NFIA haploinsufficiency: case series and literature review

Background NFIA-related disorder (OMIM #613735) is an autosomal dominant neurodevelopmental disorder characterized by a variable degree of cognitive impairment and non-specific dysmorphic features. To date, fewer than thirty patients affected by this disorder have been described. Methods Our study included three children with NFIA haploinsufficiency recruited from three medical genetics centers. Clinical presentations were recorded on a standardized case report form. Results All patients presented a variable degree of intellectual disability. None of the individuals in our cohort had urinary tract malformations. Three novel mutations, c.344G>A, c.261T>G, and c.887_888del are reported here. Conclusion NFIA haploinsufficiency can be suspected through careful observation of specific dysmorphisms, including macrocephaly and craniofacial abnormalities. Instrumental tests such as MRI and renal ultrasound provide further diagnostic clues, while genetic testing can confirm the diagnosis.


Introduction
The NFIA (nuclear factor I/A) gene encodes a transcription factor belonging to the nuclear factor I family and plays key roles in central nervous system (CNS) development, including axonal growth, glial cell differentiation, and neuronal migration (1).Initially, the phenotypic presentation resulting from deletions affecting NFIA was referred to as chromosome 1p32-p31 deletion syndrome.However, the observation of overlapping phenotypes in patients with chromosome translocations that disrupt NFIA has demonstrated that the loss of function of NFIA is responsible for most of the phenotypes associated with the 1p32-p31 deletion (2)(3)(4).Haploinsufficiency of NFIA causes a syndrome characterized by brain malformations, most commonly abnormalities of the corpus callosum, with or without urinary tract defects (BRMUTD, OMIM#613735).Additional features include macrocephaly, seizures, developmental delay, ventriculomegaly, and hypotonia (5,6).To date, fewer than thirty patients affected by this disorder have been described.Herein, we report three additional patients showing a variable degree of intellectual disability (ID) and CNS abnormalities, all carrying a heterozygous de novo mutation in the NFIA gene.Our findings provide further evidence that the NFIA gene has a role in development of the CNS.

Materials and methods
Our analysis included three molecularly confirmed NFIA haploinsufficiency patients, aged between 3 and 13 years, none of whom have been previously described.Genomic DNA of the patients was extracted from peripheral blood using standard methods.Information on the age and sex of the children was obtained from their medical records.DNA samples were collected as parent/child trios.Exome sequencing (ES) was performed for Patient 1 and his parents using a Twist Custom Panel (Twist Bioscience, San Francisco, CA, USA) on a NovaSeq 6,000 platform (Illumina Inc., San Diego, CA, USA).For patients 2 and 3, we performed ES combined with variant analysis within a panel of neurodevelopmental disorders-related genes, as previously described (7).All patients were born to non-consanguineous parents.The study was conducted in accordance with the Declaration of Helsinki.Written informed consent has been obtained from the parents to publish clinical information with photographs.We summarized the molecular findings and clinical presentation of our patients in Table 1.

Patient 1
Patient 1 (P1) is a 3-year-old male who was referred to the pediatric unit for macrocephaly and developmental delay.He was born after 39 weeks of gestation by C-section because of non-reassuring fetal status.Pregnancy was uncomplicated by teratogenic exposures or maternal illness.Birth weight was 3,620 g (76th centile), his head circumference was 39.5 cm (+4.7 SD), and his birth length was 50 cm (50th centile).Regarding his developmental milestones, he started walking without support at 15 months and speaking his first words at 19 months.The Bayley Infant Development Scale (3rd edition, BSID-III) at 2 years of age, demonstrated moderate global cognitive impairment.At the time of referral, his head circumference was 57.0 cm (>97th centile).The patient presented with a high forehead, prominent occipital protuberance, short palpebral fissures, flat nasal bridge, large philtrum, macroglossia, and a crumpled helix (Figure 1).Speech consisted of about three words in an appropriate context.The brain MRI carried out at 2.5 years, showed Chiari I malformation and subependymal grey matter heterotopia (Figure 2).Trio exome analysis revealed a de novo mutation c.261T>G, p.(Tyr87*) in the NFIA gene.

Patient 2
Patient 2 (P2) (Figure 3) is the second child of nonconsanguineous parents with an unremarkable family history.She was born by vaginal delivery after an uneventful pregnancy.The birth weight was 2,945 g (12th centile), length 48 cm (14th centile), and head circumference 34.5 cm (64th centile).No apparent external malformations were observed.The brain MRI at 3 years of age (Figure 4) revealed a thin corpus callosum, dysmorphic appearance of the lateral ventricles, and a retrocerebellar arachnoid cyst.Renal ultrasound was normal.
When last examined at age 7 years, her height was 134 cm (>97th centile), her weight was 28.5 kg (90th centile).Her head circumference was within the normal range, measuring at 53 cm (63rd centile).The neurobehavioral evaluation showed a learning disability and psychopathological findings, such as attention deficit hyperactivity disorder (ADHD), bizarre behaviour and emotional dysregulation.No epileptic seizures have been observed to date.Trio ES identified a heterozygous missense variant (c.344G>A, p.Arg115Gln) in the NFIA gene.

