An Ultra-Rare Mixed Phenotype with Combined AP-4 and ERF Mutations: The First Report in a Pediatric Patient and a Literature Review.

The adaptor protein 4 (AP-4) constitutes a conserved hetero-tetrameric complex within the family of adaptor protein (AP) complex, crucial for the signal-mediated trafficking of integral membrane proteins. Mutations affecting all subunits of the AP-4 complex have been linked to autosomal-recessive cerebral palsy and a complex hereditary spastic paraparesis (HSP) phenotype. Our report details the case of a 14-year-old boy born to consanguineous parents, presenting psychomotor delay, severe intellectual disability, microcephaly, and trigonocephaly. Despite a history of febrile seizures, subsequent years were devoid of seizures, with normal EEG. Exome sequencing revealed pathogenic variants in both the AP4B1 and ERF genes. Significantly, the patient exhibited features associated with AP4B1 mutations, including distinctive traits such as cranial malformations. The ERF gene variant, linked to craniosynostosis, likely contributes to the observed trigonocephaly. This case represents the initial documentation of a concurrent mutation in the AP4B1 and ERF genes, underscoring the critical role of exome analysis in unraveling complex phenotypes. Understanding these complex genotypes offers valuable insights into broader syndromic conditions, facilitating comprehensive patient management.

Towards a Long-Read Sequencing Approach for the Molecular Diagnosis of RPGRORF15 Genetic Variants.

Sequencing of the low-complexity ORF15 exon of RPGR, a gene correlated with retinitis pigmentosa and cone dystrophy, is difficult to achieve with NGS and Sanger sequencing. False results could lead to the inaccurate annotation of genetic variants in dbSNP and ClinVar databases, tools on which HGMD and Ensembl rely, finally resulting in incorrect genetic variants interpretation. This paper aims to propose PacBio sequencing as a feasible method to correctly detect genetic variants in low-complexity regions, such as the ORF15 exon of RPGR, and interpret their pathogenicity by structural studies. Biological samples from 75 patients affected by retinitis pigmentosa or cone dystrophy were analyzed with NGS and repeated with PacBio. The results showed that NGS has a low coverage of the ORF15 region, while PacBio was able to sequence the region of interest and detect eight genetic variants, of which four are likely pathogenic. Furthermore, molecular modeling and dynamics of the RPGR Glu-Gly repeats binding to TTLL5 allowed for the structural evaluation of the variants, providing a way to predict their pathogenicity. Therefore, we propose PacBio sequencing as a standard procedure in diagnostic research for sequencing low-complexity regions such as RPGRORF15, aiding in the correct annotation of genetic variants in online databases.

Pulp chamber features, prevalence of abscesses, disease severity, and PHEX mutation in X-linked hypophosphatemic rickets.

INTRODUCTION: Rickets, growth failure, and recurrent periapical abscesses with fistulae are main signs in patients with X-linked hypophosphatemic rickets (XLH). Prevalence of abscesses, pulp chamber features, biochemical findings, disease severity, and PHEX gene mutation were examined. MATERIALS AND METHODS: Pulp chambers size, shape, and morphology were assessed by orthopantomography in XLH patients (n = 24, age 5.8 ± 1.6 years) and in sex and age-matched healthy controls (n = 23, age 6.2 ± 1.4 years). XLH patients received conventional treatment (3.5 ± 1.9 years). Pulp chamber features were assessed in teeth of primary dentition and in the permanent left mandibular first molar and compared with those of controls. Rickets severity score was assessed at wrist, knee, and ankle. RESULTS: The mean pulp chamber area/tooth area ratio, mean pulp chamber height/pulp chamber width ratio, and prominence of pulp horns into the tooth crown in primary and secondary molars were significantly higher in patients than in controls and in patients suffered abscesses than in patients without abscesses. Sixteen patients (67%) had a history of abscesses; incisors were affected more than canines and molars. Severity of rickets and mean serum parathyroid hormone (PTH) levels were significantly higher, and mean serum 1,25-dihydroxyvitamin D [1,25(OH)2D] levels significantly lower in patients suffered abscesses than in patients without abscesses. PHEX gene mutations were not correlated with dental phenotype and disease severity. CONCLUSION: Enlarged pulp chambers with altered shape and morphology affected the majority of XLH patients predisposing to recurrent periapical abscesses with fistulae. Dental phenotype was associated with severity of rickets, high serum PTH, and low serum 1,25(OH)2D levels.

Familial Interstitial 6q23.2 Deletion Including Eya4 Associated With Otofaciocervical Syndrome.

