De novo variants in FRYL are associated with developmental delay, intellectual disability, and dysmorphic features

Xueyang Pan; Alice M Tao; Shenzhao Lu; Mengqi Ma; Shabab B Hannan; Rachel Slaugh; Sarah Drewes Williams; Lauren O'Grady; Oguz Kanca; Richard Person; Melissa T Carter; Konrad Platzer; Franziska Schnabel; Rami Abou Jamra; Amy E Roberts; Jane W Newburger; Anya Revah-Politi; Jorge L Granadillo; Alexander PA Stegmann; Margje Sinnema; Andrea Accogli; Vincenzo Salpietro; Valeria Capra; Lina Ghaloul-Gonzalez; Martina Brueckner; Marleen EH Simon; David A Sweetser; Kevin E Glinton; Susan E Kirk; Baylor College of Medicine Center for Precision Medicine Models; Michael F Wangler; Shinya Yamamoto; Wendy K Chung; Hugo J Bellen

doi:10.1016/j.ajhg.2024.02.007

. 2024 Mar 12;111(4):742–760. doi: 10.1016/j.ajhg.2024.02.007

De novo variants in FRYL are associated with developmental delay, intellectual disability, and dysmorphic features

Xueyang Pan ^1,², Alice M Tao ³, Shenzhao Lu ^1,², Mengqi Ma ^1,², Shabab B Hannan ^1,², Rachel Slaugh ⁴, Sarah Drewes Williams ⁵, Lauren O'Grady ^6,⁷, Oguz Kanca ^1,², Richard Person ⁸, Melissa T Carter ⁹, Konrad Platzer ¹⁰, Franziska Schnabel ¹⁰, Rami Abou Jamra ¹⁰, Amy E Roberts ^11,¹², Jane W Newburger ^11,¹³, Anya Revah-Politi ¹⁴, Jorge L Granadillo ⁴, Alexander PA Stegmann ¹⁵, Margje Sinnema ¹⁵, Andrea Accogli ^16,¹⁷, Vincenzo Salpietro ¹⁸, Valeria Capra ¹⁹, Lina Ghaloul-Gonzalez ^5,²⁰, Martina Brueckner ^21,²², Marleen EH Simon ²³, David A Sweetser ^6,²⁴, Kevin E Glinton ^1,²⁵, Susan E Kirk ^26,²⁷; Baylor College of Medicine Center for Precision Medicine Models, Michael F Wangler ^1,², Shinya Yamamoto ^1,^2,²⁸, Wendy K Chung ^29,^∗, Hugo J Bellen ^1,^28,^∗∗

¹Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA

²Jan & Dan Duncan Neurological Research Institute, Texas Children’s Hospital, Houston, TX, USA

³Vagelos School of Physicians and Surgeons, Columbia University, New York, NY, USA

⁴Division of Genetics and Genomic Medicine, Department of Pediatrics, Washington University in St. Louis School of Medicine, St. Louis, Missouri, USA

⁵Division of Genetic and Genomic Medicine, Department of Pediatrics, UPMC Children’s Hospital of Pittsburgh, Pittsburgh, PA, USA

⁶Division of Medical Genetics & Metabolism, Massachusetts General for Children, Boston, MA, USA

⁷MGH Institute of Health Professions, Charlestown, MA, USA

⁸GeneDx, LLC, Gaithersburg, MD, USA

⁹Department of Genetics, Children’s Hospital of Eastern Ontario, Ottawa, ON, Canada

¹⁰Institute of Human Genetics, University of Leipzig Medical Center, Leipzig, Germany

¹¹Department of Cardiology, Boston Children’s Hospital, Boston, MA, USA

¹²Department of Medicine, Division of Genetics, Boston Children’s Hospital, Boston, MA, USA

¹³Department of Pediatrics, Harvard Medical School, Boston, MA, USA

¹⁴Institute for Genomic Medicine and Precision Genomics Laboratory, Columbia University Irving Medical Center, New York, NY, USA

¹⁵Department of Clinical Genetics, Maastricht University Medical Center, Maastricht, the Netherlands

¹⁶Division of Medical Genetics, Department of Medicine, McGill University Health Center, Montreal, QC, Canada

¹⁷Department of Human Genetics, McGill University, Montreal, QC, Canada

¹⁸Department of Neuromuscular Disorders, University College London Institute of Neurology, Queen Square, London, UK

¹⁹Unit of Medical Genetics and Genomics, IRCCS Istituto Giannina Gaslini, Genoa, Italy

²⁰Department of Human Genetics, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA, USA

²¹Department of Pediatrics, Yale University School of Medicine, New Haven, CT, USA

²²Department of Genetics, Yale University School of Medicine, New Haven, CT, USA

²³Department of Medical Genetics, University Medical Center Utrecht, Utrecht, the Netherlands

²⁴Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, USA

²⁵Department of Genetics, Texas Children’s Hospital, Houston, TX, USA

²⁶Section of Hematology/Oncology, Department of Pediatrics, Baylor College of Medicine, Houston, TX, USA

²⁷Texas Children’s Cancer and Hematology Center, Houston, TX, USA

²⁸Department of Neuroscience, Baylor College of Medicine, Houston, TX, USA

²⁹Departments of Pediatrics, Boston Children’s Hospital, Harvard Medical School, Boston, MA, USA

^∗

Corresponding author wendy.chung@childrens.harvard.edu

^∗∗

Corresponding author hbellen@bcm.edu

PMCID: PMC11023917 PMID: 38479391

Summary

FRY-like transcription coactivator (FRYL) belongs to a Furry protein family that is evolutionarily conserved from yeast to humans. The functions of FRYL in mammals are largely unknown, and variants in FRYL have not previously been associated with a Mendelian disease. Here, we report fourteen individuals with heterozygous variants in FRYL who present with developmental delay, intellectual disability, dysmorphic features, and other congenital anomalies in multiple systems. The variants are confirmed de novo in all individuals except one. Human genetic data suggest that FRYL is intolerant to loss of function (LoF). We find that the fly FRYL ortholog, furry (fry), is expressed in multiple tissues, including the central nervous system where it is present in neurons but not in glia. Homozygous fry LoF mutation is lethal at various developmental stages, and loss of fry in mutant clones causes defects in wings and compound eyes. We next modeled four out of the five missense variants found in affected individuals using fry knockin alleles. One variant behaves as a severe LoF variant, whereas two others behave as partial LoF variants. One variant does not cause any observable defect in flies, and the corresponding human variant is not confirmed to be de novo, suggesting that this is a variant of uncertain significance. In summary, our findings support that fry is required for proper development in flies and that the LoF variants in FRYL cause a dominant disorder with developmental and neurological symptoms due to haploinsufficiency.

Keywords: FRYL, furry, Drosophila, developmental delay, intellectual disability, rare disease

We report fourteen individuals with de novo variants in FRYL who present with neurodevelopmental features. Nine variants correspond to premature stop codons, frameshifts, or splicing variants. Modeling in Drosophila supports an essential developmental function for the fly FRYL ortholog; genomic integration of three missense variants caused a loss-of-function phenotype.

Introduction

FRYL (FRY-like transcription coactivator, also known as AF4p12) belongs to an evolutionarily conserved protein family, which is named after the fruit fly Furry (Fry) protein,¹ the first member identified in the family. Furry proteins are large proteins (FRYL has 3,013 amino acid residues) with a number of conserved regions (Figure 1A), including a FRY N-terminal domain (FND) predicted to fold into a large number of HEAT/Armadillo repeats² and a C-terminal region predicted to have two leucine zipper motifs and a coiled-coil motif (only present in vertebrate orthologs).³ The other conserved regions do not show similarity to any well-characterized motif.² In model organisms, Furry family proteins have diverse functions, including cell polarity maintenance,⁴^,⁵ cell morphogenesis,¹^,⁶ arborization and tiling of dendrites,²^,⁷^,⁸ and transcriptional regulation.³

Locations of predicted LoF and missense FRYL variants identified in this study

(A) Schematic of human FRYL and its alignment to human FRY and fly Fry. The Fry N-terminal (Pfam: #14222), cell morphogenesis central (Pfam: #PF14225), and cell morphogenesis C-terminal domains (Pfam: #PF14228) are identified in the Pfam project.⁹ The C-terminal leucine zipper and coiled-coil motifs are predicted by Goto et al.,³ which are only present in human FRY and FRYL but not fly Fry. Five FRYL missense variants identified in this study are shown above the protein, and nine predicted LoF variants are shown below the protein.

(B) The conservation of the five amino acid residues affected by the *FRYL* missense variants. The “--” symbols indicate that no aligned amino acid residue is found in the corresponding protein. All the residues are conserved in the Fryl proteins across vertebrate species as well as human FRY. In *Drosophila*, which is used as model organism in this study, four residues are conserved while the Arg110 residue is not. The variants identified in the five affected individuals correspond to amino acid changes, which are shown on the bottom row.

