Phenotypic Spectrum of NFIA Haploinsufficiency: Two Additional Cases and Review of the Literature

The NFIA (nuclear factor I/A) gene encodes for a transcription factor belonging to the nuclear factor I family and has key roles in various embryonic differentiation pathways. In humans, NFIA is the major contributor to the phenotypic traits of “Chromosome 1p32p31 deletion syndrome”. We report on two new cases with deletions involving NFIA without any other pathogenic protein-coding gene alterations. A cohort of 24 patients with NFIA haploinsufficiency as the sole anomaly was selected by reviewing the literature and public databases in order to analyze all clinical features reported and their relative frequencies. This process was useful because it provided an overall picture of the phenotypic outcome of NFIA haploinsufficiency and helped to define a cluster of phenotypic traits that can facilitate clinicians in identifying affected patients. NFIA haploinsufficiency can be suspected by a careful observation of the dysmorphisms (macrocephaly, craniofacial, and first-finger anomalies), and this potential diagnosis is strengthened by the presence of intellectual and developmental disabilities or other neurodevelopmental disorders. Further clues of NFIA haploinsufficiency can be provided by instrumental tests such as MRI and kidney urinary tract ultrasound and confirmed by genetic testing.

NFIA is a gene located on chromosome 1p31.3 that encodes for a transcription factor belonging to the nuclear factor I family of dimeric DNA binding proteins, along with NFIB, NFIC, and NFIX [8]. This protein has three domains: (1) an N-terminal highly conserved region, whose function is not known; (2) a DNA binding/dimerization domain (MH1), and (3) a C-terminal transactivation and/or repression domain (CTF_NFI). The MH1 domain binds to a nucleotide consensus sequence within the promoter region of several genes, whereas the CTF_NFI domain activates basal transcription factors at transcription start sites by displacing repressive histones from target genes and interacting with other coactivator proteins [9]. NFIA gives rise to 31 transcript variants, but the functional significance of each isoform has not been yet clarified. The MANE transcript (matched annotation from NCBI and EMBL-EBI) is NFIA-207 (ENST00000403491.8), whichis encoded by 11 exons, extending from position 61,548,233 to 61,928,460 (GRCh37/hg19), and has ubiquitous expression (https://www.gtexportal.org/, accessed on 20 September 2022).
According to in vitro analyses and studies of animal models, NFIA has a key role in various differentiation pathways during embryogenesis. It is a crucial regulator of articular cartilage differentiation [10] and controls the adipogenic and myogenic gene program to ensure brown and beige adipocyte differentiation [11]. Nfia knock-out mice show hydrocephalus and agenesis of the corpus callosum, and, at reduced penetrance, exhibit abnormalities of the ureteropelvic and ureterovesical junctions, as well as bifid and megaureters [1,8]. NFI proteins play an important role in the development of the central nervous system (CNS), including axon guidance with outgrowth and in glial or neuronal cell differentiation and migration [8,[12][13][14][15].
In this paper, we focused on cases with NFIA haploinsufficiency as the sole anomaly, excluding all those patients with other pathogenic protein-coding gene alterations. We report on two new individuals and reviewed the literature and public databases to better delineate the phenotypic spectrum of NFIA haploinsufficiency and its contribution to "Chromosome 1p32p31 deletion syndrome".

Molecular Analysis
Array CGH was performed. Genomic DNA of the patients was isolated from peripheral blood by standard methods. DNA from healthy subjects was used as controls. Test and reference DNA were differentially labeled with Cy5-dCTP or with Cy3-dCTP using random primer labeling and applied to 60K arrays, according to the manufacturer's protocol (case 1: Agilent, Santa Clara, CA, USA; case 2: Technogenetics, Milan, Italy). For each case, the array-CGH was repeated twice in order to confirm the results. Since quality of the experiment influences the Copy Number Variants (CNVs) (i.e., number and type of calls and probes included/excluded in each call), we elaborated only experiments that met the 'excellent' criteria as determined by the QC report. The CNV analysis was performed according to the guidelines of the Italian Society of Human Genetics (https://www.sigu.net, accessed on 20 September 2022) and to the American College of Medical Genetics guidelines [16].

