A Multi-Institutional Description of Processes and Outcomes of Postbaccalaureate Research Education Programs in the Mid-Atlantic Region.

ABSTRACT: Outcome data from 6 National Institutes of Health-funded Postbaccalaureate Research Education Programs (PREPs) in the Mid-Atlantic region were combined to give a multi-institutional perspective on their scholars' characteristics and progress through biomedical research training. The institutions hosting these programs were Johns Hopkins University School of Medicine, the Medical University of South Carolina, the University of Maryland School of Medicine, the University of North Carolina at Chapel Hill, Virginia Commonwealth University, and Virginia Polytechnic Institute and State University. The authors summarize the institutional pathways, demographics, undergraduate institutions, and graduate institutions for a total of 384 PREP scholars who completed the programs by June 2021. A total of 228 (59.4%) of these PREP scholars identified as Black or African American, 116 (30.2%) as Hispanic or Latinx, and 269 (70.0%) as female. The authors found that 376 of 384 scholars (97.9%) who started PREP finished their program, 319 of 376 (84.8%) who finished PREP matriculated into PhD or MD/PhD programs, and 284 of 319 (89.0%) who matriculated have obtained their PhD or are successfully making progress toward their PhD.

rRNA transcription is integral to phase separation and maintenance of nucleolar structure.

Transcription of ribosomal RNA (rRNA) by RNA Polymerase (Pol) I in the nucleolus is necessary for ribosome biogenesis, which is intimately tied to cell growth and proliferation. Perturbation of ribosome biogenesis results in tissue specific disorders termed ribosomopathies in association with alterations in nucleolar structure. However, how rRNA transcription and ribosome biogenesis regulate nucleolar structure during normal development and in the pathogenesis of disease remains poorly understood. Here we show that homozygous null mutations in Pol I subunits required for rRNA transcription and ribosome biogenesis lead to preimplantation lethality. Moreover, we discovered that Polr1a-/-, Polr1b-/-, Polr1c-/- and Polr1d-/- mutants exhibit defects in the structure of their nucleoli, as evidenced by a decrease in number of nucleolar precursor bodies and a concomitant increase in nucleolar volume, which results in a single condensed nucleolus. Pharmacological inhibition of Pol I in preimplantation and midgestation embryos, as well as in hiPSCs, similarly results in a single condensed nucleolus or fragmented nucleoli. We find that when Pol I function and rRNA transcription is inhibited, the viscosity of the granular compartment of the nucleolus increases, which disrupts its phase separation properties, leading to a single condensed nucleolus. However, if a cell progresses through mitosis, the absence of rRNA transcription prevents reassembly of the nucleolus and manifests as fragmented nucleoli. Taken together, our data suggests that Pol I function and rRNA transcription are required for maintaining nucleolar structure and integrity during development and in the pathogenesis of disease.

Dynamic regulation and requirement for ribosomal RNA transcription during mammalian development.

Ribosomal RNA (rRNA) transcription by RNA polymerase I (Pol I) is a critical rate-limiting step in ribosome biogenesis, which is essential for cell survival. Despite its global function, disruptions in ribosome biogenesis cause tissue-specific birth defects called ribosomopathies, which frequently affect craniofacial development. Here, we describe a cellular and molecular mechanism underlying the susceptibility of craniofacial development to disruptions in Pol I transcription. We show that Pol I subunits are highly expressed in the neuroepithelium and neural crest cells (NCCs), which generate most of the craniofacial skeleton. High expression of Pol I subunits sustains elevated rRNA transcription in NCC progenitors, which supports their high tissue-specific levels of protein translation, but also makes NCCs particularly sensitive to rRNA synthesis defects. Consistent with this model, NCC-specific deletion of Pol I subunits Polr1a, Polr1c, and associated factor Tcof1 in mice cell-autonomously diminishes rRNA synthesis, which leads to p53 protein accumulation, resulting in NCC apoptosis and craniofacial anomalies. Furthermore, compound mutations in Pol I subunits and associated factors specifically exacerbate the craniofacial anomalies characteristic of the ribosomopathies Treacher Collins syndrome and Acrofacial Dysostosis-Cincinnati type. Mechanistically, we demonstrate that diminished rRNA synthesis causes an imbalance between rRNA and ribosomal proteins. This leads to increased binding of ribosomal proteins Rpl5 and Rpl11 to Mdm2 and concomitantly diminished binding between Mdm2 and p53. Altogether, our results demonstrate a dynamic spatiotemporal requirement for rRNA transcription during mammalian cranial NCC development and corresponding tissue-specific threshold sensitivities to disruptions in rRNA transcription in the pathogenesis of congenital craniofacial disorders.

