHH domain, frameshift variants, nonsense variants, homozygous microdeletions, or a homozygous PRUNE1 splice site variant (c.521–2A>G) (Table S1) (Alfadhel et al., 2018; Alhaddad et al., 2018; Costain et al., 2017; Fujii et al., 2020; Hartley, Simard, Ly, Del Bigio, & Frosk, 2019; Iacomino et al., 2018; Imagawa et al., 2018; Karaca et al., 2015; Karakaya et al., 2017; Nistala et al., 2021; Okur et al., 2019; Papuc et al., 2019; Zollo et al., 2017). These findings suggest that NMIHBA syndrome is due to partial or complete loss of PRUNE1 function. PRUNE1-related NMIHBA syndrome is reported in a wide range of ethnicities, show no sexual predominance, and occur equally among the offspring of both consanguineous and non-consanguineous unions, although this is due largely to the influence of founder effects (Fig. S1a). (Costain et al., 2017; Hartley, Simard, Ly, Del bigio, & Frosk, 2019)

Although the full phenotypic spectrum remains to be elucidated, atypical presentations have been reported that may be related to the type and severity of the PRUNE1 variant. These atypical features include spinal motor neuron involvement, muscle atrophy, elevated creatine kinase (Hartley et al., 2019; Iacomino et al., 2018; Karakaya et al., 2017; Okur et al., 2019), developmental regression, and the absence of absolute microcephaly (Table 1, Fig. S1b–c) (Alhaddad et al., 2018; Costain et al., 2017). Individuals with variants affecting the DHHA2 domain (c.874_875InsA, c.889C>T, and c.933G>A) or disrupting splicing (c.521–2A>G and c.933G>A) are less likely to exhibit absolute microcephaly, but may have clonus and abnormal muscle biopsies (Fig. S1c). However, developmental delay, axial hypotonia, peripheral spasticity, and non-verbal status were present in all patients regardless of PRUNE1 variant type (Fig. S1c). Structural brain, musculoskeletal, and eye abnormalities were the next most common features reported in affected individuals (Fig. S1d–e). Together, our findings reveal that PRUNE1 variants are associated with a heterogenous phenotypic spectrum. Pathogenic PRUNE1 variants disrupting the DHHA2 domain or intronic and synonymous single nucleotide variants (SNV) perturbing splicing are often found to be associated with various atypical clinical features (Fig. S1c).

Table 1:

Neurologic characteristics of proband compared to previously reported cases of PRUNE1 pathogenic variants and the corresponding protein domains

Feature PRUNE1 c.933G>A (p.Ala259_Thr311del)
(Proband)		 PRUNE1 c.889C>T (p.Arg297Trp) PRUNE1 c.874_875insA (p.His292Glnfs*3) PRUNE1 c.521–2A>G PRUNE1 variants outside of DHHA2
Zygosity Homozygous Homozygous Homozygous Homozygous Homozygous or Bi-allelic
Variant type(s) Synonymous Missense Frameshift Splice acceptor Missense; Nonsense; Frameshift; CNV
Located in exon 7 Yes Yes Yes No No
Corresponding protein domain DHHA2 DHHA2 DHHA2 DHH DHH
Number of reported individuals 1 2 1 10 27
Consanguinity 1/1 2/2 1/1 0/10 18/27
Microcephaly 0/1 2/2 1/1 0/10 26/27
Axial hypotonia 1/1 2/2 1/1 10/10 25/25
Spastic quadriparesis 0/1 2/2 1/1 7/10 25/25
Spastic paraparesis 1/1 0/2 0/1 0/10 0/12
Delayed development 1/1 2/2 1/1 10/10 27/27
Non-ambulatory 1/1 2/2 1/1 10/10 27/27
Non-verbal 1/1 2/2 1/1 10/10 27/27
Seizures 0/1 0/2 1/1 8/10 17/27
Abnormal EEG n/a 0/2 n/a 2/3 10/11
Abnormal muscle biopsy n/a 0/2 n/a 6/7 8/10
Brain anomalies 0/1 2/2 1/1 8/10 27/27
Delayed myelination 0/1 0/2 1/1 3/10 12/23
Thin corpus callosum 0/1 0/2 0/1 2/10 9/21
Cerebral atrophy 0/1 0/2 1/1 7/10 17/25
Cerebellar atrophy 0/1 0/2 1/1 1/10 13/25
White matter disease 0/1 2/2 1/1 3/10 7/23
Associated studies Current (Zollo et al., 2017) (Karakaya et al., 2017) (Costain et al., 2017; Hartley et al., 2019) Table S1
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Abbreviations: EEG (electroencephalogram), n/a (not available), DHH (aspartic acid-histidine-histidine) domain, DHHA2 (DHH associated) domain

Synonymous SNVs are often predicted to be non-deleterious, but increasing evidence demonstrate the pathogenic role of synonymous variants in human disease through disruption of a wide-range of transcriptional and translational processes (Hunt, Simhadri, Iandoli, Sauna, & Kimchi-Sarfaty, 2014; Spencer, Siller, Anderson, & Barral, 2012). Single nucleotide substitutions within the intronic or exonic segments of the splice site can result in alternative splicing events that lead to either NMD or generation of a dysfunctional protein (Faustino & Cooper, 2003; Maquat & Carmichael, 2001; Maquat & Serin, 2001).

In conclusion, we report the case of a male with a homozygous PRUNE1 c.933G>A variant leading to an in-frame skipping of the penultimate exon. This event would remove part of the DHHA2 domain, which may contribute to the atypical presentation compared to PRUNE1 variants affecting the DHH domain (Table 1). To the best of our knowledge, this is the first synonymous variant and the third variant known to date affecting the DHHA2 domain in NMIBHA (Table 1, Fig. 1f). Our case further expands the understanding of the phenotypic heterogeneity associated with NMIHBA and highlights the utility of fibroblast transcriptomic analysis when a causative synonymous variant is suspected.