Humans have two Furry paralogs, FRY and FRYL, which share 61% identity and 75% similarity. In mammalian cells, FRY has been shown to regulate microtubule bundling and spindle organization during mitosis.¹⁰^,¹¹^,¹² Variants in FRY (MIM: 614818) are associated with cancer susceptibility and progression in patient-derived cancer cell lines and carcinogen-induced mammalian cancer models.¹³^,¹⁴ In humans, homozygous variants in FRY have been observed in individuals with intellectual disability in three consanguineous families.¹⁵^,¹⁶^,¹⁷ However, due to the insufficient number of affected individuals and the consanguineous nature of the families, the association between FRY variants and the intellectual disability phenotype has not been robustly established. FRYL was first identified in a FRYL-mixed lineage leukemia (MLL) fusion gene in an individual with lymphoblastic leukemia.¹⁸ Full-length FRYL was later found to interact with the active intracellular domain of NOTCH1 (ICN1).¹⁹ ICN1 translocates into the nucleus and functions with other coactivators to activate transcription of target genes;²⁰ however, how FRYL regulates this process is unknown. Most Fryl homozygous mutant mice die shortly after birth, and rare escapers survive less than a year and present with growth retardation and defects in kidney development.²¹ Morpholino-mediated knockdown of fryl in zebrafish causes cardiac and craniofacial defects during development.²² One individual with a heterozygous de novo variant in FRYL was identified in the Deciphering Developmental Disorders (DDD) study who presented with short stature and craniofacial and cardiac defects.²²

Here, we report thirteen individuals who have de novo heterozygous variants in FRYL and one individual with heterozygous FRYL variant that is not confirmed to be de novo. The individuals present with developmental delay (DD), intellectual disability, dysmorphic features, and other congenital anomalies in cardiovascular, skeletal, gastrointestinal, renal, and urogenital systems. Using fruit flies, we find that the fly FRYL ortholog furry (fry) is required for fly development, consistent with previous reports.¹^,⁶ Using fry knockin alleles analogous to human missense variants, we provide evidence that three out of the five missense variants found in affected individuals cause an LoF. These data support that haploinsufficiency in FRYL likely underlies a disorder with developmental and neurological symptoms.

Material and methods

Recruitment and sequencing of individuals

The cohort of individuals described in this study was recruited through the Pediatric Cardiac Genomics Consortium (individuals 3 and 9), the SPARK consortium (individual 12), and matchmaking via GeneMatcher (11 individuals).²³ Clinical data were obtained after written informed consent was provided by a parent or legal guardian at the institution at which they were enrolled for all individuals. The study of the individuals was approved by the Columbia University Institutional Review Board. The clinical data were supplied by the clinicians at each of the centers and then reviewed centrally by two clinicians who harmonized the data across the cases in collaboration with the clinicians at each site. The clinical features of the individuals and their variants in FRYL are summarized in Figures 1 and 2 and Tables 1 and 2.

Dysmorphic facial features of individuals with heterozygous variants in *FRYL*

(A and B) Facial images of individual 1 showing bitemporal narrowing, tall forehead, hypertelorism, epicanthal folds, upslanting palpebral fissures, flat nasal bridge with short upturned nose with bulbous tip, long deeply grooved philtrum, and cleft chin.

(C and D) Facial images of individual 6 showing hypertelorism, epicanthal folds, flat nasal bridge, and ear pit (indicated by the arrow).

Table 1.

Predicted LoF and deleterious missense variants in FRYL

	Variant		Inheritance	Method used to detect variant	Chr 4 coordinates (hg19/hg38)	gnomAD frequency	ACMG classification	Polyphen-2	Mutation taster		PROVEAN	CADD	REVEL	M-CAP
Predicted LoF

Individual 1	c.1129_1130delGT	p.Val377Asnfs^∗24	de novo	whole-exome sequencing	48597923 48595905	–	likely pathogenic (PVS1, PS2, PM2)	N/A	D	N/A	N/A	N/A	N/A	N/A
Individual 2	c.1224del	p.Lys409Argfs^∗15	de novo	whole-exome sequencing	48597631 48595613	–	likely pathogenic (PVS1, PS2, PM2)	N/A	D	N/A	N/A	N/A	N/A	N/A
Individual 3	c.1855_1858del	p.Val619Ilefs^∗7	de novo	whole-exome sequencing	48584642 48582624	–	likely pathogenic (PVS1, PS2, PM2)	N/A	D	N/A	N/A	33	N/A	N/A
Individual 4	c.1987-1G>A	splice site acceptor variant	de novo	whole-exome sequencing	48583623 48581606	–	likely pathogenic (PVS1, PS2, PM2)	N/A	N/A	N/A	N/A	34	N/A	N/A
Individual 5	c.2210_2211dupTA	p.Ser738^∗	de novo	whole-exome sequencing	48582930 48580913	–	likely pathogenic (PVS1, PS2, PM2)	N/A	D	N/A	N/A	N/A	N/A	N/A
Individual 6	c.4663C>T	p.Arg1555^∗	de novo	exome sequencing gene panels for intellectual disabilities, MCAs, and CHD	48551611 48549594	–	likely pathogenic (PVS1, PS2, PM2)	D	D	D	N/A	40	N/A	N/A
Individual 7	c.4724del	p.Cys1575Serfs^∗24	de novo	whole-exome sequencing	48551550 48549532	–	likely pathogenic (PVS1, PS2, PM2)	N/A	D	N/A	N/A	N/A	N/A	N/A
Individual 8	c.5474_5475delAT	p.Tyr1825Cysfs^∗22	de novo	whole-exome sequencing	48545941 48543923	–	likely pathogenic (PVS1, PS2, PM2)	N/A	D	N/A	N/A	N/A	N/A	N/A
Individual 9	c.8079_8080del	p.Gln2693Hisfs^∗4	de novo	whole-exome sequencing	48514563 48512545	–	likely pathogenic (PVS1, PS2, PM2)	N/A	D	N/A	N/A	34	N/A	N/A

Missense

Individual 10	c.328C>T	p.Arg110Cys	de novo	whole-exome sequencing	48621374 48619357	–	likely pathogeni (PVS1, PS2, PM2)	D	D	D	N	32	0.405	0.024
Individual 11	c.4882T>C	p.Phe1628Leu	de novo	whole-exome sequencing	48550713 48548696	–	likely pathogenic (PVS1, PS2, PM2)	D	D	D	D	28.5	0.589	0.015
Individual 12	c.6884T>C	p.Phe2295Ser	de novo	whole-exome sequencing	48533192 48531175	–	likely pathogenic (PVS1, PS2, PM2)	D	D	D	D	29.8	0.768	0.19
Individual 13	c.7190G>T	p.Ser2397Ile	not paternal (mother never tested)	whole-exome sequencing	48529621 48527604	4.02 × 10⁻⁶	VUS (PP3)	D	D	D	D	25.5	0.428	0.095
Individual 14	c.8852A>G	p.Tyr2951Cys	de novo	whole-exome sequencing	48501629 48499612	–	likely pathogenic (PVS1, PS2, PM2)	D	D	D	D	27.4	0.639	0.096

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CHD, congenital heart disease; D, deleterious; MCA, multiple congenital anomalies; N, neutral; N/A, not available; VUS, variant of uncertain significance.

Table 2.

Detailed clinical findings of patients with heterozygous predicted LoF and missense FRYL variants