Bioinformatic Analyses
CNVs classification was performed using the Database of Genomic Variants (DGV) (http://projects.tcag.ca/variation, accessed on 20 September 2022) and the University of California Santa Cruz (UCSC) Genome Browser (https://genome.ucsc.edu, accessed on 20 September 2022).PubMed and Online Mendelian Inheritance in Man (OMIM) were also consulted for evaluating genotype-phenotype association.
NFIA transcripts were identified using Ensembl. Data on the expression profiles were assessed using GTEx (https://www.gtexportal.org/, accessed on 20 September 2022) and the UCSC Genome Browser. Patient 1 (P1) is a 2-year-old male toddler who was referred to the pediatric unit at the age of 10 months for macrocephaly.
On physical examination he presented with macrocephaly, scaphocephaly, a high and wide forehead with evident frontal bossing, and low-set ears. He showed brachydactyly with bilateral proximally placed first fingers and short lower limbs. The eye examination revealed the presence of a mild strabismus, whereas hearing screening, blood tests, and metabolic and hormonal work up were all normal. EEG revealed minor, nonspecific discharges in the fronto-central-parietal regions. No epileptic seizures have been recorded. A cranial ultrasound, performed at the age of 3 months, revealed sagittal synostosis, whereas the metopic, lambdoid, and coronal sutures were still open. A brain MRI showed diffuse corpus callosum hypoplasia and a dysmorphic aspect of the cerebral ventriculus with widening of the anterior portions of the horns. No Chiari I malformation was detected.
At the first evaluation, the patient showed normal psychomotor development, but, at follow-up (age 2 years), he presented mild/moderate DD with severe delay in language (absence of speech) and hyperactivity disorder. An appearance of congestion of the optic disc was reported in anocular ultrasound, but the ophthalmological examination was negative. A brain MRI showed no alteration of the cranial nerves but confirmed the dysmorphic aspects already described previously; in particular, it confirmed the hypoplasia of the cerebral commissural system (corpus callosum and anterior commissure) with dysmorphic appearance of the lateral ventricles. An EEG showed focal abnormalities in posterior-temporal regions, with more in the right hemisphere. However, the patient did not present with epileptic seizures. Slight facial dysmorphisms were shown, such as a high forehead, downslanting palpebral fissures, low-set and posteriorly rotated ears, a small and poorly structured philtrum, an open bite, hypotrophy of thenar and hypothenar eminences, and bilateral proximally placed first fingers. No skin dyschromia was revealed. An MRI showed a thin corpus callosum, Chiari malformation type I, hypoplastic rectus sinus, and a vicariant vessel along the right profile of the trunk. Neither ventricles anomies nor craniosynostosis were detected. Heart, lung, and abdomen ultrasound was normal. No epilepsy or EEG anomalies were reported. The patient was no longer available for follow up.
P2 showed a 27 kb deletion, starting from position 61,818,169 and ending at position 61,845,524 (GRCh37/hg19). The deleted region includes part of exon 5 and the entirety of exon 6 of NFIA (ENST00000403491.8; RefSeq ID NM_001134673). A segregation pattern could not beestablished.
The pathological traits of these patients have been grouped into macroareas: dysmorphisms (Table 2), neurological/behavioral abnormalities, renal/urinary trait alterations (Table 3), and CNS malformations (Table 4). In each table are listed only those patients where the clinical traits were evaluated. The few additional defects not included in these categories are reported in Table S1.

Discussion
Here, we report on two new individuals carrying microdeletions solely affecting NFIA without any other flanking protein-coding sequences. In order to further define the phenotypic effects of NFIA haploinsufficiency, we reviewed the literature and public databases excluding those patients without detailed clinical information or individuals with pathogenic deletions, translocations, inversions, or genetic variants disrupting additional protein-coding sequences [1][2][3][4][5][6][7]23,[26][27][28][29] (Table S2). This careful selection allowed for the identification of a cohort of 24 patients with pathogenic alterations solely involving NFIA.