Patient 3
Patient 3 (P3) is the male, first child of healthy nonconsanguineous parents.The family history was uneventful for disabilities.We first evaluated the patient at the age of 5.1 years for macrocephaly and developmental delay.The weight was 21.8 kg (75-90th centile), height 116 cm (95th centile), and

Discussion
The NFIA gene belongs to a family of genes known as NFI, which also consists of NFIB, NFIC, and NFIX in vertebrates (8).Lu et al. (9) initially proposed the hypothesis that NFIA haploinsufficiency might be responsible for CNS malformations, as well as ureteral and renal defects.These authors also demonstrated ventricular enlargement, callosal agenesis, and  In this case series, we described the clinical and molecular characteristics of three patients with NFIA haploinsufficiency.Similar to previous reports, the patients in our study presented with CNS abnormalities and a variable degree of intellectual disability.Table 2 provides a comparison of our cohort and previously reported patients with NFIA mutations.Hereafter, the major clinical characteristics have been grouped into macroareas.

Dysmorphisms
Among the physical anomalies, macrocephaly and a high forehead are recurrent signs.In our cohort, P1 and P3 exhibited the typical facial features of NFIA haploinsufficiency.

Developmental progress/learning difficulties
The developmental milestones, especially speech, were delayed in our cohort; P2 and P3 began using single words by 2 years of age, while P1 had only a few words by the age of three.All children achieved walking.In no instances did patients lose skills or regress.All patients received special education at school.

CNS defects
CNS defects, most commonly abnormalities of the corpus callosum, are a prominent characteristic of NFIA haploinsufficiency.In addition to these, a range of less frequent and heterogeneous CNS anomalies have been reported, including cortical malformations, non-progressive ventriculomegaly, hydrocephalus, Chiari type I malformation, and tethered spinal cord.

Seizures
Seizures have been observed in approximately 15% of the NFIA patients (14).In our study, none of the patients experienced seizures.EEG was performed for P2 and P3, and the results were normal for both.

Neurodevelopment/behavior
Developmental delay and/or ID, ranging from very mild to severe, are invariably present in this disorder.Beside ID and developmental delay, patients may exhibit other neurodevelopmental disorders, including hyperactivity disorder and oppositional defiant disorder (ODD) (14).In our cohort, one patient (P2) had a diagnosis of ADHD.

Urinary trait defects
Renal/urinary defects are considered a key feature of NFIArelated disorder (9).The most common among these defects are vesicoureteral reflux and hydronephrosis, which can occur unilaterally or bilaterally.Other phenotypes associated with NFIA-related disorder include pyelonephritis, ureterovesical junction diverticulum, dysplastic kidneys, and renal cysts.
According to Revah-Politi et al., approximately 50% of patients diagnosed with NFIA-haploinsufficiency exhibited genitourinary defects (15).However, a subsequent literature review indicated a lower prevalence of urinary tract abnormalities (<20%).In our cohort, no patients presented with genitourinary abnormalities.Nevertheless, a surveillance of kidneys and urinary trait is recommended when NFIA-haploinsufficiency is detected.

Musculoskeletal abnormalities
Although musculoskeletal manifestations are not considered a typical sign of NFIA-related disorder, these features are frequently found in patients with the NFIA gene mutation.Bilateral proximally placed first fingers seem to be a frequently   reported dysmorphism in patients with NFIA-related disorder (14).
In our study, two patients (P2 and P3) presented with pes planus, while another with congenital talipes valgus (P1).Interestingly, it was demonstrated that NFIA and GATA-binding protein 3 regulate the differentiation of embryonic articular cartilage in chicken (22).The musculoskeletal manifestations associated with NFIA haploinsufficiency need further characterization to provide more definitive insights and treatment options for patients.

Conclusions
NFIA haploinsufficiency can be suspected through careful observation of specific dysmorphisms, including macrocephaly and craniofacial abnormalities.Instrumental tests, such as MRI and renal ultrasound, can offer further diagnostic clues, with definitive diagnosis achievable through genetic testing.
Three novel mutations, c.344G>A, c.261T>G, and c.887_888del are reported here.Like other authors, we did not identify a predictive value for specific mutations.However, the mutation c.344G>A might correlate with a milder phenotype.With the current use of clinical exome sequencing, it is expected that more patients with point mutations or microdeletions in NFIA will be diagnosed.Currently, a specific treatment for NFIA-related disorder is lacking, and symptomatic supportive treatment remains the main approach in clinical practice.
head circumference 56.5 cm (>97th centile).Facial anomalies included frontal bossing, high forehead, down-slanting palpebral fissures, a pointed chin, anteverted nostrils, and crumpled helix in the left ear.Ophthalmologic examination, auditory brainstem response, electroencephalography, echocardiography and renal ultrasound were unremarkable.Brain MRI revealed a hypoplastic corpus callosum with enlarged ventricles and agenesis of the septum pellucidum.Metabolic work up, karyogram, chromosomal microarray, and genetic studies for Sotos syndrome were all normal.At the age of 13, the family pediatrician required new medical advice for dysmorphisms.We performed trio ES.This analysis revealed a de novo heterozygous frameshift mutation (c.887_888del, p.Gly296Alafs*16) in the NFIA gene.

FIGURE 4 Brain
FIGURE 4 Brain MRI of patient 2 at 3 years of age.(A) Mid-sagittal T1-weighted image showing hypoplasia of the corpus callosum.(B) Axial T2-weighted image at the level of the lateral ventricles.

-
: not evaluated; *: deduced by the picture; CNS: central nervous system; NDDs: neurodevelopmental disorders.The bracketed numbers denote the total number of patients in which the feature/data has been evaluated.Dini et al. 10.3389/fped.2023.1292654

TABLE 1
Clinical characteristics of patients in our cohort.

TABLE 2
Frequency of specified features in our patients compared with cumulative data (ours and previously reported cases).