	Individual 1	Individual 2	Individual 3	Individual 4	Individual 5	Individual 6	Individual 7	Individual 8	Individual 9	Individual 10	Individual 11	Individual 12	Individual 13	Individual 14	Summary
Variant class	predicted LoF	predicted LoF	predicted LoF	predicted LoF	predicted LoF	predicted LoF	predicted LoF	predicted LoF	predicted LoF	missense	missense	missense	missense	missense	9 LoF (64%), 5 missense (36%)
Variant in FRYL	p.Val377Asnfs^∗24	p.Lys409Argfs^∗15	p.Val619Ilefs^∗7	c.1987-1G>A	p.Ser738^∗	p.Arg1555^∗	p.Cys1575Serfs^∗24	p.Tyr1825Cysfs^∗22	p.Gln2693Hisfs^∗4	p.Arg110Cys	p.Phe1628Leu	p.Phe2295Ser	p.Ser2397Ile	p.Tyr2951Cys	–
Sex	F	F	M	M	M	M	F	M	M	M	M	M	F	M	4 F (28%), 10 M (72%)
Current age (years)	3.25	19	10	deceased (0.3)	12	2.2	6	20	8.8	15	12	8	21	9	average: 11.3
Prenatal complications	none	premature placental abruption	none	none	pre-eclampsia, HELLP syndrome, premature delivery (26 weeks)	short long bones	none	none	umbilical cord around neck at birth, forceps-assisted delivery, and torticollis	increased nuchal translucency	none	none	premature delivery (33 weeks)	C-section due to maternal hypertension and failure to progress, maternal diabetes	–
Birth weight (kg) (Z score)	2.14 (−1.65)	3.35 (-0.30)	3.35 (+0.53)	3.19 (+0.12)	0.62 (−1.35)	2.2 (−2.98)	2.88 (−0.73)	2.89 (−0.70)	2.1 (−3.37)	3.8 (+1.58)	3.89 (+1.12)	NR	NR	2.84 (−0.85)	2/14 (14%) with Z score < −2 average: −0.72
Birth length (cm) (Z score)	44 (−1.65)	49 (−1.23)	NR	51.5 (+0.9)	30.5 (−1.43)	NR	47 (−1.5)	NR	41 (−4.7)	NR	NR	NR	NR	47 (−1.5)	1/14 (7%) with Z score < −2 average: −1.59
Birth OFC (cm) (Z score)	31.5 (−1.65)	36.5 (1.23)	NR	35.5 (+1.5)	NR	NR	33.5 (+0.24)	NR	30.5 (−1.79)	NR	NR	NR	NR	33 (−0.08)	0/14 (0%) with Z score < −2 average: −0.09
Current weight (kg) (Z score)	10.9 (−4.00)	38.5 (−3.52)	20 (−0.30)	N/A	61.5 (+1.76)	9.7 (−2.0)	11.9 (-3.7)	90.2 (+1.32)	34.6 (+1.46)	56.9 (+2.4)	36.3 (−0.70)	NR	75.2 (+1.09)	37 (+1.12)	4/13 (31%) with Z score < −2 average: −0.42
Current length (cm) (Z score)	87 (−1.88)	159.6 (−1.33)	109.2 (−1.21)	N/A	147 (−0.28)	79 (−1.4)	98 (−3.6)	169.7 (-0.96)	131 (+0.09)	155.2 (-0.9)	146.1 (−0.42)	NR	155.5 (−1.19)	130 (−0.58)	1/13 (8%) with Z score ≤ −2 average: −1.1
Current OFC (cm) (Z score)	45.9 (-1.28)	52.2 (−2.6)	NR	N/A	52.3 (−1.02)	47.5 (−0.7)	48.7 (−1.2)	55.8 (+0.49)	NR	56.9 (+1.3)	NR	NR	53.5 (−0.76)	NR	1/13 (0%) with Z score ≤ −2 average: −0.72
Developmental delay and/or intellectual disability	+	+	+	N/A	+	+	+	+	+	+	+	+	+	+	13/13 (100%)
Speech and language	delayed	delayed	unknown	N/A	delayed	delayed	delayed	delayed	delayed	delayed	delayed	non-verbal	unknown	delayed	11/13 (85%)
Autism	–	–	–	N/A	+	N/A	–	+	unknown	+	+	+	–	–	5/12 (42%)
Psychiatric	–	dissociative motor disorder, borderline personality disorder, recurrent depressive disorders, eating disorder	–	N/A	poor socialization, avoidant/restrictive food intake disorder (ARFID)	anxiety	–	ADHD	behavioral disturbances	tics	behavioral and sleep disturbances	unknown	adjustment disorder with depressed mood	ADHD	9/13 (69%)
Hypotonia	–	+	–	–	–	+	+	–	–	–	+	unknown	–	+	5/14 (36%)
Seizure	staring spells daily lasting 1–2 min	–	–	Seizures noted after cardiac arrest and prolonged hypotensive episode at 28 days. Recurrent seizure at 2 months. Right and left frontal and temporal sharps and rare spikes, poorly formed sleep architecture on EEG.	generalized epilepsy, onset age 2	–	–	–	focal seizures, epileptiform discharges in the right central region	–	–	–	–	–	4/14 (29%)
Brain MRI/CT findings	intraventricular hemorrhage and small subdural collections	normal	–	–	normal	–	normal	normal	mild thinning of the corpus callosum and mild diffuse cerebral volume loss	–	cortical dysplasia, incomplete inversion of the hippocampus	–	–	–	–
Cardiovascular	RV tumor, partial AVSD, ASD, PPS	PDA, PFO, pulmonary artery stenosis	TOF with PA	HLHS, AS, MS, LA egress obstruction, IAA	–	VSD, PPS	–	–	dextrocardia, common atrium, CAVC defect, IAA, PDA	–	–	–	dextrocardia, interrupted IVC, ASD, VSD, PDA	–	7/14 (50%)
Genitourinary	–	urinary incontinence	hydrocele	–	undescended testes, horseshoe kidney	hypospadias	–	–	hypospadias, undescended testes, chordee	–	–	–	–	undescended testes	6/14 (43%)
Gastrointestinal	–	–	–	jejunal atresia, microcolon	–	–	–	–	jejunal atresia, midline liver	–	–	–	midline liver, right-sided stomach	–	3/14 (21%)
Dysmorphic features	upslanting palpebral fissures, bitemporal narrowing, tall forehead, epicanthal folds, flat nasal bridge, short upturned nose, long well-grooved philtrum, cleft chin	long face, down-slanted palpebral fissures, retrognathy, low-set ears (can be attributed to SF3B4 variant)	–	slightly triangular face, mild hypertelorism, low-set small ears with upturned lobules	sacral dimples, mildly anteverted nares	epicanthal folds, low nasal bridge, small ear tags, ear pit, nail dysplasia	frontal bossing, deep-set eyes, down-slanting palpebral fissure, nail dystrophy	deep-set eyes, up-slanting palpebral fissures, slightly posteriorly rotated ears	down-slanting palpebral fissures, flattened nasal bridge	deep-set eyes, up-slanting palpebral fissures, large ears, short philtrum	frontal bossing, anterior hairline	–	almond-shaped eyes, small hyperpigmented macule under right breast	–	11/14 (79%)
Additional phenotypes	pulmonary hypertension	hearing impairment, median cleft palate, dental crowding (can be attributed to SF3B4 variant)	–	non-union of the sternum, unknown coagulopathy, chylous effusions	–	–	pectus carenatus, interstitial lung disease	–	unknown coagulopathy, spleen on right, polysplenia syndrome	–	–	–	dermatomyositis, stage 4B mixed cellularity Hodgkin lymphoma, functional asplenia	neonatal thrombocytopenia	–
Additional genetic test results	PTPN11 VUS (c.879C>G [GenBank: NM_002834.5]; p.His293Gln), paternally inherited (asymptomatic father)	de novo pathogenic variant in SF3B4 (c.417C>T [GenBank: NM_005850.5]; p.Asp140Leufs^∗3) for acrofacial dysostosis 1, Nager type, AD (MIM: 154400); (PVS1, PS2_Moderate, PS3, PS4_Supporting, PM2_Supporting); PS3 based on Cassina et al. and Wai et al.²⁴^,²⁵	–	–	pathogenic variant in DHCR7 (c.452G>A [GenBank: NM_001360.3]; p.Trp151^∗) for Smith-Lemli-Opitz syndrome, AR (MIM: 270400), maternally inherited	de novo VUS in maternal UPD 22	344 kb duplication in 19q13.41pat classified as likely benign	pathogenic variant in SLC6A19 (c.1173 + 2T>G [GenBank: NM_001003841.3]) for Hartnup disorder, AR (MIM: 234500), heterozygous, maternally inherited	Heterozygous likely pathogenic variant in DNAH11 (c.2152_2153delGT [GenBank: NM_001277115.2] p.Val718Glnfs^∗3) for PCD, AR (MIM: 617577), VUS in FOXG1 (c.1425C>G [GenBank: NM_005249.4]; p.Phe475Leu), and 197 kb duplication at 10q21.3 classified as a VUS	likely benign variant in ABCD1 (c.1447A>G [GenBank: NM_000033.3]; p.Thr483Ala) for X-linked adrenoleukodystrophy (MIM: 300100), maternally inherited, metabolic analysis normal (C26:0 Lyso PC and VLCFA)	–	–	VUS in RSPH1 (c.916G>C [GenBank: NM_080860.2]; p.Asp306His) and VUS in DNAH1 (c.11534G>T [GenBank: NM_001277115.1]; p.Arg3845Leu)	de novo heterozygous pathogenic variant in SDHA (c.223C>T [GenBank: NM_004168.4]; p.Arg75^∗) for pheochromocytoma and paraganglioma, AD (MIM: 614165)	–

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Abbreviations: AD, autosomal dominant; ADHD, attention deficit hyperactivity disorder; AR, autosomal recessive; AS, aortic stenosis; ASD, atrial septal defect; AV, atrioventricular; AVSD, atrioventricular septal defect; CAVC, complete atrioventricular canal; EEG, electroencephalogram; HELLP, hemolysis, elevated liver enzymes, and low platelets; HLHS, hypoplastic left heart syndrome; IAA, interrupted aortic arch; IVC, inferior vena cava; LA, left atrium; LoF, loss of function; LSVC, left superior vena cava; LA, leftatrium; LV, left ventricle; MS, mitral stenosis; N/A, not applicable; NR, not reported; OT, occupational therapy; PA, pulmonary atresia; PC, phosphatidylcholine; PCD, primary ciliary dyskinesia PDA, patent ductus arteriosus; PFO, patent foramen ovale; PPS, peripheral pulmonary stenosis; PT, physical therapy; RA, right atrium; RV, right ventricle; TOF, tetralogy of fallot; UPD, uniparental disomy; VSD, ventricular septal defect; VUS, variant of uncertain significance.

Whole-exome sequencing of all but one family (individual 6) was performed to detect genetic variants. Individual 6 was tested by exome sequencing targeting gene panels for intellectual disabilities, multiple congenital anomalies (MCA), and congenital heart disease (CHD). Trio sequencing was included in the analysis to identify de novo variants, except one family (individual 13) for whom only paternal exome sequencing was completed due to lack of availability of the mother. The gnomAD database was used to assess variant frequency in the general population.²⁶ In silico algorithms, including PolyPhen-2,²⁷ MutationTaster,²⁸ SIFT,²⁹ PROVEAN,³⁰^,³¹ CADD,³² REVEL,³³ and M-CAP,³⁴ were employed to predict the pathogenicity of the variants. The American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) guideline for variant interpretation was also employed to evaluate the pathogenicity of the variants.³⁵

Drosophila stocks and maintenance

Flies were cultured using standard fly food at 25°C on a 12-h light/dark cycle. The fry knockin lines and fry^GF^P line were generated in house for this study (see methods below). The ubxFLP; ; ubi-GFP FRT80B and vas-phiC31; ; VK33 lines were from the Bellen lab stock. All other fly lines were obtained from the Bloomington Drosophila Stock Center (BDSC) (see Table S1).