Whole-Exome Sequencing Identifies the VHL Mutation (c.262T > C, p.Try88Arg) in Non-Obstructive Azoospermia-Associated Cystic Renal Cell Carcinoma

Von Hippel-Lindau (VHL) genes are intimately involved in renal cell carcinoma (RCC), including clear cell RCC (ccRCC) pathogenesis. However, the contribution of pathogenic VHL mutations to ccRCC remains poorly understood. We report a xanthoderm with non-obstructive azoospermia (NOA)-associated cystic ccRCC, and the missense VHL mutation (c.262T > C, p.Try88Arg). In a 34-year-old patient, a urologic physical examination identified hard epididymis, and imaging tests revealed deferens-associated NOA, as well as multi-organ hydatid cysts, including bilateral epididymal cysts, bilateral testicular cysts, bilateral renal cysts, and pancreatic cysts. Five years later, ccRCC was developed based on clinical and radiologic evidence. Two different prediction models of protein structure and multiple sequence alignment across species were applied to assess the pathological effects of the VHL mutation. The reliability of the assessment in silico was determined by both the cellular location and protein levels of the mutant products, using IF and Western blot, respectively. Our study shows that the missense VHL mutation (c.262T > C, p.Try88Arg) plays a deleterious role in pVHL functions, as predicted by multiple sequence alignment across species. While a structural analysis identified no significant structural alterations in pVHL, the detrimental effects of this mutation were determined by exogenous expression, evidenced by a markedly different spatial distribution and reduced expression of mutant pVHL. This is the first report of the VHL gene mutation (c.475T > C, p.Try88Arg) in a xanthoderm.

A single amino acid mutation in the mouse MEIG1 protein disrupts a cargo transport system necessary for sperm formation.

Mammalian spermatogenesis is a highly coordinated process that requires cooperation between specific proteins to coordinate diverse biological functions. For example, mouse Parkin coregulated gene (PACRG) recruits meiosis-expressed gene 1 (MEIG1) to the manchette during normal spermiogenesis. Here we mutated Y68 of MEIG1 using the CRISPR/cas9 system and examined the biological and physiological consequences in mice. All homozygous mutant males examined were completely infertile, and sperm count was dramatically reduced. The few developed sperm were immotile and displayed multiple abnormalities. Histological staining showed impaired spermiogenesis in these mutant mice. Immunofluorescent staining further revealed that this mutant MEIG1 was still present in the cell body of spermatocytes, but also that more MEIG1 accumulated in the acrosome region of round spermatids. The mutant MEIG1 and a cargo protein of the MEIG1/PACRG complex, sperm-associated antigen 16L (SPAG16L), were no longer found to be present in the manchette; however, localization of the PACRG component was not changed in the mutants. These findings demonstrate that Y68 of MEIG1 is a key amino acid required for PACRG to recruit MEIG1 to the manchette to transport cargo proteins during sperm flagella formation. Given that MEIG1 and PACRG are conserved in humans, small molecules that block MEIG1/PACRG interaction are likely ideal targets for the development of male contraconception drugs.

Expanding the phenotype for the recurrent p.Ala391Glu variant in FGFR3: Beyond crouzon syndrome and acanthosis nigricans.

BACKGROUND: Craniosynostosis, or premature fusion of the skull sutures, is a group of disorders that can present in isolation (nonsyndromic) or be associated with other anomalies (syndromic). Delineation of syndromic craniosynostosis is confounded due to phenotypic overlap, variable expression as well as molecular heterogeneity. We report on an infant who presented at birth with multisuture synostosis, turribrachycephaly, midface hypoplasia, beaked nose, low set ears, a high palate and short squat appearing thumbs, and great toes without deviation. The additional MRI findings of choanal stenosis and a Chiari I malformation suggested a diagnosis of Pfeiffer syndrome. First tier molecular testing did not reveal a pathogenic variant. METHODS: Whole exome sequencing on DNA samples from the proband and her unaffected parents was utilized to delineate the variant causative for the Pfeiffer syndrome diagnosis. RESULTS: On whole exome sequencing, a de novo NM_000142.4:c.1428C>A missense variant causing a p.Ala391Glu amino acid change in FGFR3 has been identified. The p.Ala391Glu change has been predominantly identified in patients with Crouzon syndrome with acanthosis nigricans. CONCLUSIONS: This finding illustrates the first reported case of a child with an overlap with Pfeiffer syndrome to have the p.Ala391Glu variant.

