Further characterization of Borjeson‐Forssman‐Lehmann syndrome in females due to de novo variants in PHF6

Abstract While inherited hemizygous variants in PHF6 cause X‐linked recessive Borjeson‐Forssman‐Lehmann syndrome (BFLS) in males, de novo heterozygous variants in females are associated with an overlapping but distinct phenotype, including moderate to severe intellectual disability, characteristic facial dysmorphism, dental, finger and toe anomalies, and linear skin pigmentation. By personal communication with colleagues, we assembled 11 additional females with BFLS due to variants in PHF6. We confirm the distinct phenotype to include variable intellectual disability, recognizable facial dysmorphism and other anomalies. We observed skewed X‐inactivation in blood and streaky skin pigmentation compatible with functional mosaicism. Variants occurred de novo in 10 individuals, of whom one was only mildly affected and transmitted it to her more severely affected daughter. The mutational spectrum comprises a two‐exon deletion, five truncating, one splice‐site and three missense variants, the latter all located in the PHD2 domain and predicted to severely destabilize the domain structure. This observation supports the hypothesis of more severe variants in females contributing to gender‐specific phenotypes in addition to or in combination with effects of X‐inactivation and functional mosaicism. Therefore, our findings further delineate the clinical and mutational spectrum of female BFLS and provide further insights into possible genotype–phenotype correlations between females and males.

in females contributing to gender-specific phenotypes in addition to or in combination with effects of X-inactivation and functional mosaicism. Therefore, our findings further delineate the clinical and mutational spectrum of female BFLS and provide further insights into possible genotype-phenotype correlations between females and males.

K E Y W O R D S
Borjeson-Forssman-Lehmann syndrome, de novo, PHF6, X-chromosomal 1 | INTRODUCTION X-linked recessive Borjeson-Forssman-Lehmann syndrome (BFLS, OMIM#301900) was first described in 1962. 1 In 2002, variants in the gene encoding PHD finger protein 6 (PHF6) were identified as the underlying cause. 2 PHF6 contains two extended atypical PHD-like zinc finger domains (PHD1 and PHD2), two nuclear and one nucleolar localization sequences 2 and is assumed to play a role in transcription, ribosomal RNA transcription and neuronal migration. [3][4][5] Affected males present with developmental delay, moderate to severe intellectual disability (ID), truncal obesity, hypogonadism, tapering fingers, toe anomalies, and a typical facial gestalt with long ears or prominent earlobes and prominent cheekbones. 6,7 Some of the female carriers in these families show mild aspects of BFLS such as learning difficulties, mild facial features or toe and finger anomalies. [7][8][9] Skewing of Xinactivation (XI) in female carriers in these reports was inconsistent and did not correlate with clinical findings. [9][10][11] In 2013, a series of seven females with de novo variants in PHF6 was reported. 12 Affected individuals presented with a neurodevelopmental disorder overlapping with BFLS in males, but also displaying additional distinct features. 12 Next to moderate to severe intellectual disability, a characteristic facial gestalt with long shaped ears, bitemporal narrowing, prominent supraorbital ridges, high eyebrows, a short nose, and a bulbous nasal tip was delineated. Furthermore, oligomenorrhea, more prominent finger and toe deformities, dental anomalies, and linear skin hyperpigmentation occurred. In accordance with streaky skin pigmentation, skewed XI in blood samples and random XI in fibroblasts indicated functional mosaicism of the active and inactivated mutant allele. 12 Up to now, a total of twelve female individuals with such de novo germline deletions, duplications or single nucleotide variants in PHF6 were reported. 10,[12][13][14][15][16][17] Male individuals with BFLS predominantly harbor missense variants and only a few truncating variants distributed all over the gene/ protein, 2 while in females with de novo variants, mostly deletions, (likely) truncating aberrations and only one missense variant located within the second PHD zinc finger domain were identified to date. 12,17,18 Observing differential cellular localization between "male" and "female" variants in vitro and predicting more severe effects of the single female missense variant compared to male missense variants in the PHD2 zinc finger on domain stability, a possible genotypephenotype correlation between nature and localization of variants and gender-specific phenotypic manifestation was recently suggested. 18 We now further delineate the mutational and clinical spectrum of female BFLS by assembling 11 additional cases with aberrations in PHF6. Variants occurred de novo in 10 individuals, of whom one was only mildly affected and transmitted it to her more severely affected daughter. Identification of three further missense variants within the PHD2 domain and subsequent structural modeling support the previously suspected genotype-phenotype correlation.

