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. Author manuscript; available in PMC: 2023 Apr 1.
Published in final edited form as: Hum Mutat. 2022 Jan 30;43(4):461–470. doi: 10.1002/humu.24332

Delineation of a novel neurodevelopmental syndrome associated with PAX5 haploinsufficiency

Yoel Gofin 1,2, Tianyun Wang 3, Madelyn A Gillentine 3,4, Tiana M Scott 5, Aliska M Berry 1, Mahshid S Azamian 1,2, Casie Genetti 6, Pankaj B Agrawal 6,7, Jonathan Picker 6, Monica H Wojcik 7,8,9, Mauricio R Delgado 10,11, Sally Ann Lynch 12, Stephen W Scherer 13,14, Jennifer L Howe 13, Carlos A Bacino 1,2, Stephanie DiTroia 7,9,15, Grace E VanNoy 9,15, Anne O’Donnell-Luria 7,9,15, Seema R Lalani 1,2, William D Graf 16, Jill A Rosenfeld 1,17, Evan E Eichler 3,18, Rachel K Earl 19,20,21, Daryl A Scott 1,2,22,*
PMCID: PMC8960338  NIHMSID: NIHMS1772796  PMID: 35094443

Abstract

PAX5 is a transcription factor associated with abnormal posterior midbrain and cerebellum development in mice. PAX5 is highly loss-of-function intolerant and missense constrained and has been identified as a candidate gene for autism spectrum disorder (ASD). We describe 16 individuals from 12 families who carry deletions involving PAX5 and surrounding genes, de novo frameshift variants that are likely to trigger nonsense-mediated mRNA decay, a rare stop-gain variant, or missense variants that affect conserved amino acid residues. Four of these individuals were published previously but without detailed clinical descriptions. All these individuals have been diagnosed with one or more neurodevelopmental phenotypes including delayed developmental milestones (DD), intellectual disability (ID) and/or ASD. Seizures were documented in four individuals. No recurrent patterns of brain MRI findings, structural birth defects, or dysmorphic features were observed. Our findings suggest that PAX5 haploinsufficiency causes a neurodevelopmental disorder whose cardinal features include DD, variable ID, and/or ASD.

Keywords: PAX5, developmental delay, intellectual disability, autism spectrum disorder, seizures

The human Paired Box (PAX) family of transcription factors has nine members whose characteristic features include a conserved N-terminal DNA-binding motif, the paired domain, which was originally described in the Drosophila protein Paired (Underhill, 2000). All nine PAX genes have been associated with a human disease or disease susceptibility. Currently, the only disease association known for PAX5—also known as B-cell lineage-specific activator protein—is susceptibility to acute lymphoid leukemia (ALL) [MIM# 615545] (Gu et al., 2019; Mullighan et al., 2007; Passet et al., 2019; Shah et al., 2013).

During mice embryogenesis, PAX5 is transiently expressed in the mesencephalon and spinal cord with a spatial and temporal expression pattern that is distinct from that of other PAX genes (Adams et al., 1992). Pax5−/− mice have abnormal posterior midbrain and cerebellum development (Bouchard, Pfeffer, & Busslinger, 2000; Reimer, Urbánek, Busslinger, & Ehret, 1996; Urbánek, Wang, Fetka, Wagner, & Busslinger, 1994) and conditional ablation of Pax5 in GABAergic neurons causes marked enlargement of the lateral ventricles (Ohtsuka et al., 2013). PAX2/5 deficient mice have a missing mesencephalon/metencephalon suggesting that these genes play an important role in defining the midbrain vesicle (Schwarz et al., 1999). Taken together, these studies suggest that PAX5 plays a key role in normal murine CNS development.

PAX5 is highly conserved between humans and mice (Adams et al., 1992). In humans, PAX5 transcripts have been shown to be significantly downregulated in bipolar disorder postmortem hippocampal extracts (Benes et al., 2007). More recently, PAX5 was identified as a candidate gene for autism spectrum disorder (ASD) in large sequencing studies (Feliciano et al., 2019; Iossifov et al., 2012; O’Roak et al., 2014; Satterstrom et al., 2020; Stessman et al., 2017; Turner , Coe, et al., 2017; Werling et al., 2018; Wilfert et al., 2021; C Yuen et al., 2017).