A schizophrenia associated CMYA5 allele displays differential binding with desmin.

CMYA5 is a candidate gene for schizophrenia because of the genetic association of variant rs10043986 (C > T) to this severe mental disorder. Studies of CMYA5 and its gene product, myospryn, in the brain and neuronal cells have not been previously reported. The SNP rs10043986 changes the 4,063rd amino acid from Pro to Leu, which is likely to alter protein function. To understand its potential role in the brain, we examined the neuronal expression of myospryn and its binding partner, desmin, an intermediate filament (IF) protein, and investigated how the two alleles of myospryn affect its binding to desmin. Myospryn and desmin are shown to be expressed in the brain and myospryn is shown to localize to the cytoplasm and nucleus of myoblast, neuroblastoma, and glioblastoma cell lines. Peripherin and vimentin, known brain IF proteins, have high protein similarity to desmin but were found not to interact with myospryn using yeast two-hybrid (Y2H). Using a quantitative Y2H assay and surface plasmon resonance, the T allele (Leu) of rs10043986 was found to have stronger binding to desmin than the C allele (Pro). Based on findings described in this report, we hypothesize that the interaction between myospryn to IF provides structural support and efficient rearrangement of the cytoskeleton network during early neuritogenesis.

Genomic analyses in African populations identify novel risk loci for cleft palate.

Orofacial clefts are common developmental disorders that pose significant clinical, economical and psychological problems. We conducted genome-wide association analyses for cleft palate only (CPO) and cleft lip with or without palate (CL/P) with ~17 million markers in sub-Saharan Africans. After replication and combined analyses, we identified novel loci for CPO at or near genome-wide significance on chromosomes 2 (near CTNNA2) and 19 (near SULT2A1). In situ hybridization of Sult2a1 in mice showed expression of SULT2A1 in mesenchymal cells in palate, palatal rugae and palatal epithelium in the fused palate. The previously reported 8q24 was the most significant locus for CL/P in our study, and we replicated several previously reported loci including PAX7 and VAX1.

Gene expression changes between patent and fused cranial sutures in a nonsyndromic craniosynostosis population.

OBJECTIVE: Craniosynostosis is a premature fusion of 1 or more cranial sutures. It may occur with additional morphological abnormalities (syndromic) or in isolation. Studies suggest that dysregulation of normal cell proliferation, differentiation, and migration has a role in isolated or nonsyndromic craniosynostosis but the molecular mechanisms remain unknown. The aim of this research is to identify genes differentially expressed in prematurely fused human suture compared to patent suture in nonsyndromic craniosynostosis. METHODS: Bone fragments from synostosed and patent sutures of 7 infants with nonsyndromic craniosynostosis were collected during surgical release of fused sutures. RNA was isolated from the fragments (7 patent and 7 fused) and global gene expression profiled using the Illumina WGE-DASL assay and HumanRef 8.0 Beadchip. RESULTS: Comparison of mRNA expression in fused and patent suture identified 68 genes significantly differentially expressed and having fold changes ≤ -2.0 and ≥ 2.0 with a false discovery rate adjusted P value at .10 and 136 with adjusted P value of 0.15. SFRP2 (secreted frizzled-related protein 2) demonstrated the largest decrease in fused sutures. Analysis including only sagittal fused sutures revealed a set of 35 overlapping genes that may be involved in suture patency over all suture types. SPHKAP (sphingosine kinase type 1-interacting protein), a modulator of TGFß signaling, was significant in the sagittal subset. CONCLUSION: Differentially expressed genes were identified in fused suture relative to patent in a nonsyndromic craniosynostosis population. SFRP2 is likely important in suture patency. Genes having significant roles in osteoblastogenesis as negative regulators of canonical Wnt pathway were significantly downregulated.

Genetic studies in Drosophila and humans support a model for the concerted function of CISD2, PPT1 and CLN3 in disease.