Generation of the fry^GFP allele

The fry^GFP allele was generated by a well-established recombination-mediated cassette exchange (RMCE)-based strategy (Figure S1).³⁶ Females carrying hs-flippase (FLP), vas-phiC31, and flippase recognition target (FRT)-attB-EGFP-FlAsH-StrepII-TEVcs-3xFlag (GFSTF)-attB-white⁺-FRT cassette with correct phase (phase I) were crossed to fry^MI12326 males. Upon heat shock, the FRT-flanked cassette was flipped out as a circular DNA in the presence of Flippase, and the GFSTF sequence was swapped into the fry^MI12326 Minos-mediated integration cassette (MiMIC) site by phiC31-mediated RMCE. The progenies with fry^GFP allele were selected by the loss of white and yellow markers. The resulting fry^GFP allele was verified by PCR using the GFSTF_ver_L and GFSTF_ver_R primers listed in Table S2. The publicly available fly lines used in this protocol are listed in Table S1.

Generation of fry knockin lines by prime editing

Two knockin alleles, fry^p.Phe2024Leu and fry^p.Phe2746Ser, were generated by prime editing following a published protocol with adaptations.³⁷

The prime editing guide RNAs (pegRNAs) and nicking single-guide RNAs (sgRNA) were designed with the help from the PrimeDesign platform (http://primedesign.pinellolab.org/).³⁸ The pegRNAs were set to have 13 bp primer-binding sites (PBSs) and 16 bp reverse transcribed (RT) templates. All the protospacer adjacent motifs (PAMs) of the pegRNAs were disrupted by the edit to prevent repeated editing. To increase editing efficiency, second sgRNAs were designed to nick the opposite strand within 43 bp (for fry^p.Phe2024Leu) or 38 bp (for fry^p.Phe2746Ser) of the pegRNA-induced nicking. The pegRNA and nicking sgRNA sequences are listed in Table S3.

The pegRNA-tRNA-sgRNA sequences were synthesized and cloned into a pCFD5-white-NS vector by NEB HiFi Assembly (New England Biolabs) following the published cloning protocol.³⁷ The pCFD5-white-NS vector is modified from the pCFD5-NS vector (Addgene #149546) by replacing the vermillion selection marker by a mini-white sequence. Using the pCFD5-white-NS constructs, both pegRNA and sgRNA were expressed by a single Drosophila U6-3 promoter.³⁷^,³⁹ The constructs were injected into vas-phiC31 embryos and inserted into the VK33 docking site using phiC31-mediated transgenesis.⁴⁰

The induction of prime editing and the isolation of variant alleles were achieved following the published protocol.³⁷ The pegRNA-sgRNA-expressing flies were crossed to nos-GAL4 upstream activation sequence ((UAS)-PE2 flies to induce prime editing, and the progenies were cultured at 29°C and heat shocked at 37°C. In each trial, ten stocks were established for each variant, and flies with desired editing were selected by sequencing the modified locus using primers listed in Table S2. Two variants (fry^p.Phe2024Leu and fry^p.Phe2746Ser) were successfully generated using this method in two trials. No successful editing was achieved for the other two variants (fry^p.Ser2910Ile and fry^p.Tyr3410Cys), therefore an alternative method was employed (see below).

Generation of fry knockin lines by an RMCE- and single-strand annealing-based strategy

This gene knockin strategy is established based on previously developed methods.⁴¹^,⁴²^,⁴³ All knockin alleles were successfully generated using this strategy. The schematic of the strategy is illustrated in Figure S2. All primers used in this protocol are listed in Table S2.

To generate the RMCE donor constructs, a 6.2 kb genomic sequence covering all the desired fry variants was amplified from the genomic DNA of fry^MI02265 flies using fry genomic primers. The genomic sequence was cloned in to a pDONR223 vector (Invitrogen) by BP cloning, and variants were introduced into the genomic sequence by Q5 site-directed mutagenesis (New England Biolabs). Following mutagenesis, the variant genomic sequences were amplified by PCR, and AatII and AscI sites were added. The PCR products were treated with AatII and AscI and ligated into AatII- and AscI-digested pTarget-RMCE vector (Drosophila Genomics Resource Center #1534).⁴¹

The donor constructs were injected into vas-phiC31; ; fry^MI02265 embryos where the donor sequences (6.2 kb genomic sequence plus mini-white marker) were swapped into the fry^MI02265 MiMIC site by RMCE. Successfully conversed flies (fry^MI02265_RMCE) were selected by the loss of yellow⁺ present in the MiMIC cassette and the gain of the white⁺ present in the RMCE donor.⁴¹^,⁴⁴ The correct orientation of the donor sequence was verified by PCR using the RMCE_ori primers.

Next, the fry^MI02265-RMCE flies were crossed to the hs-I-CreI flies to induce double-strand breaks (DSBs) and the single-strand annealing (SSA) process (Figure S2). The expression of I-CreI was induced by heat shock at 37°C for 20 min. Potential variant alleles were isolated by selecting the white⁻ flies. Ten stocks were isolated for each of the fry^p.Phe2024Leu, fry^p.Phe2746Ser, and fry^p.Ser2910Ile variants, and fifteen stocks were isolated for the fry^p.Tyr3410Cys variant. The correct variants (fry^Variant) were selected by sequencing the modified locus. In every case, we found at least one stock with the expected variant.

Immunofluorescence staining and confocal microscopy

Immunostaining of the larval CNS and adult brain samples was performed following a previously published method.⁴⁵ Briefly, samples were fixed in 4% paraformaldehyde for 1 h and washed in 0.2% Triton X-100 in PBS (PBT). Samples were next incubated with anti-Elav (DSHB #7E8A10; 1:500) or anti-Repo (DSHB #8D12; 1:50) antibody. Fluorescent secondary antibodies were used at 1:200 (Jackson ImmunoResearch). Finally, samples were clarified using RapidClear reagent (SUNJin lab) and mounted on slides. Confocal images of whole-mount samples were taken using a Zeiss LSM710 confocal microscope.

Drosophila behavioral assays

The fly behavioral assays were performed following previously published methods.⁴⁶^,⁴⁷ To measure negative geotaxis, flies were transferred to a clean vial for at least 20 min prior to the experiment. During the test, flies were tapped to the bottom of the vial and their negative geotaxis climbing ability was measured. In each measurement, flies were allowed to climb for 30 s, after which the climbing distances were measured (18 cm is maximum). To perform bang-sensitive paralytic analyses, adult flies were transferred to a clean vial and vortexed at maximum speed for 10 s, after which the time required for flies to stand on their feet was counted (30 s is maximum). To perform heat shock assay, flies were transferred to an empty vial and submerged in a 42°C water bath for 30 s. The percentage of flies that were unable to keep an upright position was quantified.

Developmental time course measurement

Flies were cultured in an egg laying cage for two days before embryo collection. Fly embryos were collected on grape juice plates supplemented with yeast paste every 2 h. Twenty-four hours after egg laying (AEL), the hatched L1 larvae with desired genotype were transferred to fly food. Thirty larvae were transferred to one culturing vial to avoid crowding during the culture. From 60 h AEL, newly molted L3 larvae were counted and then transferred to new fly food every 4 h. The time from embryo to L2-L3 molting was recorded. The L3 larvae were further cultured into pupae, and the number of newly formed pupae were recorded every 4 h until all larvae pupariated. The time from L2-L3 molting to pupariation was recorded. Since the timing of eclosion of Drosophila is largely influenced by the circadian clock,⁴⁸ the time course of eclosion was not recorded and studied.

Electroretinogram recording

Electroretinograms (ERGs) were recorded following an established method.⁴⁹ Briefly, flies were anesthetized by CO₂ and fixed to a slide with office glue. A recording electrode filled with 150 mM NaCl was placed on the surface of the compound eye and a ground electrode inserted into the upper torso. ERGs were recorded during pulses of light stimulation and later analyzed using the LabChart 8 software.

Statistical analysis

Statistical analyses were carried out using the Student’s unpaired two-tailed t test for comparison of two groups. Multiple comparisons within the group were tested against the corresponding control. Calculated p values of less than 0.05 were considered significant. All statistical analyses were performed using GraphPad Prism, version 9.5.0 (GraphPad Software).

Results

Individuals with heterozygous variants in FRYL exhibit neurodevelopmental defects, dysmorphic facial features, and congenital anomalies

Fourteen unrelated individuals were found to harbor heterozygous variants in FRYL (GenBank: NM_015030.2). Thirteen out of the 14 variants were confirmed to be de novo and absent from gnomAD. The variant found in individual 13, c.7190G>T (p.Ser2397Ile), could not be confirmed de novo due to the lack of availability of the mother; however, the variant was not paternally inherited. This variant was present in gnomAD at a frequency of 4.02 × 10⁻⁶. Population genetic data from the gnomAD database suggest that FRYL is intolerant to LoF (pLI = 1) and missense variants (Z score = 3.3). Moreover, only two individuals with a microdeletion covering coding exons of FRYL were documented in the Database of Genomic Variants (DGV). Among the 14 identified variants, nine are predicted LoF variants as they encode premature stop codons, frameshifts, or splicing defects. The five remaining are missense variants (Table 1). All the predicted LoF variants are predicted to undergo nonsense mediated decay (NMD).⁵⁰^,⁵¹ All missense variants were predicted to be deleterious by multiple computational prediction tools, and all except the p.Ser2397Ile variant are classified as likely pathogenic by the ACMG/AMP guideline for variant interpretation (Table 1).³⁵

The mapping locations of the missense variants are shown in Figure 1A. The affected amino acids are highly conserved across vertebrate species, and four out of five are conserved in Drosophila (Figure 1B). One missense variant, c.328C>T (p.Arg110Cys), which is not conserved in flies, is located within the FND, a highly evolutionarily conserved domain found in all Fry and Fryl proteins. Another missense variant, c.4882T>C (p.Phe1628Leu), maps to the central cell morphogenesis region, which is conserved across many proteins involved in the process of cell morphogenesis. The other three variants (c.6884T>C [p.Phe2295Ser], c.7190G>T [p.Ser2397Ile], and c.8852A>G [p.Tyr2951Cys]) do not map to known domains.

