Review of the phenotypic spectrum associated with haploinsufficiency of MYRF

Abstract

The myelin regulatory factor gene (MYRF) encodes a transcription factor that is widely expressed. There is increasing evidence that heterozygous loss-of-function variants in MYRF can lead to abnormal development of the heart, genitourinary tract, diaphragm, and lungs. Here, we searched a clinical database containing the results of 12,000 exome sequencing studies. We identified three previously unreported males with putatively deleterious variants in MYRF: one with a point mutation predicted to affect splicing and two with frameshift variants. In all cases where parental DNA was available, these variants were found to have arisen de novo. The phenotypes identified in these subjects included a variety of congenital heart defects (hypoplastic left heart syndrome, scimitar syndrome, septal defects, and valvular anomalies), genitourinary anomalies (ambiguous genitalia, hypospadias, and cryptorchidism), congenital diaphragmatic hernia, and pulmonary hypoplasia. The phenotypes seen in our subjects overlap those described in individuals diagnosed with PAGOD syndrome [MIM# 202660], a clinically defined syndrome characterized by pulmonary artery and lung hypoplasia, agonadism, omphalocele, and diaphragmatic defects that can also be associated with hypoplastic left heart and scimitar syndrome. These cases provide additional evidence that haploinsufficiency of MYRF causes a genetic syndrome whose cardinal features include congenital heart defects, urogenital anomalies, congenital diaphragmatic hernia, and pulmonary hypoplasia. We also conclude that consideration should be given to screening individuals with PAGOD for pathogenic variants in MYRF, and that individuals with MYRF deficiency who survive the neonatal period should be monitored closely for developmental delay and intellectual disability.

INTRODUCTION

The myelin regulatory factor gene (MYRF; MIM# 608329) encodes an endoplasmic reticulum membrane protein that undergoes auto-processing to release its N-terminal fragment which enters the nucleus, forms a homo-trimer, and functions as a transcription factor [Bujalka et al., 2013; Kim et al., 2017; Li et al., 2013]. MYRF was first noted to be a key transcription factor for oligodendrocyte differentiation and central nervous system myelination [Emery et al., 2009; Hornig et al., 2013; Kim et al., 2017; Koenning et al., 2012]. In keeping with that function, Kurahashi et al. have described nine individuals from two unrelated Japanese families with mild encephalitis/encephalopathy and reversible myelin vacuolization (MIM# 618113) who carried the same heterozygous c.1208A>G, p.(Gln403Arg) variant in MYRF [Kurahashi et al., 2018]. This variant leads to a single amino acid substitution in the highly conserved DNA-binding domain of MYRF and causes decreased transcriptional activity in luciferase assays.

MYRF is also expressed outside of the central nervous system, and there is increasing evidence that it plays a critical role in the development of various organs including the heart, lungs, diaphragm, and genitourinary tract [Chitayat et al., 2018; Homsy et al., 2015; Jin et al., 2017; Nagase et al., 1999; Pinz et al., 2018; Qi et al., 2018; Stohr et al., 2000]. Pinz et al. described two males with scimitar syndrome [MIM# 106700] and other features including penoscrotal hypospadias, cryptorchidism, pulmonary hypoplasia, tracheal anomalies, congenital diaphragmatic hernia, cleft spleen, thymic involution, and thyroid fibrosis who were found to have likely pathogenic variants in MYRF [Pinz et al., 2018]. Chitayat et al. then described a male fetus with hypoplastic left heart sequence, atretic/dysplastic aortic, mitral, and tricuspid valves, hypoplasia of the ascending aorta, total anomalous pulmonary venous connection to the right atrium, mild pulmonary hypoplasia, intestinal malrotation, and ambiguous genitalia who also had a likely pathogenic variant in MYRF [Chitayat et al., 2018]. Based on these reports, a new genetic syndrome, cardiac-urogenital syndrome [MIM# 618280], was defined.

Qi et al. subsequently reported ten individuals with de novo missense or frameshift variants in MYRF who were identified in large-scale screens of individuals with congenital heart defects and/or congenital diaphragmatic hernia [Homsy et al., 2015; Jin et al., 2017; Qi et al., 2018]. All these individuals had congenital heart defects, six had congenital diaphragmatic hernia, and one had a right hemidiaphragm eventration. This report provided strong evidence that MYRF not only plays a role in cardiac and urogenital development, but also in diaphragm development.

Here we describe three previously unreported males with deleterious MYRF variants that are predicted to lead to a loss of MYRF activity.

MATERIALS AND METHODS

Editorial Policies and Ethical Considerations

The parents of Subjects 1 and 2 provided informed consent and they were enrolled in an institutional review board-approved research study. The clinical and molecular description of Subject 3 is based solely on anonymized clinical data. This study was conducted in accordance with the ethical standards of the institution’s committee on human research and were in keeping with international standards.