| Patient material and data
Personal communication with colleagues following the initial reports 12,16 enabled us to collect clinical and mutational details on 11 female individuals with BFLS due to variants in PHF6. The study was approved by the ethics committee of the medical faculty of the Friedrich-Alexander-University Erlangen-Nuremberg (approval 142_15B). Testing in the majority of individuals was performed in a diagnostic setting. Individual 10 was analyzed within a study to unravel the diagnosis of patients with developmental disorders. Informed consent for publication of mutational and clinical data and particularly for publication of patient photographs was obtained from the parents or legal guardians.

| PHF6 analysis and structural modeling
In four individuals, targeted analysis of PHF6 (NM_032458) based on clinical suspicion was performed by Sanger sequencing and/or MLPA, as described previously. 12 Further details on primer and probe sequences and conditions are available on request. Trio-exome sequencing was performed in one, panel sequencing in two and single exome sequencing in three individuals (Table 1). Segregation analysis in the non-trio cases was performed by Sanger sequencing or MLPA, respectively. XI analysis in blood samples was performed in seven individuals in the respective centers within routine diagnostics. VIPUR scores 19 of the three novel and one published 20 missense variants were determined as described previously. 18 VIPUR is designed to distinguish between neutral (score <0.5) and deleterious (score >0.    All but one of the individuals at informative ages presented with intellectual disability, ranging from mild/moderate (7/10) to severe/ profound (3/10). Formally tested IQs were not available. Of note, the

| Mutational spectrum
For a summary of identified variants, see Figure 2, Tables 1 and S1.
Three missense and six truncating variants, including a splice-site variant, in PHF6 were detected in the 11 herewith described individuals. The deletion of exons 6 and 7 in one individual was predicted to be frame-shifting and thus truncating. Nine of the variants were shown to have occurred de novo. In one case, maternal inheritance was excluded, and the father was not available for testing. One individual inherited the variant from her mildly affected mother, in whom the variant was shown to have occurred de novo. Sanger sequencing in blood was not indicative for mosaicism in her ( Figure S1).
While the truncating variants were distributed all over the gene/ protein, the three missense variants clustered within the PHD2 domain ( Figure 2). To our knowledge, none of the variants has been reported as pathogenic before in literature or ClinVar. They are not observed in gnomAD. The missense variants affect highly conserved amino acids and are predicted to be deleterious by at least three of the used in silico prediction programs (Table S1). According to ACMG guidelines, 21 all identified variants were classified as pathogenic or likely pathogenic (Table S1).
XI pattern in blood samples was tested in seven individuals and was skewed (>90%) in all of them. Of note, both the mildly affected mother and the more severely affected daughter of the familial case had a similarly skewed degree of XI of more than 90% (Table 1).