Data from the Genome Aggregation Database (gnomAD v2.1.1; https://gnomad.broadinstitute.org/) suggests that PAX5 is likely to be loss-of-function intolerant (pLI = 1) with 18.1 loss-of-function variants expected but none observed (observed [o]/expected [e] ratio = 0; 90% confidence interval = 0-0.17) (Karczewski et al., 2020). PAX5 is also missense constrained (o/e = 247.6/139 = 0.56, 90% confidence interval = 0.49-0.65; Z = 2.45), but no statistical differences are seen between the expected and observed numbers of synonymous variants (o/e = 105.9/112 = 1.06, 90% confidence interval = 0.91-1.24; Z = −0.47), indicating the model performs well for this gene. PAX5's autosomal dominant gene variation intolerance rank (GeVIR AD; http://www.gevirank.org), a continuous gene-level metric that uses the distribution pattern of variants within a gene to prioritize autosomal dominant disease candidate genes, is high (3.56, 5.01%) (Abramovs, Brass, & Tassabehji, 2020). Similarly, PAX5’s LOEUF AD rank, GeVIR’s metric for loss-of function variance tolerance, is also high (3.35; 3.64%). Taken together, these parameters suggest that PAX5 haploinsufficiency is likely to contribute to human disease.

To identify phenotypes potentially associated with PAX5 haploinsufficiency, we searched clinical and public databases for individuals who carried deletions involving PAX5. Subjects 1-5 were found to carry deletions that include all of PAX5 as well as surrounding genes (Table 1A; Figure 1A; Supp. Mat.—Clinical Summaries). All of these individuals underwent array-based copy number variant (CNV) analysis for neurodevelopmental phenotypes that included delayed developmental milestones (DD), intellectual disability (ID) and/or ASD. Subject 4 also had a history of seizures. Subjects 1-3 are full siblings with Subjects 2 and 3 being fraternal twins.

Table 1A:

Subjects with deletions involving PAX5

Subject 1 Subject 2 Subject 3 Subject 4 Subject 5
Alternative ID N/A N/A N/A N/A DECIPHER 323105
Sex Male Male Male Male Male
Age at Last Assessment 14 y 20 y 20 y 17 y 8 y
Indication(s) for CNV Analysis ASD ASD ASD DD, ID, ASD, seizures DD, ID, normal brain MRI
Deletion (hg19) chr9:36,426,622-38,787,479 chr9:36,426,622-38,787,479 chr9:36,442,195-39,156,958 (min); 36,419,493-40,774,118 (max) chr9:36,088,563-39,092,820; de novo chr9:35,059,633-37,660,586
Approximate Size (Mb) 2.4 2.4 2.7-4.4 3.0 2.6
Protein-Coding Genes Affected MELK, PAX5, ZCCHC7, GRHPR, ZBTB5, POLR1E, FBXO10, TOMM5, FRMPD1, TRMT10B. EXOSC3, DCAF10, SLC25A51, SHB, ALDH1B1, IGFBPL1, ANKRD18A MELK, PAX5, ZCCHC7, GRHPR, ZBTB5, POLR1E, FBXO10, TOMM5, FRMPD1, TRMT10B. EXOSC3, DCAF10, SLC25A51, SHB, ALDH1B1, IGFBPL1, ANKRD18A MELK, PAX5, ZCCHC7, GRHPR, ZBTB5, POLR1E, FBXO10, TOMM5, FRMPD1, TRMT10B. EXOSC3, DCAF10, SLC25A51, SHB, ALDH1B1, IGFBPL1, ANKRD18A, CNTNAP3, SPATA31A1, CNTNAP3C, SPATA31A2, LOC100653002, SPATA31A3, ZNF658 RECK, GLIPR2, CCIN, CLTA, GNE, RNF38, MELK, PAX5, ZCCHC7, GRHPR, ZBTB5, POLR1E, FBXO10, TOMM5, FRMPD1, TRMT10B. EXOSC3, DCAF10, SLC25A51, SHB, ALDH1B1, IGFBPL1, ANKRD18A, CNTNAP3 VCP, FANCG, PIGO, STOML2, FAM214B, UNC13B, LOC100509263, RUSC2, FAM166B, TESK1, CD72, SIT1, ARHGEF39, CA9, TPM2, TLN1, CREB3, GBA2, RGP1, MSMP, NPR2, SPAG8, HINT2, FAM221B, TMEM8B, OR13J1, HRCT1, SPAAR, OR2S2, RECK, GLIPR2, CCIN, CLTA, GNE, RNF38, MELK, PAX5, ZCCHC7, GRHPR, ZBTB5, POLR1E, FBXO10, TOMM5, FRMPD1,
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ASD = autism spectrum disorder, DD = delayed developmental milestones, ID = intellectual disability, y = year