Supplementary Material

Figure S1

Figure S1: Demographics, PRUNE1 variant frequencies, and clinical features a: Multiple ethnicities are affected by PRUNE1 disorders and no sexually dimorphic preference is observed. Affected individuals are found both within consanguineous and non-consanguineous unions. b: Regression is noted in almost 1/3 of individuals with PRUNE1 syndromes, particularly among individuals with splice and frameshift variants. c: i) c.521–2A>G and c.316G>A variants are the most reported variants, and only 10% of variants are predicted to affect the DHHA2 domain. ii) Splice and frameshift variants are associated with more atypical features such as abnormal muscle biopsy, tongue fasciculations, and absence of microcephaly. d: Cerebral and cerebellar atrophy, as well as delayed myelination are the most common structural brain abnormalities noted by magnetic resonance imaging (MRI). e: Musculoskeletal abnormalities are reported in nearly 50% of affected individuals, while eye abnormalities are noted in nearly 30% of individuals with an identified PRUNE1-related disorder.

Table S1

Table S1: Summary of molecular and clinical findings associated with identified PRUNE1 causative variants Meta-analysis of clinical and molecular findings in 40 affected individuals with sequence confirmation of PRUNE1 variants and phenotypes reported in the literature. Findings are compared to our study proband for a total analysis of 41 affected individuals. ABBREVIATIONS: years (yr), month (mo), male (M), female (F), not available (n/a), magnetic resonance imaging (MRI), electroencephalogram (EEG)

Table S2

Table S2: Undiagnosed Diseases Network Member Affiliations and Contact Info

Acknowledgements.

We thank our patient, his family, and the clinical staff at the Undiagnosed Diseases Network, Texas Children’s Hospital, and Baylor College of Medicine (BCM) for participating in this study.

Research Funding.

Research reported in this manuscript was supported by the NIH Common Fund, through the Office of Strategic Coordination/Office of the NIH Director under Award Number(s) U01HG007709 and U01HG007942. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. C.L.M. and H.T.C. are supported by The Robert and Janice McNair Foundation. D.R.M, L.B., H.D., S.L., R.L., J.A.R., M.A., C.A.B., B.H.L., and H.T.C. are supported by the NIH grant U01HG007709. H.T.C.’s research efforts are also supported by the Burroughs Wellcome Fund, Child Neurology Society and Foundation, Wallace Endowment Award, The Gordon and Mary Cain Foundation, Annie and Bob Graham, and NIH DP5OD026428.

Conflict of Interest.

Funding organization(s) played no role in the study design, data collection, analysis, and interpretation; writing; or the decision to submit the report. The Department of Molecular and Human Genetics at BCM derives revenue from Baylor Genetics Laboratory genetic testing. D.R.M has consulted for Illumina and is currently employed by Invitae.

Footnotes

Supplemental Materials. One supplemental figure and two supplemental tables.

Data Availability.

Raw data were generated at Ambry Genetics and Baylor College of Medicine’s Laboratory for Translational Genomics. Derived data supporting the findings of this study are available from the corresponding author (H.T.C.) on request. The accession number for the variant reported to ClinVar is SCV001432740.1: NM_021222.3(PRUNE1):c.933G>A(p.Thr311=).

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Associated Data

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

Supplementary Materials

Figure S1

Figure S1: Demographics, PRUNE1 variant frequencies, and clinical features a: Multiple ethnicities are affected by PRUNE1 disorders and no sexually dimorphic preference is observed. Affected individuals are found both within consanguineous and non-consanguineous unions. b: Regression is noted in almost 1/3 of individuals with PRUNE1 syndromes, particularly among individuals with splice and frameshift variants. c: i) c.521–2A>G and c.316G>A variants are the most reported variants, and only 10% of variants are predicted to affect the DHHA2 domain. ii) Splice and frameshift variants are associated with more atypical features such as abnormal muscle biopsy, tongue fasciculations, and absence of microcephaly. d: Cerebral and cerebellar atrophy, as well as delayed myelination are the most common structural brain abnormalities noted by magnetic resonance imaging (MRI). e: Musculoskeletal abnormalities are reported in nearly 50% of affected individuals, while eye abnormalities are noted in nearly 30% of individuals with an identified PRUNE1-related disorder.

NIHMS1997271-supplement-Figure_S1.tif (3.5MB, tif)
Table S1

Table S1: Summary of molecular and clinical findings associated with identified PRUNE1 causative variants Meta-analysis of clinical and molecular findings in 40 affected individuals with sequence confirmation of PRUNE1 variants and phenotypes reported in the literature. Findings are compared to our study proband for a total analysis of 41 affected individuals. ABBREVIATIONS: years (yr), month (mo), male (M), female (F), not available (n/a), magnetic resonance imaging (MRI), electroencephalogram (EEG)

NIHMS1997271-supplement-Table_S1.xlsx (29.1KB, xlsx)
Table S2

Table S2: Undiagnosed Diseases Network Member Affiliations and Contact Info

NIHMS1997271-supplement-Table_S2.xlsx (34KB, xlsx)

Data Availability Statement

Raw data were generated at Ambry Genetics and Baylor College of Medicine’s Laboratory for Translational Genomics. Derived data supporting the findings of this study are available from the corresponding author (H.T.C.) on request. The accession number for the variant reported to ClinVar is SCV001432740.1: NM_021222.3(PRUNE1):c.933G>A(p.Thr311=).

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