| Structural modeling of the missense variants
We used the VIPUR score, 19 integrating sequence analysis and structural modeling, to assess the effect of the identified missense variants on the three-dimensional protein structure. All three missense variants in our cohort showed a high VIPUR score of >0.86, thus predicting a strong destabilizing effect on the protein structure ( Figure 2, Table S1). Also, the published missense variant c.823G > A, recessive neurodevelopmental disorder (NDD) and de novo variants in females with a comparable severe but distinct NDD were identified.
Female variant carriers in the X-linked recessive families are mostly asymptomatic but may display mild and infrequent clinical aspects (Table 1). While the male BFLS phenotype has been known for several decades, 1,2 the distinct female phenotype associated with de novo variants in PHF6 was only delineated in 2013. 12 Thus, the available information on the latter is still limited and based on twelve published cases so far. 10,[12][13][14][15][16][17] By reporting on eleven further individuals with the female form of BFLS, we further characterize the phenotypic and mutational spectrum.
With this study, we confirm the very distinct phenotype of BFLS in females caused by de novo variants in PHF6 to include variable intellectual disability, a characteristic facial gestalt, acral and dental anomalies and linear skin hyperpigmentation. While the variable degree of intellectual disability is comparable to that of affected male individuals, some of the facial aspects, as well as the presence of dental and pigmentation abnormalities are rather specific for the female phenotype. Finger and toe abnormalities are similarly frequent in both novel and published females and males with BFLS (>90%). However, in males, mainly tapering of fingers has been observed, while finger deformities in females are more prominent and diverse with tapering, campto-, clino-, brachydactyly, and hypoplastic nails (Table 1). While sandal gaps have been reported more frequently in males, syndactyly of toes and hypoplastic toe nails occurs more frequently in females ( Table 1) While general or truncal obesity was described in more than 90% of males with BFLS, 1,6,9 this has been only observed in 20% of females with BFLS both in the published and the herewith reported individuals (Table 1). In addition, hypogonadism has been described as one of the prominent features in males with BFLS, 1,9 and variable endocrinological abnormalities were observed in individuals carrying PHF6 variants. 23 Oligomenorrhea, frequently observed in females with de novo variants in PHF6, might also reflect hypogonadism. 12 Streaky skin pigmentation has been observed in the majority of previously reported affected females [12][13][14][15][16][17] but not in the unaffected carrier females in X-recessive families. [7][8][9] Of note, in this study, only half of the females showed skin pigmentation anomalies, and the presence of these was not correlating with the severity of disease manifestation.
Random XI might be another indicator of functional mosaicism, supported by a previous report showing skewed XI in blood samples but random XI in fibroblasts. 12 In blood, there is a high frequency of skewed XI both in asymptomatic carriers and symptomatic females. 6,9,12,16,23 For two affected females with de novo variants preferential inactivation of the mutant allele in blood was demonstrated (so far unpublished data, Supplementary Figure S2) 24 the frequency of truncating variants is still significantly higher in females with BFLS than in males. 18 Furthermore, "male" and "female" missense variants behave differently in in vitro assays and regarding protein domain stability. 18 While a missense variant in the PHD2 domain identified in a female was predicted to have a strong destabilizing effect, male missense variants in the same domain were predicted to have a milder effect. 18 Missense variants from males in the PHD1 domain were shown to have a strong destabilizing effect 18 and were shown to result in reduced protein expression, 24 but were so far exclusively observed in X-linked recessive families and not as de novo variants in females with the full clinical picture of BFLS. Missense variants in the PHD1 domain were therefore postulated to be less deleterious than missense variants in the PHD2 domain, and within the PHD2 domain "female" missense variants to be more deleterious than "male" missense variants. 18 By now, we find further evidence for this previously discussed genotype-phenotype correlation.
Three additional de novo missense variants identified in females were all located in the PHD2 domain and predicted to result in severe destabilization of the domain, similar to the previously published "female" variant 18 and more severe compared to "male" missense variants in the same domain. 18 This is also supported by another published missense variant detected in a female, c.823G > A, p.(Gly275Arg), 20 which is also located in the PHD2 domain and predicted to have a strong destabilizing effect. Surprisingly, however, this variant was transmitted from an asymptomatic mother to a more severely affected daughter. 20 There is another case of female-to-female transmission of a truncating variant in the literature 23 and additionally in the herewith reported family 2 (see also above). Similar degrees of XI skewing in blood in mildly affected mothers and more severely affected daughters 20,23 do not provide an explanation for the phenotypic differences.
In family 2 and in one of the published cases 20 the variant was shown to be de novo in the mothers, however, without indication of mosaicism for the variant in them. Still, this cannot be excluded as an explanation for the mild presentation. Post-zygotic mosaicism associated with a milder phenotypic presentation has been reported in a female individual before. 15 In total, the phenotypic manifestation of BFLS in females cannot be attributed to a single factor but seems to result from a complex