Exome Sequencing and In Silico Prediction of the Effects of Sequence Variants

Exome sequencing studies were performed on a clinical basis at Baylor Genetics. MutationTaster (http://www.mutationtaster.org/) was used to predict the effects of sequence variants on protein function. This program takes into account both the potential effects of amino acid substitution and effects on splice junctions. Human Splicing Finder version 3.1 (http://www.umd.be/HSF3/) was also used to predict the effects of sequence variants on splicing.

RESULTS

We searched a clinical database containing the results of 12,000 exome sequencing studies. We identified three previously unreported males (Subjects 1–3) with putatively deleterious variants in MYRF. Their clinical phenotypes and molecular findings are summarized in Table 1 and are described in detail below. All MYRF variants reported are based on MYRF transcript variant 2 (NM_001127392.2).

Table 1.

Summarized genotype and phenotype information for individuals currently reported in the literature carrying variants in MYRF with structural birth defects affecting the heart, lungs, diaphragm, and/or genitourinary system

	Genotype^	Sex	Congenital heart disease	Genitourinary anomalies	Diaphragm anomalies	Pulmonary hypoplasia	Other	Outcome
Pinz et al. Subject 1	c.2336+1G>A	M	Scimitar syndrome	Hypospadias, micropenis, cryptorchidism	-	+	Speech delay	Alive at 18 months old
Pinz et al. Subject 2	c.2518C>T p.(Arg840*)	M	Scimitar syndrome	Persistent urachus	CDH	+	-	Died at 10 days
Chitayat et al. Subject 1	c.1254_1255dupGA p.(Thr419Argfs*14)	M	Hypoplastic left heart syndrome	Ambiguous genitalia	-	+	Intestinal malrotation	Pregnancy terminated at 19 weeks
Qi et al. Subject 1	c.235dupG p.(Gly81Trpfs*45)	M	Tetralogy of Fallot, ASD, VSD	Cryptorchidism	Left CDH	-	-	Unknown
Qi et al. Subject 2	c.1303G>A p.(Gly435Arg)	F	VSD	No internal sex organs, blind-ending vagina	Left CDH	-	Accessory spleen	Unknown
Qi et al. Subject 3	c.2036T>C p.(Val679Ala)	M	ASD, VSD	Unknown	Left CDH	-		Deceased
Qi et al. Subject 4	c. 2084G>A p.(Arg695His)	M	Hypoplastic left heart syndrome	Ambiguous genitalia, cryptorchidism	CDH	-	Intellectual disability and motor delay	Alive at 2 years old
Qi et al. Subject 5	c.1904–1G>A	F	Scimitar syndrome	Unknown	Right CDH	-	-	Deceased
Qi et al. Subject 6	c.1904–1G>A	F	Hypoplastic left heart syndrome	Unknown	-	-	-	Deceased
Qi et al. Subject 7	c.1786C>T p.(Gln596*)	M	Dextrocardia	Female-appearing external genitalia	-	+	-	Unknown
Qi et al. Subject 8	c.1160T>C p.(Phe387Ser)	M	Aortic arch hypoplasia, coarctation of aorta, hypoplastic left heart syndrome	Ambiguous genitalia, hypospadias, cryptorchidism	-	-	-	Unknown
Qi et al. Subject 9	c.1209G>C p.(Gln403His)	M	Scimitar syndrome, aortic arch hypoplasia, ASD, bicuspid aortic valve, hypoplastic left heart syndrome, mitral stenosis, VSD	Cryptorchidism	Right hemidiaphragm eventration	+	-	Unknown
Qi et al. Subject 10	c.1435C>G p.(Leu479Val)	M	Bicuspid aortic valve, coarctation of aorta	Female-appearing external genitalia	-	-	Short stature	Unknown
This report Subject 1	c.3118A>G p.(Arg1040Gly)	M	Scimitar syndrome, hypoplastic left heart syndrome, mitral valve atresia, hypoplastic aortic valve, VSD, ASD, PDA	-	Right-sided diaphragm elevation	+	-	Alive at 3 months
This report Subject 2	c.3239dupA p.(Glu1081Glyfs*5)	M	Hypoplastic left heart syndrome	Hypospadias, cryptorchidism, chordee	Left-sided CDH	+	-	Died at 4 weeks
This report Subject 3	c.350_366delinsT p.(Gly117Valfs*31)	M	-	Ambiguous genitalia, hypospadias, horseshoe kidney	CDH	-	-	Unknown
Percent			15/16 (94%)	12/16 (75%)	10/16 (62.5%)	7/16 (44%)

Based on transcript NM_001127392.2. + = reported, - = not reported, ASD = atrial septal defect, CDH = congenital diaphragmatic hernia, F = female, M = male, PDA = patent ductus arteriosus, VSD = ventricular septal defect