The precise localization of NFIA pathogenic variants is also reported. Since alteration of this gene hasoften been referred to alternative transcripts, we have converted each of them according to the NFIA-207 (ENST00000403491.8; RefSeq ID NM_001134673) ( Table S3). As shown in Figure 1, SNVs do not have one hotspot and are spread over various functional domains; some microdeletions are intragenic and involve only few exons, while others disrupt all of the coding sequence with theirregulatory elements (NFIA-AS2, NFIA-AS1) and further noncoding RNAs (LINC01748, LOC101926964).
The precise localization of NFIA pathogenic variants is also reported. Since alteration of this gene hasoften been referred to alternative transcripts, we have converted each of them according to the NFIA-207 (ENST00000403491.8; RefSeq ID NM_001134673) ( Table  S3). As shown in Figure 1, SNVs do not have one hotspot and are spread over various functional domains; some microdeletions are intragenic and involve only few exons, while others disrupt all of the coding sequence with theirregulatory elements (NFIA-AS2, NFIA-AS1) and further noncoding RNAs (LINC01748, LOC101926964). To better evaluate the phenotypic outcome of NFIA haploinsufficiency, the pathological traits of the selected patients have been grouped into macroareas: dysmorphisms (Table 2), neurological/behavioral abnormalities, renal/urinary trait To better evaluate the phenotypic outcome of NFIA haploinsufficiency, the pathological traits of the selected patients have been grouped into macroareas: dysmorphisms (Table 2), neurological/behavioral abnormalities, renal/urinary trait alterations (Table 3), and CNS malformations (Table 4). In each table are listed only those patients where the clinical traits were evaluated. The few additional defects not included in these categories are reported in Table S3 and CNS malformations ( Table 4).
The major clinical characteristics are summarized in Table 5 along with their frequency. The prevalence of each single trait was calculated both by the ratio between the presence of a clinical sign on the number of patients where this clinical sign has been evaluated and the ratio of all the 26 selected patients. Dysmorphisms (Table 2). Among physical anomalies, macrocephaly is a trait almost invariably present (except P2); high forehead and a low-set ears are other recurrent signs, often associated with nonspecific facial-dysmorphic features.
Craniofacial anomalies, including asymmetries and craniosynostosis, represent a distinctive trait described in most patients. Metopic, lambdoid, or sagittal craniosynostosis were not observed in four cases (P4, P5, P6, P8). Three of them are members of the same family; thus, it was hypothesized that these anomalies could be the consequence of additional familiar genetic modifiers; our report of an additional case with craniosynostosis (P1) strengthens this correlation with haploinsufficiency. It is worth noting that in chicken, NFIA participates in osteoblast differentiation by interacting with the Wnt and Ihh pathways [10].
Bilateral proximally placed first fingers seem to be another characteristic dysmorphism, present in our patients (P1, P2) and in four other cases (P6, P8, P11, P12). This sign may have been overlooked, and it is possible that its frequency is underestimated.
Macrocephaly, craniofacial abnormalities, and first-finger anomalies are quite peculiar traits, and their concurrence can represent a first diagnostic clue for NFIA haploinsufficiency.
Neurological/behavioral abnormalities (Table 3). DD and/or ID are invariably present in this disorder, ranging from very mild to severe (details are available in Table S4). Beside ID and DD, this cohort shows the concurrence of other NDDs, including hyperactivity disorder (P1), ODD (P2), ADHD-Combined Type (P4), PDDNOS (P4), as well as neurological signs such as facial asymmetry (P9), mild spasticityat lower limbs (P9), extremely low PSI (P16), and epilepsy (P16, P17, P18, P21).It is worth noting that the comorbidity is an almost constant feature of NDDs because different NDDs can represent the variable expressivity of the same genetic alteration [30]. The comorbidity of this cohort strengthens this hypothesis. Hypotonia, exotropia, and hearing impairment are other neurological signs reported.