Clinical information about individuals with heterozygous FRYL variants is summarized in Table 2. The individuals include four females and nine males ranging in current age from 2.2 to 21 years, with a mean age of 11.3 years. Additionally, one male (individual 4) had a complex congenital heart disease (hypoplastic left heart syndrome [HLHS], aortic and mitral stenosis, left atrium egress obstruction, and interrupted aortic arch). He died at 4 months of age from cardiac failure and tachyarrhythmia secondary to hyperkalemia. This individual is included in the summary of features observed at birth but is excluded from the summary of non-congenital clinical features, such as autism, DD and/or intellectual disability, and behavioral differences, as he was not old enough at time of death to display these features.

Neurodevelopmental issues, including DD and/or intellectual disability, were observed in all 13 individuals in the cohort. A total of 10 individuals were noted to have delayed development of speech and language, and one other individual was non-verbal at 8 years of age (11/13, 85%). Autism spectrum disorder was observed in five individuals among those who were over the age of 3 and old enough to be diagnosed (5/12, 42%). Other behavioral challenges were reported in nine individuals (9/13, 69%), including attention deficit hyperactivity disorder (ADHD) in two individuals and general behavioral disturbances in two individuals, as well as dissociative motor disorder, borderline personality disorder, depressive disorders, eating disorder, poor socialization, anxiety, tics, and adjustment disorder with depressed mood in different individuals.

Five of the probands were noted to have hypotonia, and an additional three were noted to have a confirmed history of seizures. Individual 5 was diagnosed with generalized epilepsy at 2 years of age. Individual 9 had focal seizures with epileptiform discharges on electroencephalogram (EEG). Individual 4 had seizures after a prolonged hypotensive episode and cardiac arrest at 28 days of life, followed by another possible seizure at 2 months. This individual had epileptiform discharges on EEG at 2 months. A fourth individual, individual 1, had clinical seizure-like activity, with daily staring spells lasting 1–2 min, but has not yet had a diagnostic EEG. Among the six individuals who had neuroimaging (brain magnetic resonance imaging [MRI] or computed tomography [CT]), two had abnormal structural results. Individual 9 had mild thinning of the corpus callosum with mild diffuse cerebral volume loss, and individual 11 had cortical dysplasia.

The affected individuals were smaller than average at birth when adjusted for gestational age. The average Z score for birth length was −1.59, with one individual being significantly below average with a Z score of −4.7. At most recent follow-up visits, there was a trend toward shorter stature; the average Z score for current height was −1.1. Two individuals also had low birth weights with a Z score of ≤ −2, and four individuals (4/13, 31%) had a low weight (Z score ≤ −2) at their most recent visit.

Dysmorphic facial features were observed in a majority of individuals (11/14, 79%), with facial features of individuals 1 and 6 shown in Figure 2. Common facial features included upslanting (3/11) or downslanting palpebral fissures (3/11), deep-set eyes (3/11), epicanthal folds (2/11), flattened nasal bridge (3/11), and frontal bossing (2/11). Five individuals had variable ear features, including low-set ears, ear tags, slightly posteriorly rotated ears, and large ears.

Congenital heart defects were also common within the cohort (7/14, 50%). The congenital heart disease was often complex and required cardiac surgery (Table 2). There were two cases of heterotaxy syndrome. These probands were also both found to have a midline liver, and one had a right-sided spleen, whereas another had a right-sided stomach. Individual 1 was also diagnosed prenatally with a non-obstructive right ventricular tumor that has since regressed.

Other common features among these fourteen individuals included genitourinary and gastrointestinal abnormalities. Genitourinary abnormalities were reported in six individuals (6/14, 43%) and included undescended testes in three individuals, hypospadias in two individuals, and hydrocele, chordee, horseshoe kidney, and urinary incontinence. Gastrointestinal anomalies were seen in three individuals (21%). They include midline livers in the setting of heterotaxy syndrome, jejunal atresia, microcolon, and right-sided stomach.

Skeletal abnormalities of the chest were noted in two individuals: one with non-union of the sternum and the other with pectus carinatum. Two individuals were diagnosed with a coagulopathy of unknown etiology. Individual 13 had a history of stage 4B mixed cellularity Hodgkin lymphoma, which was in remission at the time of her most recent visit, as well as a history of dermatomyositis. Other medical issues observed in one individual each included: pulmonary hypertension, chylous effusions, interstitial lung disease, polysplenia syndrome, functional asplenia, and neonatal thrombocytopenia.

Two individuals in the cohort (individuals 2 and 14) were found to have de novo pathogenic variants in other disease-associated genes in addition to their variants in FRYL. Individual 2 was reported to have a de novo pathogenic variant in SF3B4 (MIM: 605593), which is associated with autosomal dominant acrofacial dysostosis 1, otherwise known as Nager syndrome (MIM: 154400). This condition typically affects development of the face (down-slanted palpebral fissures, retrognathia, low-set ears, cleft palate, dental crowding observed in this individual) as well as pre-axial upper limb anomalies, such as radial hypoplasia or aplasia, small or absent thumbs, or radioulnar synostosis (none of which were observed in this individual).⁵² Typically, individuals with Nager syndrome have normal cognitive function and normal cardiac structure, although there are occasional reports of individuals with Nager syndrome with congenital heart defects, such as patent ductus arteriosus, ventricular septal defect, or tetralogy of Fallot.⁵³ Individual 2 presented with intellectual disability, hypotonia, and congenital heart defects, which cannot be explained by the SF3B4 variant. Individual 14 has a de novo pathogenic variant in SDHA (MIM: 600857) associated with an increased risk of pheochromocytoma and paraganglioma (MIM: 614165). However, SDHA is not associated with neurobehavioral phenotypes. Therefore, dual diagnoses were made for these two individuals.

Fly fry is the ortholog of human FRYL

To determine whether the FRYL variants are associated with the clinical features in the affected individuals, we used Drosophila melanogaster as the model organism. Drosophila fry is the ortholog of human FRYL and FRY with DIOPT scores⁵⁴ of 15/18 and 14/18, respectively. Fry and FRYL share 35% identity and 52% similarity (Figure S3). Most of the conserved domains in FRYL are also found in Fry, except for the leucine zipper/coiled-coil motifs (Figure 1A).² Among the five missense variants identified in the affected individuals, four (p.Phe1628Leu, p.Phe2295Ser, p.Ser2397Ile, and p.Tyr2951Cys) affect conserved amino acid residues in Fry, while the p.Arg110Cys variant affects a residue that is not conserved (Figure 1B).

Loss of fry causes lethality at an early developmental stage and morphological defects in mutant clones

Since most probands present with DD and various congenital anomalies, we first sought to determine whether fry is required for development. We first generated a fry^T2A-GAL4 allele by RMCE of a MiMIC insertion in fry^MI12326 allele (Figure 3A).³⁶^,⁴⁴^,⁵⁵^,⁵⁶ In this process, a splice acceptor (SA)-T2A-GAL4-polyA cassette is integrated in a coding intron shared by all fry transcripts.⁵⁵^,⁵⁷ The fry^T2A-GAL4 allele is predicted to be a severe loss-of-function (LoF) allele since the SA induces the inclusion of the artificial exon during transcription and the poly(A) sequence results in early transcription termination (Figure 3A).⁵⁷ We also used a previously reported fry¹ allele, which is a null allele caused by a frameshift that affects amino acid 403 and beyond,¹ as well as a deficiency that encompasses fry (Df(3L)BSC669, marked as fry^Df hereafter).

*Fry* is essential for fly development, and the loss of *fry* causes morphological defects in the wings and compound eyes

(A) Schematic of the generation of the *fry*^T2A-GAL4 allele. The *fry*^T2A-GAL4 allele is predicted to be a severe LoF allele. It also induces the expression of GAL4 protein under the control of the endogenous regulatory elements of *fry*, which can be used to determine the expression pattern using a fluorescent protein.

(B) Flies heterozygous for a severe LoF allele (*fry*¹) and a deficiency allele (*fry*^Df) are lethal at early developmental stages. The *fry*^T2A-GAL4 allele fails to complement both *fry*¹ and *fry*^Df alleles. The stage-specific lethality rates are indicated in the table. Lethality caused by the loss of *fry* is rescued by the introduction of a genomic rescue (GR) construct.

(C) *Fry*¹ homozygous mutant clones were generated in the fly wings using the FRT/FLP system. The loss of *fry* causes the clustered wing hair (furry) phenotype in clones.

(D) *Fry*¹ homozygous mutant clones were generated in the fly compound eyes using the FRT/FLP system. Homozygous *fry* mutant clones are not present, and the eyes are small and rough, indicating that loss of *fry* causes cell lethality in the developing eye. Scale bar, 200 μm.