Wolfram syndrome (WFS) is a progressive neurodegenerative disease characterized by diabetes insipidus, diabetes mellitus, optic atrophy, and deafness. WFS1 and WFS2 are caused by recessive mutations in the genes Wolfram Syndrome 1 (WFS1) and CDGSH iron sulfur domain 2 (CISD2), respectively. To explore the function of CISD2, we performed genetic studies in flies with altered expression of its Drosophila orthologue, cisd2. Surprisingly, flies with strong ubiquitous RNAi-mediated knockdown of cisd2 had no obvious signs of altered life span, stress resistance, locomotor behavior or several other phenotypes. We subsequently found in a targeted genetic screen, however, that altered function of cisd2 modified the effects of overexpressing the fly orthologues of two lysosomal storage disease genes, palmitoyl-protein thioesterase 1 (PPT1 in humans, Ppt1 in flies) and ceroid-lipofuscinosis, neuronal 3 (CLN3 in humans, cln3 in flies), on eye morphology in flies. We also found that cln3 modified the effects of overexpressing Ppt1 in the eye and that overexpression of cln3 interacted with a loss of function mutation in cisd2 to disrupt locomotor ability in flies. Follow-up multi-species bioinformatic analyses suggested that a gene network centered on CISD2, PPT1 and CLN3 might impact disease through altered carbohydrate metabolism, protein folding and endopeptidase activity. Human genetic studies indicated that copy number variants (duplications and deletions) including CLN3, and possibly another gene in the CISD2/PPT1/CLN3 network, are over-represented in individuals with developmental delay. Our studies indicate that cisd2, Ppt1 and cln3 function in concert in flies, suggesting that CISD2, PPT1 and CLN3 might also function coordinately in humans. Further, our studies raise the possibility that WFS2 and some lysosomal storage disorders might be influenced by common mechanisms and that the underlying genes might have previously unappreciated effects on developmental delay.

Human polynucleotide phosphorylase (hPNPaseold-35): should I eat you or not--that is the question?

RNA degradation plays a fundamental role in maintaining cellular homeostasis whether it occurs as a surveillance mechanism eliminating aberrant mRNAs or during RNA processing to generate mature transcripts. 3'-5' exoribonucleases are essential mediators of RNA decay pathways, and one such evolutionarily conserved enzyme is polynucleotide phosphorylase (PNPase). The human homologue of this fascinating enzymatic protein (hPNPaseold-35) was cloned a decade ago in the context of terminal differentiation and senescence through a novel "overlapping pathway screening" approach. Since then, significant insights have been garnered about this exoribonuclease and its repertoire of expanding functions. The objective of this review is to provide an up-to-date perspective of the recent discoveries made relating to hPNPaseold-35 and the impact they continue to have on our comprehension of its expanding and diverse array of functions.

GLRB is the third major gene of effect in hyperekplexia.

Glycinergic neurotransmission is a major inhibitory influence in the CNS and its disruption triggers a paediatric and adult startle disorder, hyperekplexia. The postsynaptic α(1)-subunit (GLRA1) of the inhibitory glycine receptor (GlyR) and the cognate presynaptic glycine transporter (SLC6A5/GlyT2) are well-established genes of effect in hyperekplexia. Nevertheless, 52% of cases (117 from 232) remain gene negative and unexplained. Ligand-gated heteropentameric GlyRs form chloride ion channels that contain the α(1) and ß-subunits (GLRB) in a 2α(1):3ß configuration and they form the predominant population of GlyRs in the postnatal and adult human brain, brainstem and spinal cord. We screened GLRB through 117 GLRA1- and SLC6A5-negative hyperekplexia patients using a multiplex-polymerase chain reaction and Sanger sequencing approach. The screening identified recessive and dominant GLRB variants in 12 unrelated hyperekplexia probands. This primarily yielded homozygous null mutations, with nonsense (n = 3), small indel (n = 1), a large 95 kb deletion (n = 1), frameshifts (n = 1) and one recurrent splicing variant found in four cases. A further three cases were found with two homozygous and one dominant GLRB missense mutations. We provide strong evidence for the pathogenicity of GLRB mutations using splicing assays, deletion mapping, cell-surface biotinylation, expression studies and molecular modelling. This study describes the definitive assignment of GLRB as the third major gene for hyperekplexia and impacts on the genetic stratification and biological causation of this neonatal/paediatric disorder. Driven principally by consanguineous homozygosity of GLRB mutations, the study reveals long-term additive phenotypic outcomes for affected cases such as severe apnoea attacks, learning difficulties and developmental delay.