Figure 1: PAX5 copy number and sequence variants.

Figure 1:

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A) Schematic showing the location of deletions seen in Subjects 1-5. Subjects 1-3 are brothers whose deletions were defined clinically. Hence, the differences in the reported deletion sizes between Subjects 1 and 2 and Subject 3 are most likely due to differences in array platforms and/or the reporting practices of the different laboratories. The minimal and maximal deletions for Subject 3 are shown in black and gray, respectively. The minimal region of overlap is delineated by red lines. This region spans ~1.2 Mb (chr9:36,426,622-37,660,586; hg19) and includes PAX5 (red bar), eight other protein-coding genes (MELK, ZCCHC7, GRHPR, ZBTB5, POLRIE, FBXO10, TOMM5, and FRMPD1; blue bars), and three genes that encode microRNAs (hsa-mir-4475, hsa-mir-4540, and hsa-mir-4476; not shown). B) A schematic showing the location at which PAX5 sequence variants have their effects on the PAX5 protein. The location of frameshift variants predicted to trigger nonsense mediated mRNA decay and the stop-gain variant predicted to result in the generation of a truncated protein are shown in red. Missense variants found in Subjects 9-16 are shown in blue. The variant in Subject 9 may also affect splicing. The inherited missense variants reported by Wilfert et al. are shown in green (Wilfert et al., 2021). The germline p.(Gly183Ser) variant reported Shah et al. that segregated with B-ALL in two kindreds is shown in black (Shah et al., 2013). C) The missense variants seen in Subjects 9-16 are rare and affect amino acids that are conserved in vertebrates. Amino acids displayed in red are not conserved between humans and the other species indicated. Amino acids displayed in blue are located in the highly conserved paired domain.

The smallest region of overlap between the deletions found in Subjects 1-5 spans ~1.2 Mb (chr9:36,426,622-37,660,586; hg19). It includes PAX5, eight other protein-coding genes (MELK, ZCCHC7, GRHPR, ZBTB5, POLRIE, FBXO10, TOMM5, FRMPD1), and three genes that encode microRNAs (hsa-mir-4475, hsa-mir-4540, and hsa-mir-4476). Although the haploinsufficiency of PAX5 appears to be the most likely amongst these genes to be deleterious based on its constraint scores (pLI score, o/e ratio, GeVIR AD rank, and LOEUF AD rank, etc.; Supp. Table S1), haploinsufficiency of other genes may have also contributed to the phenotypes of Subjects 1-5.

To determine if haploinsufficiency of PAX5 alone is sufficient to cause neurodevelopmental phenotypes, we searched for individuals with putatively deleterious PAX5 sequence variants (Table 1B, Figure 1B-C, Supp. Mat.—Clinical Summaries). Subjects 6 and 7 carry de novo frameshift variants that are predicted to cause non-sense mediated mRNA decay. Subject 8 carries a stop-gain variant that is likely to generate a truncated protein. Subjects 9-16 carry rare missense variants (gnomAD allele frequencies ≤ 1.2x10−5) that affect amino acid residues that are conserved in vertebrates (Figure 1B, C). The missense variants of Subjects 9-13 affect the highly conserved paired domain. The missense variant of Subject 9 (NM_016734.3 c.46G>C, p.Gly16Arg) is located on the first exon’s last nucleotide and may also have an effect on splicing, with a SpliceAI score of 0.7 for donor loss (Jaganathan et al., 2019). All of these variants have high CADD scores (29.1-38) (Kircher et al., 2014; Rentzsch, Schubach, Shendure, & Kircher, 2021) and are predicted to be “Deleterious” by MutationTaster (https://www.genecascade.org/MutationTaster2021/, 2021 version). Subjects 6, 7, 9 and 12 were previously published without detailed clinical information (C Yuen et al., 2017; Feliciano et al., 2019; O’Roak et al., 2014).