Subject 1

Subject 1 is a male born at 38 weeks gestation. He was known to have a cardiac malformation based on ultrasound, but the pregnancy had otherwise been uncomplicated. Birth weight was 2889g (16^th centile), length was 48cm (16^th centile), and head circumference was 33cm (11^th centile). A postnatal echocardiogram showed hypoplastic left heart syndrome with mitral valve atresia, hypoplasia of the aortic valve annulus, a muscular ventricular septal defect, an atrial septal defect, patent ductus arteriosus, and partial anomalous pulmonary venous return to the inferior vena cava. He was also been noted to have right-sided pulmonary hypoplasia with concomitant right-sided diaphragmatic elevation consistent with a diagnosis of scimitar syndrome. Additional imaging studies, including a renal ultrasound and brain MRI, were normal. No other anomalies were noted on physical examination. He underwent the first steps of surgical repair at 4 weeks of life and was eventually discharged home at 8 months of age.

Array-based copy number variant (CNV) analysis revealed two changes. The first was a 1.5 kb loss on chromosome 3q13.2, including the last 2 exons of CD96 [MIM# 606037]. CD96 has been proposed as a gene responsible for an autosomal dominant form of C syndrome (Opitz trigonocephaly) (MIM# 211750), but has a predicted loss-of-function intolerance (pLI) score in the gnomAD database of 0.0 [http://gnomad.broadinstitute.org/ accessed 11/9/2018]. The second change was a 25 kb loss on chromosome 8q21.3, which included exon 3 of CNGB3 (MIM# 605080). This gene has been associated with two autosomal recessive conditions, achromatopsia 3 (MIM# 262300) and juvenile macular degeneration (MIM# 248200).

Exome sequencing demonstrated a heterozygous de novo c.3118A>G, p.(Arg1040Gly) missense variant in MYRF (NM_001127392.2). This variant is not reported in gnomAD and is highly conserved down to mice (Figure 1). The 3118 position is also located just two base pairs away from the exon 23/intron 23 junction (Figure 1) and may result in breakage of the wild-type donor splice site based on an in silico analysis using Human Splicing Finder version 3.1. This variant is also predicted to be “disease causing” by MutationTaster which takes into consideration both conservation and possible effects on splicing.

Figure 1. The heterozygous de novo c.3118A>G, p.(Arg1040Gly) missense variant in MYRF seen in Subject 1 affects a conserved amino acid and may also affect splicing.

A) The arginine at position 1040 is conserved down to mice. B) The c.3118A>G change occurs close to the exon 23/intron 23 junction and is predicted to disrupt the associated donor splice site.

Subject 2

Subject 2 was a male born at 37 weeks gestation. He was prenatally diagnosed with hypoplastic left heart syndrome, and a left-sided congenital diaphragmatic hernia. The pregnancy had been complicated by maternal kidney infection requiring treatment with a course of antibiotics. Birth weight was 2685g (7^th centile), length was 54.5cm (86^th centile), and head circumference was 33.5cm (2^nd centile). His physical exam was also notable for genitourinary anomalies, including bilateral undescended testes, hypospadias, and chordee.

He underwent repair of his diaphragmatic hernia at 4 days of life, however, he continued to require high ventilatory support likely due to the degree of his pulmonary hypoplasia. He continued to decline clinically, and eventually the decision was made to withdraw care. The patient passed away at 1 month of life in the cardiovascular intensive care unit.

Prenatal array-based CNV analysis was nondiagnostic. Exome sequencing revealed a heterozygous de novo c.3239dupA, p.(Glu1081Glyfs*5) frameshift variant in MYRF (NM_001127392.2) which was confirmed by Sanger sequencing. This variant was not reported in gnomAD, and is predicted to be disease causing by MutationTaster.

Subject 3

Subject 3 is a male referred for clinical exome sequencing due to congenital diaphragmatic hernia, ambiguous genitalia, surgically repaired hypospadias, and a possible history of horseshoe kidney and hydronephrosis. Testing revealed that he carried a c.350_366delinsT, p.(Gly117Valfs*31) variant in MYRF (NM_001127392.2). This variant was not detected in a maternal sample, but no paternal sample was available for analysis. This variant was not reported in gnomAD, and is predicted to be disease causing by MutationTaster.

DISCUSSION

Including the patients we report here, there are now sixteen reported cases of individuals carrying variants in MYRF with structural birth defects affecting the heart, lungs, diaphragm, and genitourinary system (Table 1; Figure 2). Loss-of-function variants—stop-gain, frameshift, and variants that may affect splicing, including the c.3118A>G, p.(Arg1040Gly) variant carried by Subject 1—are distributed throughout the MYRF gene. Based on their locations, these variants are likely to trigger nonsense-mediated mRNA decay. In contrast, single amino acid changes are clustered in the DNA binding domain and the peptidase S74 domain. This suggests that loss of MYRF function is the likely mode of action of these variants. It follows that haploinsufficiency of MYRF causes a genetic syndrome whose cardinal features include congenital heart defects, pulmonary hypoplasia, congenital diaphragmatic hernia, and genitourinary anomalies.