Mutations in the GlyT2 gene (SLC6A5) are a second major cause of startle disease.

Hereditary hyperekplexia or startle disease is characterized by an exaggerated startle response, evoked by tactile or auditory stimuli, leading to hypertonia and apnea episodes. Missense, nonsense, frameshift, splice site mutations, and large deletions in the human glycine receptor α1 subunit gene (GLRA1) are the major known cause of this disorder. However, mutations are also found in the genes encoding the glycine receptor ß subunit (GLRB) and the presynaptic Na(+)/Cl(-)-dependent glycine transporter GlyT2 (SLC6A5). In this study, systematic DNA sequencing of SLC6A5 in 93 new unrelated human hyperekplexia patients revealed 20 sequence variants in 17 index cases presenting with homozygous or compound heterozygous recessive inheritance. Five apparently unrelated cases had the truncating mutation R439X. Genotype-phenotype analysis revealed a high rate of neonatal apneas and learning difficulties associated with SLC6A5 mutations. From the 20 SLC6A5 sequence variants, we investigated glycine uptake for 16 novel mutations, confirming that all were defective in glycine transport. Although the most common mechanism of disrupting GlyT2 function is protein truncation, new pathogenic mechanisms included splice site mutations and missense mutations affecting residues implicated in Cl(-) binding, conformational changes mediated by extracellular loop 4, and cation-π interactions. Detailed electrophysiology of mutation A275T revealed that this substitution results in a voltage-sensitive decrease in glycine transport caused by lower Na(+) affinity. This study firmly establishes the combination of missense, nonsense, frameshift, and splice site mutations in the GlyT2 gene as the second major cause of startle disease.

Pathophysiological mechanisms of dominant and recessive GLRA1 mutations in hyperekplexia.

Hyperekplexia is a rare, but potentially fatal, neuromotor disorder characterized by exaggerated startle reflexes and hypertonia in response to sudden, unexpected auditory or tactile stimuli. This disorder is primarily caused by inherited mutations in the genes encoding the glycine receptor (GlyR) alpha1 subunit (GLRA1) and the presynaptic glycine transporter GlyT2 (SLC6A5). In this study, systematic DNA sequencing of GLRA1 in 88 new unrelated human hyperekplexia patients revealed 19 sequence variants in 30 index cases, of which 21 cases were inherited in recessive or compound heterozygote modes. This indicates that recessive hyperekplexia is far more prevalent than previous estimates. From the 19 GLRA1 sequence variants, we have investigated the functional effects of 11 novel and 2 recurrent mutations. The expression levels and functional properties of these hyperekplexia mutants were analyzed using a high-content imaging system and patch-clamp electrophysiology. When expressed in HEK293 cells, either as homomeric alpha1 or heteromeric alpha1beta GlyRs, subcellular localization defects were the major mechanism underlying recessive mutations. However, mutants without trafficking defects typically showed alterations in the glycine sensitivity suggestive of disrupted receptor function. This study also reports the first hyperekplexia mutation associated with a GlyR leak conductance, suggesting tonic channel opening as a new mechanism in neuronal ligand-gated ion channels.

Regulation of the mouse Treacher Collins syndrome homolog (Tcof1) promoter through differential repression of constitutive expression.

Treacher Collins syndrome is an autosomal-dominant mandibulofacial dysostosis caused by haploinsufficiency of the TCOF1 gene product treacle. Mouse Tcof1 protein is approximately 61% identical and 71% similar to treacle, and heterozygous knockout of Tcof1 causes craniofacial malformation. Tcof1 expression is high in developing neural crest, but much lower in other tissues. To investigate this dual regulation, highly conserved regions upstream of TCOF1 homologs were tested through deletion and mutation reporter assays, and conserved predicted transcription factor binding sites were assessed through chromatin binding studies. Assays were performed in mouse P19 embryonic carcinoma cells and in HEK293 cells to determine differential activation in cell types at different stages of differentiation. Binding of Cebpb, Zfp161, and Sp1 transcription factors was specific to the Tcof1 regulatory region in P19 cells. The Zfp161 binding site demonstrated P19 cell-specific repression, while the Sp1/Sp3 candidate site demonstrated HEK293 cell-specific activation. Moreover, presence of c-myb and Zfp161 transcripts was specific to P19 cells. A minimal promoter fragment from -253 to +43 bp directs constitutive expression in both cell types, and dual regulation of Tcof1 appears to be through differential repression of this minimal promoter. The CpG island at the transcription start site remains unmethylated in P19 cells, 11.5 dpc mouse embryonic tissue, and adult mouse ear, which supports constitutive activation of the Tcof1 promoter.