Table 1B:

Subjects with sequence variants in PAX5

Subject 6 Subject 7 Subject 8 Subject 9 Subject 10 Subject 11 Subject 12 Subject 13 Subject 14 Subject 15 Subject 16
Alternative ID Yuen et al., 2017, Subject 1-0255-003 O'Roak et al., 2014, Subject 12858.p1 O'Roak et al. 2014, Subject 220-9860-201 Feliciano et al. 2019, Subject SP0016232
Sex Male Female Female Female Male Male Male Male Female Male Female
Age at last assessment 12 y 4 y 5 y 5 y 9 y 13 y 13 y 24 y 20 m 9 y 7 y
Variant position (chr9, hg19) 37,020,769 37,015,071 36,840,604 37,033,983 37,020,688 37,020,688 37,015,066 37,006,526 36,966,665 36,882,051 36,882,051
cDNA change [NM_016734.3] c.76dupG c.333delC c.1129C>T c.46G>C c.157G>C c.157G>C c.338A>T c.419G>A c.661C>T c.962C>A c.962C>A
Predicted protein change p.(Val26Glyfs*49);
NSMD		 p.(Trp112Glyfs*47);NNSMD p.(Arg377*) p.(Gly16Arg) p.(Asp53His) p.(Asp53His) p.(Gln113Val) p.(Arg140Gln) p.(Arg221Trp) p.(Pro321His) p.(Pro321His)
CADD score N/A N/A 40 38 30 30 31 32 31 29.1 29.1
MutationTaster prediction Deleterious Deleterious Deleterious Deleterious Deleterious Deleterious Deleterious Deleterious Deleterious Deleterious Deleterious
gnomAD allele frequency 0 0 0.00000531 0 0.00000398 0.00000398 0 0 0 0.0000123 0.0000123
Inheritance De novo De novo ? De novo Maternal Maternal De novo De novo De novo ? ?
Developmental delay + + + − + + + − + + +
Intellectual disability − − + − +* + + + N/A + +
Autism spectrum disorder + + + + − + + + N/A − −
Seizures − − − − − − − + + + −
Brain MRI ND ND ND ND Pituitary cyst, arachnoid cyst, mild bilateral under opercularization, T2 hyperintensity of the posterior centrum semi-ovale. ND ND Normal Normal Thin corpus callosum, paucity of periventricular white matter Thin corpus callosum
Other neurological features ADHD, aggression Hypotonia, mild scoliosis Pprofound left-sided sensorineural hearing loss Hypotonia, anxiety, obsessive behavior, sensorineural hearing loss ADHD, distal muscle atrophy in upper and lower extremities, hammer toes and high arches,
EMG = chronic motor-sensory polyneuropathy		 Mild distal muscle atrophy with pes cavus
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N/A = not applicable, ND = not done or not reported, NSMD = nonsense mediated mRNA decay, y = year, m = month, + = phenotype reported, − = negative, ? = not known

*

Not formally tested but has difficulty keeping up with assignments in regular classes

Similar to what was observed in individuals with deletions involving PAX5, Subjects 6-16 all had DD, ID and/or ASD. Subjects 13-15 also had seizures. Subjects 6 and 7 were reported to have normal intelligence (IQ = 80 and 91, respectively), and Subject 9 had a verbal IQ score in the low normal range (VIQ = 72). This suggests that the cognitive effects of deleterious PAX5 variants are variable.

Data from the International Mouse Phenotyping Consortium (IMPC; http://www.mousephenotype.org/) indicate that early adult Pax5+/− mice of both sexes are hyperactive and have abnormal auditory brainstem responses. The majority of our subjects were not reported to have related phenotypes, but Subjects 6 and 15 were noted to have attention deficit hyperactivity disorder (ADHD) and Subject 11 and 13 have sensorineural hearing loss.