Renal/urinary trait and CNS defects (Tables 3 and 4). Renal/urinary tract and CNS defects are considered to bethe key features of "Chromosome 1p32-p31 deletion syndrome", whichis also referred to as "brain malformations with or without urinary tract defects" (BRMUTD) (OMIM # 613735).
In cases with NFIA haploinsufficiency, the frequency of renal/urinary tract abnormalities is probably lower than previously supposed [1], and in this cohort, they were excluded in twelve cases ( Table 3). The defects described are heterogeneous, even when intrafamilial (i.e., P5 versus P7). Hydronephrosis and renal cysts are the most common clinical signs, even if each of them is reported only in three patients (P4, P7, P15 and P5, P9, P17, respectively). The absence of recurrent clinical features, along with their low frequency, make the renal/urinary tract anomalies not very helpful for the identification of this disorder. As experiments in knocked-out mice highlighted the involvement of NFIA in the embryonic development of the excretory apparatus [1], a surveillance of kidneys and urinary trait is mandatory when haploinsufficiency of this gene is detected.
On the other hand, CNS alterations represent a key trait of NFIA haploinsufficiency. Corpus callosum and cerebral ventricles anomalies are the most distinctive signs: the corpus callosum can be thin, hypoplastic, absent, or dysgenetic, and the presence of ventriculomegaly or hydrocephalus is quite constant. Along with these defects, a constellation of less common and heterogeneous CNS anomalies has been described, such as cortical malformations (P11, P12, P15, P21, P26), hypoplasia or atrophy of brain areas (P5, P11, P12, P19), decreased periventricular white matter (P3, P11, P25), partial incomplete inversion of the left hippocampi (P5, P11), and cyst presence (P11, P15, P25). P2 shows the presence of cerebral vascular malformations that have never been reported before. All these anomalies confirm the key role of NFIA in the differentiation of multiple brain areas during embryonic development [12][13][14][15].
From Tables 1-4, a strict correlation between the genetic variants and clinical signs does not emerge: greater deletions do not correspond to a more severe phenotype; moreover, familialcases that share the same anomaly show a clinical spectrum, confirming a variable expressivity of some phenotypic traits.

Conclusions
NFIA is a gene with a key role in various differentiation pathways during embryogenesis; therefore, its haploinsufficiency is thought to determine a pleiotropic effect.
In this article, two new cases with deletions solely involving NFIA are reported, and the literature and public databases were reviewed in order to evaluate the phenotypic outcome of NFIA haploinsufficiency. First, all those patients with NFIA alterations and pathogenic genetic variants disrupting additional protein-coding sequences were excluded, since a thoughtful selection of cases is the most important step to accurately delineate genotype-phenotype correlations of a genetic disorder. Second, all the clinical signs and their relative frequencies in the 24 selected cases were analyzed. This process was useful to have an overall picture of phenotypic outcome of NFIA haploinsufficiency and to define a cluster of phenotypic traits that can facilitate clinicians in identifying the affected patients ( Table 5). The potential diagnosis of NFIA haploinsufficiency can be advanced by a careful observation of the dysmorphisms (macrocephaly, craniofacial, and first-finger anomalies) and be strengthened by the presence of ID/DD or other NDDs. Instrumental tests such as MRI and kidney urinary tract ultrasound can provide key elements to further strengthen this hypothesis, which can be confirmed by genetic testing. Additional cases with welldescribed phenotypes will be helpful to better elucidate the full clinical spectrum.
The present analysis confirms that NFIA is responsible for the main phenotypic traits of Chromosome 1p32p31 deletion syndrome, even if other genes can contribute to modify the severity of some signs, such as ID/DD [6]. Less common traits of this syndrome, such as cryptorchidism, inguinal hernia, anomalies of external genitalia, syringomyelia, congenital heart defects, and Juvenile Moyamoya [1][2][3][4][5][6][7], were never reported in this selected cohort with only NFIA haploinsufficiency, and they are probably caused by dosage alterations of different deleted genes.