Figure 2. The locations of the variants in MYRF are shown in relation to its protein domains.

MYRF variants identified in Subjects 1–3 (S1–3) are shown in red. Variants previously reported in individuals with congenital anomalies affecting the heart, lungs, diaphragm and genitourinary system are shown in black. The variant that has been shown to cause mild encephalitis/encephalopathy with reversible myelin vacuolization (MIM# 618113) is shown in blue. Stop-gain, frameshift (shown above the protein) and variants that could affect splicing are located throughout MYRF. Based on their locations, these variants are likely to trigger nonsense-mediated mRNA decay. In contrast, single amino acid changes (shown below the protein) are clustered in the DNA binding domain and the peptidase S74 domain.

Congenital heart defects (CHD) are the most common type of anomaly seen in MYRF deficiency with 15/16 (94%) of individuals being affected. The most common forms of congenital heart defects seen in these individuals were hypoplastic left heart syndrome (7/16, 44%) and scimitar syndrome (5/16, 31%). Other forms of CHD included tetralogy of Fallot, septal defects, valvular defects, hypoplastic aortic arch, and patent ductus arteriosus.

Urogenital anomalies are the second most common structural birth defect seen among individuals with MYRF deficiency with at least 12/16 (75%) of individuals being affected. Males can present with ambiguous to completely feminized genitalia, hypospadias, cryptorchidism, micropenis, chordee, and persistent urachus. One female has been described with genital anomalies. She had no internal sex organs and a blind-ending vagina [Qi et al., 2018].

Diaphragm abnormalities, including CDH and diaphragmatic eventration, constitute the third most common structural defect with 10/16 (63%) of individuals being affected. Lung hypoplasia was seen in 7/16 (44%) of individuals. Although lung hypoplasia is common in both scimitar syndrome and in individuals with CDH, the fetus described by Chitayat et al. had neither of these defects [Chitayat et al., 2018]. This suggests that MYRF plays a role in lung development that is independent of venous drainage to the heart or diaphragm development. The high rate of diaphragmatic anomalies and lung hypoplasia underscores the importance of these phenotypes in individuals with MYRF deficiency. With that in mind, we would suggest that the current name of this disorder, cardiac-urogenital syndrome [MIM# 618280], be revised to include these features (cardiac-urogenital-diaphragm-lung syndrome; CUDL syndrome), or be changed to place greater emphasis on the gene rather than the phenotype (MYRF-related congenital anomalies syndrome). The use of a more inclusive name for this disorder may make it more likely that laboratory and clinical geneticists will consider this genetic syndrome even in cases where cardiac or urogenital features are not present.

The principle features seen in MYRF deficiency have significant overlap with PAGOD syndrome [MIM# 202660], a clinically defined syndrome characterized by pulmonary artery and lung hypoplasia, agonadism, omphalocele, and diaphragmatic defects [Delgado-Luengo et al., 2016; Gavrilova et al., 2009; Gil et al., 2014; Kennerknecht et al., 1993; Kim et al., 2007; Takahashi et al., 2014]. Other congenital heart defects seen in individuals who have been diagnosed with PAGOD syndrome include hypoplastic left heart and scimitar syndrome which are commonly seen in MYRF deficiency [Kim et al., 2007; Takahashi et al., 2014]. Although PAGOD syndrome has been hypothesized to be an autosomal or x-linked disorder, it seems likely that some individuals who have been given this diagnosis, particularly those without a family history, may actually have MYRF deficiency.

Congenital anomalies were not reported in the nine individuals described by Kurahashi et al. who had mild encephalitis/encephalopathy with reversible myelin vacuolization and carried heterozygous c.1208A>G, p.(Gln403Arg) variants in MYRF [Kurahashi et al., 2018]. In contrast, Qi et al. patient 9 had abnormalities of the heart, diaphragm, lungs, and genitourinary system and carried a p.(Gln403His) variant that affected the same amino acid [Qi et al., 2018]. One possible explanation for this difference is that the substitution of a histidine residue at this position generates a protein whose function is more severely compromised than that generated by an arginine substitution at the same location. If this is the case, it is possible that individuals who survive into adulthood with strong loss-of-function variants in MYRF may experience encephalitis/encephalopathy and myelin vacuolization. With this in mind, individuals with loss-of-function variants in MYRF who survive the neonatal period should be monitored closely for signs of developmental delay and intellectual disability.