Why do faculty leave? Reasons for attrition of women and minority faculty from a medical school: four-year results.

PURPOSE: Faculty attrition, particularly among female and minority faculty, is a serious problem in academic medical settings. The reasons why faculty in academic medical settings choose to leave their employment are not well understood. Further, it is not clear if the reasons why women and minority faculty leave differ from those of other groups. METHODS: One hundred sixty-six medical school faculty who left the School of Medicine (SOM) between July 1, 2001, and June 30, 2005, completed a survey about their reasons for leaving. RESULTS: The three most common overall reasons for leaving the institution included career/professional advancement (29.8%), low salary (25.5%), and chairman/departmental leadership issues (22.4%). The ranking of these reasons varied slightly across racial and gender groups, with women and minority faculty also citing personal reasons for leaving. Women and minority faculty were at lower academic ranks at the time they left the SOM compared with male and majority groups. Although salary differences were not present at the time of initial hire, sex was a significant predictor of lower salary at the start of the new position. Opportunity for advancement and the rate of promotion were significantly different between women and men. Job characteristics prior to leaving that were rated most poorly were protected time for teaching and research, communication across the campus, and patient parking. Harassment and discrimination were reported by a small number of those surveyed, particularly women and minority faculty. CONCLUSIONS: The majority of reasons for faculty attrition are amenable to change. Retaining high-quality faculty in medical settings may justify the costs of faculty development and retention efforts.

A homozygous mutation in a novel zinc-finger protein, ERIS, is responsible for Wolfram syndrome 2.

A single missense mutation was identified in a novel, highly conserved zinc-finger gene, ZCD2, in three consanguineous families of Jordanian descent with Wolfram syndrome (WFS). It had been shown that these families did not have mutations in the WFS1 gene (WFS1) but were mapped to the WFS2 locus at 4q22-25. A G-->C transversion at nucleotide 109 predicts an amino acid change from glutamic acid to glutamine (E37Q). Although the amino acid is conserved and the mutation is nonsynonymous, the pathogenesis for the disorder is because the mutation also causes aberrant splicing. The mutation was found to disrupt messenger RNA splicing by eliminating exon 2, and it results in the introduction of a premature stop codon. Mutations in WFS1 have also been found to cause low-frequency nonsyndromic hearing loss, progressive hearing loss, and isolated optic atrophy associated with hearing loss. Screening of 377 probands with hearing loss did not identify mutations in the WFS2 gene. The WFS1-encoded protein, Wolframin, is known to localize to the endoplasmic reticulum and plays a role in calcium homeostasis. The ZCD2-encoded protein, ERIS (endoplasmic reticulum intermembrane small protein), is also shown to localize to the endoplasmic reticulum but does not interact directly with Wolframin. Lymphoblastoid cells from affected individuals show a significantly greater rise in intracellular calcium when stimulated with thapsigargin, compared with controls, although no difference was observed in resting concentrations of intracellular calcium.

Mutations in the gene encoding GlyT2 (SLC6A5) define a presynaptic component of human startle disease.

Hyperekplexia is a human neurological disorder characterized by an excessive startle response and is typically caused by missense and nonsense mutations in the gene encoding the inhibitory glycine receptor (GlyR) alpha1 subunit (GLRA1). Genetic heterogeneity has been confirmed in rare sporadic cases, with mutations affecting other postsynaptic glycinergic proteins including the GlyR beta subunit (GLRB), gephyrin (GPHN) and RhoGEF collybistin (ARHGEF9). However, many individuals diagnosed with sporadic hyperekplexia do not carry mutations in these genes. Here we show that missense, nonsense and frameshift mutations in SLC6A5 (ref. 8), encoding the presynaptic glycine transporter 2 (GlyT2), also cause hyperekplexia. Individuals with mutations in SLC6A5 present with hypertonia, an exaggerated startle response to tactile or acoustic stimuli, and life-threatening neonatal apnea episodes. SLC6A5 mutations result in defective subcellular GlyT2 localization, decreased glycine uptake or both, with selected mutations affecting predicted glycine and Na+ binding sites.