Both fry¹ homozygous and fry¹/fry^Df compound heterozygous mutations are lethal at the embryonic, L1, or L2 larval stages (Figure 3B). The fry^T2A-GAL4 allele fails to complement the fry¹ and fry^Df alleles, and fry^T2A-GAL4 homozygous mutation is lethal at L2 and L3 larval or pupal stages (Figure 3B). These data suggest that fry^T2A-GAL4 is a strong LoF allele. The lethality associated with the fry^T2A-GAL4 homozygous mutation as well as the fry^T2A-GAL4/fry¹ and fry^T2A-GAL4/fry^Df mutations is successfully rescued by a genomic rescue (GR) construct (Dp(3:2)CH321-12N06), which carries a copy of the fry locus⁴⁰ (Figure 3B). These data demonstrate that the defects are indeed caused by the loss of fry and show that fry is essential during development.

To study the requirement of fry in the development of different tissues, we generated fry¹ homozygous clones in the wings and compound eyes using the FRT/FLP system.⁵⁸ We first generated clones in the wings using ubx-FLP. The cells in fry¹ clones form clustered wing hairs, which contrasts with the neighboring heterozygous/wild-type cells that form a single hair on each cell (Figure 3C). This result is consistent with previous reports¹^,⁶ and shows that the loss of fry causes defects in the development of the wing cells. In the compound eyes, we induced homozygous mutant clones using ey-FLP. The mutant clones are identified by their white color in contrast to the wild-type cells that are red. Normal eye size and smooth eye surface are observed when control FRT80B flies were used to generate clones (Figure 3D, left). In contrast, we did not observe homozygous mutant clones in the ey-FLP; ; fry¹ FRT80B/Rps17⁴ w⁺ FRT80B adult fly eyes. Moreover, the eyes with only wild-type cells are smaller and rough (Figure 3D, right), indicating that the homozygous mutant cells are eliminated during development.⁵⁹ This result shows that loss of fry disrupts eye development. Taken together, the results show that fry is essential for the development of multiple tissues.

Fry is predominantly expressed in neurons and localizes to the cytoplasm

FRYL is expressed in multiple tissues including the central nervous system (CNS) (GTEx).⁶⁰ Broad expression of fry is also observed in flies (FlyCellAtlas).⁶¹ In the cohort, many individuals are affected by neurological deficits, including intellectual disability, autism, and seizures. Hence, we explored the expression pattern of fry in the fly CNS. The fry^T2A-GAL4 allele carries a T2A-GAL4 sequence, which induces the expression of the GAL4 transcription factor under the control of the endogenous regulatory elements of fry. The viral T2A sequence causes a skipping of the formation of peptide bond at the C-terminus of the T2A sequence during translation, leading to the production of untagged GAL4 (Figure 3A).⁵⁵^,⁵⁷ We examined fry expression pattern by crossing the fry^T2A-GAL4 allele to a UAS-mCherry.nls (nuclear-localized mCherry fluorescent protein). By co-staining with the pan-neuronal marker Elav or the pan-glial marker Repo, we found that the mCherry signals partially overlap with anti-Elav signals (Figure 4A), but not with the anti-Repo signals (Figure 4B) in larval CNS and adult brains. Hence, fry is predominantly expressed in neurons but not in glia in the fly CNS. In the larval CNS, fry is expressed in a subset of neurons and the expression is wider and stronger in the ventral nerve cord than in the brain lobes. In the adult brain, fry is expressed in most neurons (Figure 4A). This expression pattern of fry is very similar to para, which encodes a voltage-gated sodium channel in flies.⁶² para is only expressed in functionally active neurons, and most neurons in fly larvae (80%) are not active. Hence, para is expressed in a small subset of neurons in the larval CNS but is broadly expressed in the adult.⁶² The expression pattern of fry suggests that fry is likely expressed in functionally active neurons. Given that human FRYL interacts with a nuclear-localized active form of NOTCH1,¹⁹ we next examined the subcellular localization of Fry in the fly adult brains. To achieve this, we inserted an EGFP-FlAsH-StrepII-TEVcs-3xFLAG (GFSTF) cassette into the MiMIC site in the fry^MI12326 allele via RMCE.⁵⁶ The GFSTF sequence functions as an artificial exon,⁵⁶ which inserts a GFP tag in Fry (Figure S1). The resulting fry^GFP allele reverts the lethality caused by the MiMIC insertion and the fry^GFP homozygous animals are fully viable, indicating that the GFP tag does not disrupt protein function. As shown in Figure 4C, the GFP-tagged Fry proteins mainly localize to the cytoplasm of neuronal cell bodies as well as the neuropil in the adult brains.

*Fry* is expressed in neurons in the fly CNS and localizes to the cytoplasm

(A and B) The expression of nuclear localized mCherry (mCherry.nls) was driven by the *fry*^T2A-GAL4 allele (*fry*^T2A-GAL4> mCherry.nls). The larval CNS and adult brain of *fry*^T2A-GAL4> mCherry.nls animals were immunostained with neuronal (Elav, A) or glial marker (Repo, B). Maximum projections of confocal z stack images are shown. *Fry* is expressed in neurons (A) but not in glia (B) in the fly CNS. In the larval CNS, *fry* is expressed more widely and strongly in the ventral nerve cord (yellow solid square) than in the brain lobes (yellow dashed square) (A). Single-plane, high magnification images of the regions indicated by the white dashed squares are shown on the right to visualize the colocalizations between mCherry and the immunostaining signals. Colocalizations are indicated by the arrows. Scale bars, 100 μm.

(C) The localization of Fry in neurons was visualized by the GFP-tagged protein expressed by the *fry*^GFP allele. A single-plane image of the adult brain is shown. Fry localizes to the neuropil areas and the cytoplasm in the neuron bodies. Nuclei are marked by 4',6-diamidino-2-phenylindole (DAPI) staining (magenta). High magnification images of the regions indicated by the white dashed squares are shown on the right to visualize the localization of Fryin the cell bodies. Scale bar, 100 μm.

Fry knockin alleles to study the effects of the human FRYL missense variants

To assess the function of the four FRYL missense variants, we generated fry gene knockin alleles by introducing analogous amino acid changes into the endogenous fry gene. Four FRYL variants, encoding p.Phe1628Leu, p.Phe2295Ser, p.Ser2397Ile, and p.Tyr2951Cys, correspond to the fry^p.Phe2024Leu, fry^p.Phe2746Ser, fry^p.Ser2910Ile, and fry^p.Tyr3410Cys alleles, respectively. The FRYL p.Arg110Cys variant was not modeled as the affected residue is not conserved. The p.Ser2397Ile variant is found in the gnomAD database and the variant was not confirmed de novo. However, we modeled this variant since it is predicted to be deleterious by in silico algorithms (Table 1), and the affected residue is highly conserved across species (Figure 1B).

We first used the scarless prime editing (PE) method³⁷^,⁶³ (for details see material and methods) and were able to generate two alleles (fry^p.Phe2024Leu and fry^p.Phe2746Ser) upon two trials. As we were not able to generate the other two alleles, we developed an alternative strategy, which is based on RMCE and SSA mechanisms and causes a small intronic insertion in fry (Figure S2).⁴¹^,⁴²^,⁴³ This approach is clearly more efficient and allowed us to recover all four alleles upon the first trial. Hereafter, we denominate the nature of the alleles with PE and RMCE to refer to the corresponding method: for example, fry^{p.Phe2024Leu_PE} versus fry^{p.Phe2024Leu_RMCE}. Since the RMCE-based method introduces a small intronic insertion, a fry^WT_RMCE control allele was generated, which has the same intronic insertion but no coding variant.

The fry p.Phe2746Ser variant phenocopies severe LoF variants

The fry alleles were first examined by complementation tests. Both fry^{p.Phe2746Ser_PE} and fry^{p.Phe2746Ser_RMCE} alleles fail to complement the fry¹, fry^Df, and fry^T2A-GAL4 alleles. The compound heterozygous animals die at the embryo/L1/L2 stages, and these animals are rescued by a GR construct (Figure 5A). We next tested the fry^p.Phe2746Ser alleles in the wings and compound eyes. The fry^p.Phe2746Ser homozygous wing clones lead to clustered wing hairs. It also causes aberrant compound eyes, which exhibit a rough eye phenotype and no visible homozygous clones (Figures 5B and 5C). These results show that both fry^p.Phe2746Ser PE and RMCE alleles phenocopy the fry¹ allele, suggesting that the p.Phe2746Ser variant causes a very severe LoF of Fry.

The *fry* p.Phe2746Ser variant, but not the other assayed variants, phenocopies severe LoF variants

(A) The *fry* p.Phe2746Ser variant (both PE and RMCE alleles) fails to complement the fry severe LoF variants, while the other three variants complement the severe LoF variants. The lethality of both *fry*^{p.Phe2746Ser_PE}*/fry*¹ and *fry*^{p.Phe2746Ser_RMCE}*/fry*¹ animals are rescued by the introduction of the genome rescue construct.

(B) *Fry* homozygous mutant clones were generated in the fly wings using the FRT/FLP system. Both *fry*^{p.Phe2746Ser_PE} and *fry*^{p.Phe2746Ser_RMCE} alleles cause clustered wing hair phenotype in the homozygous mutant clones, phenocopying the severe LoF *fry*¹ allele.