No common set of structural birth defects or dysmorphic features were described among individuals in this cohort. Subjects 5, 13, and 14 had normal brain MRIs. In contrast, the brain MRI of Subject 10 revealed a pars intermedia or Rathke cleft pituitary cyst, a left middle cranial fossa arachnoid cyst, mild bilateral under opercularization, and T2 hyperintensity of the posterior centra semi-ovale. The MRIs of Subjects 15 and 16, who are siblings, revealed thinning of the corpus callosum, with Subject 15 also having moderate paucity of periventricular white matter and posterior ventricular gliosis. These subjects also had signs of neuropathy not described in other members of our cohort.

The phenotypic overlap between Subjects 6 and 7, who carry de novo loss-of-function variants predicted to trigger nonsense mediated mRNA decay, and Subjects 8-16, who carry point mutations, suggests that their stop-gain and missense variants are likely to lead to a loss of PAX5 function. However, other modes of action are possible and could account for differences in phenotypic severity and/or the presence of non-recurrent features. No clear genotype/phenotype correlations can be drawn based on the relatively small number of individuals in our cohort.

Recently, Wilfert et al. demonstrated an excess of de novo PAX5 variants in a large cohort of autism trios (n = 15,182) (Wilfert et al., 2021). This analysis included data from Subjects 6, 7, 9 and 12. In a subset of 9,353 families with ASD, 14 private, inherited PAX5 variants were identified (Supp. Table S2). All were missense variants that occurred outside of the paired box domain (Figure 1B). Remarkably, all 14 of these variants were transmitted to siblings with ASD (n = 10,905), and none were transmitted to unaffected siblings (n = 5,269) (nominal p = 0.004, one-sided Fisher’s exact test). These results provide additional evidence of PAX5’s role in the development of ASD.

It is possible that the inherited PAX5 variants presented by Wilfert et al. represent autism susceptibility alleles whose effect on PAX5 function is less severe than that associated with the variants seen in Subjects 1-16. However, it is also possible that their effects are similar. If this was the case, deeper phenotyping may have revealed neurodevelopmental phenotypes in the parents who transmitted the inherited PAX5 alleles—as seen in the mother of Subjects 10 and 11 who only completed the sixth grade and struggled academically. Alternatively, PAX5 haploinsufficiency could be incompletely penetrant with the manifestation of symptoms being dependent on genetic or epigenetic modifiers, environmental exposures, and/or stochastic factors. We also recognize that the inherited variants reported by Wilfert et al. and the variants seen in Subjects 1-16, particularly the stop-gain and missense variants seen in Subjects 8-16, may have variable effects on PAX5 function ranging from partial to complete loss of function with associated differences in severity and penetrance.

As previously mentioned, PAX5 has been associated with susceptibility to ALL (Gu et al., 2019; Mullighan et al., 2007; Passet et al., 2019; Shah et al., 2013). In contrast to the neurodevelopmental phenotype presented in this report, the leukemia phenotype is commonly associated with non-germline rearrangements involving partner genes, focal intragenic amplifications, and non-silent sequence variants affecting PAX5, or the presence of the somatic p.(Pro80Arg) allele with biallelic PAX5 alterations (Gu et al., 2019; Mullighan et al., 2007; Passet et al., 2019). However, Shah et al. described a heterozygous germline PAX5 variant, c.547G>A, p.(Gly183Ser), that segregated with autosomal dominant B-ALL in two kindreds (Shah et al., 2013). Leukemic cells from all affected individuals in both families exhibited 9p deletion, with loss of heterozygosity and retention of the mutant PAX5 allele at 9p13. Functional and gene expression analysis demonstrated that the PAX5 p.(Gly183Ser) allele has significantly reduced transcriptional activity, but is not a null allele (Shah et al., 2013). While no cases of leukemia were reported in our cohort, these results suggest that individuals with neurodevelopmental phenotypes caused by a decrease in PAX5 function could be at an increased risk for ALL.

Taken together, our findings suggest that haploinsufficiency of PAX5 causes a neurodevelopmental disorder whose cardinal features include DD, variable ID, and/or ASD with no recurrent patterns of brain MRI findings, structural birth defects, or dysmorphic features. Since the phenotype associated with this disorder is not sufficiently distinct to be suspected on clinical grounds alone, diagnosis requires the identification of a damaging, loss-of-function variant in PAX5.