(C) *Fry* homozygous mutant clones were generated in the fly compound eyes using the FRT/FLP system. Both *fry*^{p.Phe2746Ser_PE} and *fry*^{p.Phe2746Ser_RMCE} alleles cause cell lethality in the homozygous mutant clones as well as small and rough eyes, phenocopying the severe LoF *fry*¹ allele.

(D) The wings of *fry*^{p.Phe2024Leu_RMCE}*/fry*¹, *fry*^{p.Ser2910Ile_RMCE}*/fry*¹, and *fry*^{p.Tyr3410Cys_RMCE}*/fry*¹ flies do not exhibit clustered wing hair phenotype.

(E) The compound eyes of *fry*^{p.Phe2024Leu_RMCE}*/fry*¹, *fry*^{p.Ser2910Ile_RMCE}*/fry*¹, and *fry*^{p.Tyr3410Cys_RMCE}*/fry*¹ flies do not exhibit any obvious morphological defect.

The fry p.Phe2024Leu and p.Tyr3410Cys variants behave as partial LoF variants

In contrast to the p.Phe2746Ser variant, the p.Phe2024Leu, p.Ser2910Ile, and p.Tyr3410Cys variants complement the lethality of the severe LoF alleles (Figure 5A) and do not cause obvious morphological defects in wings or compound eyes (Figures 5D and 5E). To determine whether these variants cause partial LoF effects, we crossed them to fry¹ flies and assessed additional phenotypes in the compound heterozygous animals. Since the studied cohort presents with DD, we first measured the time courses of development from egg laying to L2/L3 molting and the formation of puparia. Compared with the fry^WT_RMCE/fry¹ controls, the fry^{p.Phe2024Leu_RMCE}/fry¹ and fry^{p.Tyr3410Cys_RMCE}/fry¹ animals exhibit a significant developmental delay, and the delay is partially rescued by a GR construct. In contrast, the fry^{p.Ser2910Ile_RMCE}/fry¹ animals do not show any delay (Figures 6A and 6B). Given that the loss of fry causes defects in eye development (Figure 3E) and that fry is expressed in adult photoreceptors (FlyCellAtlas, Figure S4),⁶¹ we performed electroretinograms (ERGs) to assess whether the fry variants cause defects in neuronal activities. In an ERG (Figure 6C), the amplitude measures the activity of photoreceptors in response to photons upon light exposure, while the ON/OFF transients indicate the synaptic transmission between photoreceptors and the postsynaptic neurons.⁶⁴ We observed significantly reduced ON and OFF transients in the fry^{p.Phe2024Leu_RMCE}/fry¹ flies, suggesting an impaired synaptic transmission. However, no significant change was observed in the amplitude (Figures 6C–6F). No ERG defect was observed in the fry^{p.Ser2910Ile_RMCE}/fry¹ and fry^{p.Tyr3410Cys_RMCE}/fry¹ animals (Figures 6C–6F).

*Fry* p.Phe2024Leu and p.Tyr3410Cys variants behave as partial LoF variants

(A and B) The time courses from egg laying to L2/L3 molting (A) and to puparia formation (B) were measured in animals heterozygous for a tested missense allele and *fry*¹ allele. Compared with the *fry*^p.WT_RMCE/*fry*¹ controls, *fry*^{p.Phe2024Leu_RMCE}/*fry*¹ and *fry*^{p.Tyr3410Cys_RMCE}/*fry*¹ animals present with developmental delays in both measurements. The delays are rescued by the introduction of a genome rescue (GR) construct. The *fry*^{p.Ser2910Ile_RMCE}/*fry*¹ animals do not show any delay in development.

(C–F) ERGs were recorded in animals heterozygous for a tested missense allele and *fry*¹ allele (C). ON (D) and OFF (E) transients as well as amplitudes (F) were quantified. The ON and OFF transients in *fry*^{p.Phe2024Leu_RMCE}/*fry*¹ flies significantly decrease compared with the control and GR construct rescues the decreases. In contrast, *fry*^{p.Ser2910Ile_RMCE} and *fry*^{p.Tyr3410Cys_RMCE} do not cause a significant change in ERG.

(A, B, and D–F) Results are presented as means ± SEM. Numbers of animals (n values) in each group are indicated under the bars. Results in (A) and (B), as well as results in (D–F), were obtained from the same samples. Statistical analyses were performed via unpaired Student’s t test. ns, not significant; ^∗∗p < 0.01; ^∗∗∗p < 0.001; ^∗∗∗∗p < 0.0001.

Since some studied probands are affected by neurological deficits, we also explored other possible neurobehavioral phenotypes including locomotor activity and sensitivity to heat and physical stimulations and found that none of the variants cause obvious phenotypes in these assays (Figures S5A–S5C). Taken together, the results support that the fry p.Phe2024Leu and p.Tyr3410Cys variants behave as partial LoF variants, although they are weak hypomorphs. In contrast, the p.Ser2910Ile variant is not associated with any phenotype in any assay, suggesting that the variant does not cause any detrimental effect in Fry.

Discussion

Here, we describe a cohort of fourteen unrelated individuals with heterozygous variants in FRYL. The most common clinical features were DD, intellectual disability, and facial dysmorphic features. Other features include neurobehavioral differences, autism spectrum disorder, hypotonia, and seizures, as well as congenital anomalies in cardiovascular, skeletal, gastrointestinal, renal, and urogenital systems. We provide human genetic evidence in combination with evidence from fruit flies that support that the variants correspond to LoF alleles. In this cohort, nine individuals are heterozygous for variants that lead to premature stop codons, frameshifts, or splicing defects. In a previous study, one individual with a de novo heterozygous variant (c.6121C>T [p.Arg2041^∗]) in the DDD cohort was identified who had short stature and craniofacial and cardiac defects, but no other information was provided.²² In the ClinVar database, three frameshift variants and one splicing variant have been documented. The clinical features of the individuals with frameshift variants were not reported, while the individual with a heterozygous splicing variant had brisk reflexes, central hypotonia, central hypoventilation, delayed myelination, abnormal cerebral white matter morphology, and abnormal EEG (ClinVar accession: VCV001333288.1). The nine individuals reported in this study and the former two individuals described in DDD and ClinVar indicate that FRYL is haploinsufficient in humans. This is also supported by population genetic data from the gnomAD database that show that FRYL is intolerant to LoF (pLI = 1).

Five individuals in the studied cohort have a missense variant in FRYL. We modeled four out of the five variants, which share conserved amino acid residues in Fry, the fly ortholog of FRYL. By creating four fry knockin alleles, we assessed the effect of the missense variants in flies and showed that one variant (fry p.Phe2746Ser, analogous to FRYL p.Phe2295Ser) behaves as a severe LoF variant, whereas two variants (fry p.Phe2024Leu and p.Tyr3410Cys, analogous to FRYL p.Phe1628Leu and p.Tyr2951Cys, respectively) behave as partial LoF variants. The fry p.Ser2910Ile variant does not cause any phenotype in our fly assays. The corresponding FRYL p.Ser2397Ile variant is predicted to be pathogenic by multiple in silico algorithms. However, it is observed in gnomAD and was not confirmed de novo in the affected individual. Moreover, the affected individual is the only one in the cohort who did not present with DD. Due to the conflicting evidence, we argue that this variant is a variant of uncertain significance (VUS). This individual has a heterotaxy phenotype and may have variant(s) in a yet undescribed heterotaxy-associated gene. Without performing the same standardized quantitative assessments across the limited number of probands, it is difficult to compare the severity of phenotypes in the cohort, including the probands with the missense variants. The potential genotype-phenotype correlation could be studied in the future.

We frequently study human disease variants in fruit flies by integrating a T2A-GAL4⁵⁷^,⁶⁵ or Kozak-GAL4⁶⁶ to create a strong LoF allele. GAL4 is expressed in the proper spatial and temporal expression pattern and typically allows to drive the UAS-human reference cDNA and rescue the LoF phenotypes in 50%–60% of the cases.⁶⁷ Unfortunately, a human FRYL cDNA is not available because the encoded protein is very large (3,013 amino acids); this is a common issue for large genes. Therefore, we generated the four fry knockin alleles in to allow the study of the human FRYL variants. Knockin animal models have been used to study genetic disorders caused by known pathogenic variants,⁶⁸^,⁶⁹^,⁷⁰^,⁷¹ but the strategy used in these previous studies is very labor intensive and time consuming. Therefore, we first tried the recently developed PE methodology,³⁷^,⁶³ but we were only able to obtain two variants upon two trials. We then switched to an RMCE- and SSA-based mutagenesis strategy based on previously developed methods.⁴¹^,⁴²^,⁴³ The latter approach allowed us to generate the four alleles in the first trial. Importantly, a visible marker (white⁺) was employed for the selection of the positive lines in the RMCE-SSA strategy, which is not available in the PE strategy, and improved the selection process. Given that RMCE-compatible MiMIC or CRISPR-mediated integration cassette (CRIMIC) sites are available for ∼7,000 genes,⁴⁴^,⁵⁶^,⁶⁵^,⁶⁶^,⁷² this efficient gene knockin strategy can be used to assess many human variants.

Using established LoF alleles and knockin missense alleles analogous to human variants, we showed that fry is required for fly development, wing hair morphogenesis, and neuronal communication in the retina. Previous studies also showed that the loss of fry causes arborization defects of sensory dendrites,⁷^,⁸ supporting that fry plays a role in neural development. In a previous study, knock down of fryl in zebrafish causes cardiac and craniofacial defects during development.²² These observations are not inconsistent with the observations that the individuals in the cohort have neuronal issues, including intellectual disability, autism spectrum disorder, and seizures, as well as defects in morphogenesis in multiple organs. However, additional experiments will be required in model organisms to assess the roles of the Furry genes and their linkage to the human phenotypes.

Humans have two Furry proteins, FRYL and FRY. Although the homology between the two paralogs is significant, the functional redundancy between these genes is not. Biallelic variants in FRY have been observed in individuals with intellectual disability in three consanguineous families.¹⁵^,¹⁶^,¹⁷ Intellectual disability is also observed in 8/13 individuals in the studied cohort. However, no other FRYL-associated phenotypes were reported in the individuals with FRY variants. Homozygous Fryl mutant mice present with developmental retardation and kidney developmental defects, but these animals were not assessed for neurological issues and most of the animals died soon after birth.²¹ Fry mutant mice were reported in one forward genetic screen for defects in skeletal development. Mice homozygous for a frameshift Fry variant present with reduced tibia, femur, and pelvis lengths. However, no other phenotypic information was reported.⁷³ In the cohort reported here, a renal abnormality was reported in one individual (horseshoe kidney in individual 5), while skeletal defects were observed in two individuals (non-union of the sternum in individual 4 and pectus carinatum in individual 7). The molecular functions of mammalian Furry proteins are largely unknown. In non-mammalian model organisms, the best characterized activity of Furry proteins is their interactions with NDR (nuclear dbf2-related) family kinases in multiple species.¹^,²^,⁴^,⁵^,⁶^,⁷ In flies, loss of an NDR kinase tricornered (trc) phenocopies fry mutants in dendritic tiling and wing hair development, providing evidence that both genes play a role in the same pathway.⁶^,⁷ Similar genetic and physical interactions have also been reported in yeasts and worms.²^,⁴^,⁵ In humans, FRY interacts with the NDR kinase NDR1 to regulate chromosome alignment during mitosis.¹⁰ However, it is not known whether FRYL interacts with NDR kinases. The NDR1/2 encoding genes STK38 (MIM: 606964) and STK38L (MIM: 615836) have not yet been associated with inherited disorders. Both Stk38 and Stk38l homozygous mutant mice are viable⁷⁴^,⁷⁵ but show defects in retina development,⁷⁶ indicating a functional redundancy. Stk38l mutant mice also show abnormal dendrite morphology in neurons, consistent with the findings in flies that the loss of fly NDR (Trc) causes defects in dendritic tiling.⁷⁵ In summary, studies focusing on the biological functions of FRYL during development will provide a better understanding of the mechanisms underlying disease pathogenesis.

Consortia

The members of the Center for Precision Medicine Models (CPMM) at Baylor College of Medicine are Lindsay C. Burrage, Jason D. Heaney, Seon-Young Kim, Denise G. Lanza, Zhandong Liu, Dongxue Mao, Aleksander Milosavljevic, Sandesh C.S. Nagamani, Jennifer E. Posey, Uma Ramamurthy, Vivek Ramanathan, Jeffrey Rogers, Jill A. Rosenfeld, Matthew Roth, and Ramin Zahedi Darshoori.

Data and code availability

All reagents developed in this study are available upon request. The exome datasets supporting this study have not been deposited to a public database due to privacy issues. We cannot release the full exomes/genomes because the condition is so rare and the clinical characteristics so specific that the participants could be identifiable.

Acknowledgments

We thank the participants and their families for supporting this study. We thank the Bellen and Yamamoto lab members for their discussion and suggestions. We thank Ms. Wen-Wen Lin, Ying Fang, and Junyan Fang for fly embryo injection. We thank the BDSC for fly stocks, the DSHB for antibodies, and the DGRC for plasmids.

H.J.B., S.Y., and O.K. are members of the CPMM. The CPMM was supported by the Office of Research Infrastructure Programs (ORIP) of the NIH (award U54OD030165). H.J.B. was supported by the ORIP (award R24OD031447), the Huffington Foundation, and the Duncan Neurological Research Institute at Texas Children’s Hospital. The work was also supported by the Baylor College of Medicine IDDRC (P50HD103555) from the Eunice Kennedy Shriver National Institute of Child Health and Human Development of the NIH for use of the Microscopy Core facilities. W.K.C. was supported by the National Heart, Lung, and Blood Institute (NHLBI) of the NIH (award U01HL131003). A.R.-P. was supported by the National Center for Advancing Translational Sciences of the NIH (award UL1TR001873). L.O. and D.A.S. were supported by the American Institute for Neuro-Integrative Development (AIND) and The Hill Family Fund for the Diagnosis, Management of Rare and Undiagnosed Diseases at Mass General. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. A.P.A.S. and M.S. are members of the European Reference Network on Rare Congenital Malformations and Rare Intellectual Disability ERN-ITHACA (EU Framework Partnership agreement ID: 3HP-HP-FPA ERN-01-2016/739516).

Declaration of interests

W.K.C. is a member of the Board of Directors of Prime Medicine and RallyBio. R.P. is an employee of GeneDx, LLC.

Published: March 12, 2024

Footnotes

Supplemental information can be found online at https://doi.org/10.1016/j.ajhg.2024.02.007.

Contributor Information

Wendy K. Chung, Email: wendy.chung@childrens.harvard.edu.

Hugo J. Bellen, Email: hbellen@bcm.edu.

Baylor College of Medicine Center for Precision Medicine Models:

Lindsay C. Burrage, Jason D. Heaney, Seon-Young Kim, Denise G. Lanza, Zhandong Liu, Dongxue Mao, Aleksander Milosavljevic, Sandesh C.S. Nagamani, Jennifer E. Posey, Uma Ramamurthy, Vivek Ramanathan, Jeffrey Rogers, Jill A. Rosenfeld, Matthew Roth, and Ramin Zahedi Darshoori

Web resources

CADD, https://cadd.gs.washington.edu/

ClinVar, https://www.ncbi.nlm.nih.gov/clinvar/

DGV, http://dgv.tcag.ca/

DIOPT, http://www.flyrnai.org/diopt/

FlyCellAtlas, https://scope.aertslab.org/#/FlyCellAtlas/

gnomAD, https://gnomad.broadinstitute.org/

GTEx, https://gtexportal.org/

M-CAP, http://bejerano.stanford.edu/mcap/

MutationTaster, http://www.mutationtaster.org/

OMIM, https://omim.org/

PolyPhen2, http://genetics.bwh.harvard.edu/pph2/

PROVEAN, https://www.jcvi.org/research/provean/

REVEL, https://sites.google.com/site/revelgenomics/

SIFT, https://sift.bii.a-star.edu.sg/

Supplemental information

Document S1. Figures S1–S5 and Tables S1–S3

mmc1.pdf^{(2.9MB, pdf)}

Document S2. Article plus supplemental information

mmc2.pdf^{(7.6MB, pdf)}

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Associated Data

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

Supplementary Materials

Document S1. Figures S1–S5 and Tables S1–S3

mmc1.pdf^{(2.9MB, pdf)}

Document S2. Article plus supplemental information

mmc2.pdf^{(7.6MB, pdf)}

Data Availability Statement

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PERMALINK

De novo variants in FRYL are associated with developmental delay, intellectual disability, and dysmorphic features

Xueyang Pan

Alice M Tao

Shenzhao Lu

Mengqi Ma

Shabab B Hannan

Rachel Slaugh

Sarah Drewes Williams

Lauren O'Grady

Oguz Kanca

Richard Person

Melissa T Carter

Konrad Platzer

Franziska Schnabel

Rami Abou Jamra

Amy E Roberts

Jane W Newburger

Anya Revah-Politi

Jorge L Granadillo

Alexander PA Stegmann

Margje Sinnema

Andrea Accogli

Vincenzo Salpietro

Valeria Capra

Lina Ghaloul-Gonzalez

Martina Brueckner

Marleen EH Simon

David A Sweetser

Kevin E Glinton

Susan E Kirk

Michael F Wangler

Shinya Yamamoto

Wendy K Chung

Hugo J Bellen

Summary

Introduction

Figure 1.

Material and methods

Recruitment and sequencing of individuals

Figure 2.

Table 1.

Table 2.

Drosophila stocks and maintenance

Generation of the fryGFP allele

Generation of fry knockin lines by prime editing

Generation of fry knockin lines by an RMCE- and single-strand annealing-based strategy

Immunofluorescence staining and confocal microscopy

Drosophila behavioral assays

Developmental time course measurement

Electroretinogram recording

Statistical analysis

Results

Individuals with heterozygous variants in FRYL exhibit neurodevelopmental defects, dysmorphic facial features, and congenital anomalies

Fly fry is the ortholog of human FRYL

Loss of fry causes lethality at an early developmental stage and morphological defects in mutant clones

Figure 3.

Fry is predominantly expressed in neurons and localizes to the cytoplasm

Figure 4.

Fry knockin alleles to study the effects of the human FRYL missense variants

The fry p.Phe2746Ser variant phenocopies severe LoF variants

Figure 5.

The fry p.Phe2024Leu and p.Tyr3410Cys variants behave as partial LoF variants

Figure 6.

Discussion

Consortia

Data and code availability

Acknowledgments

Declaration of interests

Footnotes

Contributor Information

Web resources

Supplemental information

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

Generation of the fry^GFP allele