Supplementary Material

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supinfo1

ACKNOWLEDGEMENTS

We would like to thank Andres Hernandez-Garcia and Paula Patricia Hernandez for their help in DNA extraction and sample handling.

This study makes use of data generated by the DECIPHER community. A full list of centers who contributed to the generation of the data is available at https://deciphergenomics.org/about/stats and via email from contact@deciphergenomics.org. Funding for the DECIPHER project was provided by Wellcome (https://wellcome.org/). Subjects were also accrued with the help of GeneMatcher (Sobreira, Schiettecatte, Valle, & Hamosh, 2015), denovo-db (https://denovo-db.gs.washington.edu/denovo-db/), Simons Powering Autism Research (SPARK, https://sparkforautism.org/), Baylor Genetics (https://www.baylorgenetics.com/) and Matchmaker Exchange (https://www.matchmakerexchange.org/).

The authors wish to acknowledge the use of resources from Autism Speaks, The Centre for Applied Genomics at The Hospital for Sick Children, Toronto, Canada and the help of the Simons Foundation Powering Autism Research (SPARK) Consortium (Supp. Mat.—Consortium Data). We also thank the participating families for their time and contributions to this database, as well as the generosity of the donors who supported this program.

Sequencing and analysis for Subjects 13 and 14 were provided by the Broad Institute of MIT and Harvard Center for Mendelian Genomics (Broad CMG) and was funded by the National Human Genome Research Institute, the National Eye Institute, and the National Heart, Lung and Blood Institute grant UM1 HG008900 and in part by National Human Genome Research Institute grants R01 HG009141 and U01 HG011755. Sequencing for Subject 14 was also supported by Chan Zuckerberg Initiative DAF grant 2020-224274 by an advised fund of the Silicon Valley Community Foundation. MHW is supported by K23 HD102589.

This work was also supported, in part, by grants to E.E.E. from the National Institutes of Health, R01 MH101221, and the Simons Foundation, SFARI 608045. E.E.E. is an investigator of the Howard Hughes Medical Institute.

APPENDIX

ORCID Identification Numbers

Yoel Gofin: 0000-0003-3233-258X

Tianyun Wang: 0000-0002-5179-087X

Madelyn A. Gillentine: 0000-0002-8989-2214

Tiana M. Scott: 0000-0002-6209-8301

Mahshid S. Azamian: 0000-0002-8543-8284

Casie Genetti: 0000-0003-4173-9947

Monica H. Wojcik: 0000-0002-8162-5031

Sally Ann Lynch: 0000-0003-3540-1333

Stephen W. Scherer: 0000-0002-8326-1999

Carlos Bacino: 0000-0002-4342-5012

Stephanie DiTroia: 0000-0002-6847-6780

Grace E. VanNoy: 0000-0003-1257-9702

Anne O'Donnell-Luria: 0000-0001-6418-9592

William D. Graf: 0000-0003-0460-4605

Jill A. Rosenfeld: 0000-0001-5664-7987

Evan E. Eichler: 0000-0002-8246-4014

Daryl A. Scott: 0000-0003-1460-5169

Footnotes

WEB RESOURCES

Combined Annotation Dependent Depletion (CADD): https://cadd.gs.washington.edu/snv

Gene Variation Intolerance Ranking (GeVIR): http://www.gevirank.org

gnomAD Browser v2.1.1: https://gnomad.broadinstitute.org/

International Mouse Phenotyping Consortium (IMPC): http://www.mousephenotype.org/

MutationTaster: https://www.genecascade.org/MutationTaster2021

CONFLICT OF INTEREST

The Department of Molecular & Human Genetics at Baylor College of Medicine receives revenue from clinical genetic testing completed at Baylor Genetics Laboratories. A.O.D.L. is a paid advisor for Congenica and Tome Biosciences.

DATA AVAILABILITY STATEMENT

All previously unreported variants described in this manuscript have been submitted to ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/).

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Associated Data

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

Supplementary Materials

supinfo2
NIHMS1772796-supplement-supinfo2.xlsx (17.5KB, xlsx)
supinfo1
NIHMS1772796-supplement-supinfo1.pdf (160.2KB, pdf)

Data Availability Statement

All previously unreported variants described in this manuscript have been submitted to ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/).

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