We report on a 34-year-old woman and her mother who both have clinical features suggestive for otofaciocervical syndrome (OTFCS), a disorder characterized by a combination of facial dysmorphisms, ear abnormalities with hearing loss, and shoulder girdle anomalies. OTFCS presents overlapping features with branchiootorenal spectrum disorders, including branchiootorenal syndrome and branchiootic syndrome. These disorders have been described as clinically distinct entities, but molecular studies have shown that all the causative genes belong to the Pax-Six-Eya-Dach network (PSEDN). So far, the genetic diagnosis of OTFCS has been performed only in very few cases and involves two genes, EYA1 and PAX1; thus, it is likely that other genes have still to be identified. In the present patient, array CGH analysis showed a 3.7-Mb deletion in 6q23; a smaller 1.9-Mb deletion in the same region was detected in her mother. The minimal overlapping region harbors the EYA4 gene. The cases here described are interesting, since they all showed the typical clinical features of OTFCS, associated with a deletion in 6q23.2. Even if we cannot exclude the contribution of other genes to the phenotype, EYA4 is a good candidate for OTFCS according to its pattern of expression, its sequence similarity to EYA1, and its involvement in PSEDN.

Blepharophimosis, Ptosis, Epicanthus Inversus Syndrome: New Report with a 197-kb Deletion Upstream of FOXL2 and Review of the Literature.

Blepharophimosis, ptosis, and epicanthus inversus syndrome (BPES) is due to heterozygous FOXL2 intragenic mutations in about 70% of the patients, whereas total or partial gene deletions account for a minority of cases. Alteration of FOXL2 regulatory elements has been rarely described in patients with BPES. In this study, a prepubertal girl with BPES due to a 197-kb de novo deletion of the regulatory elements upstream of FOXL2 is reported. This girl presented with additional clinical features such as a soft cleft palate and microcephaly; thus, this copy number variant might have other somatic effects. The present deletion encompasses 2 coding genes (MRPS22 and COPB2), whose homozygous mutations have been associated with microcephaly. In our case, the sequences of the non-deleted allele were normal, ruling out a compound genetic defect. Normal levels of new biomarkers of ovarian reserve (anti-müllerian hormone, inhibin B) likely indicate an early diagnosis of type 2 BPES, but an evolutive gonadal damage will be excluded only by long-term follow-up. Additional reports of microdeletions upstream of FOXL2 are needed to better define the underlying genetic mechanism and the related phenotypic spectrum; the ability of the new hormonal markers to predict ovarian function in adolescence and adulthood should be confirmed.

Next generation sequencing technologies for a successful diagnosis in a cold case of Leigh syndrome.

BACKGROUND: Leigh Syndrome (LS, OMIM 256000) is an early-onset, progressive neurodegenerative disorder characterized by broad clinical and genetic heterogeneity; it is the most frequent disorder of mitochondrial energy production in children. LS inheritance is complex because patients may present mutations in mitochondrial DNA (mtDNA) or in nuclear genes, which predominantly encode proteins involved in respiratory chain structure and assembly or in coenzyme Q10 biogenesis. However, during the last 15 years, the discovery of several genetic mutations and improved knowledge of the natural history of LS has significantly increased our understanding of this mitochondrial disorder. CASE PRESENTATION: Here we describe a 19-year-old male with clinical and neuroimaging LS diagnosed at 3 years of age. Genetic analyses of the whole mtDNA for maternally inherited LS (MILS) and neuropathy ataxia retinitis pigmentosa (NARP) syndrome failed to reveal any pathogenic mutations. CONCLUSIONS: Recently, a missense mutation in ECHS1 and a ~ 35 kb deletion in 10q26.3 involving the region including the gene were identified by WES (whole exome sequencing), uncovering the genetic diagnosis clinically hypothesized for 15 years. We also report the long-term follow-up of this patient, showing a comparison with classical LS or other Leigh-like pictures.

Significant Improvement of Clinical Symptoms, Bone Lesions, and Bone Turnover after Long-Term Zoledronic Acid Treatment in Patients with a Severe Form of Camurati-Engelmann Disease.

Camurati-Engelmann disease (CED) is an ultrarare autosomal dominant bone dysplasia. Cortical thickening of the diaphyses of the long bones with narrowing of the medullary cavity are associated with bone pain, waddling gait, muscular weakness, easy fatigability, and a marfanoid body habitus. There is no specific treatment for CED. Nonsteroidal anti-inflammatory drugs or glucocorticoids are ineffective in improving bone lesions. A family with a mild to severe form of CED is described. Two patients received long-term bisphosphonate treatment: the 19-year-old female proband was treated with zoledronic acid for 2.2 years; the 4-year-old male proband was treated with neridronic acid for 16 months and with zoledronic acid for an additional 18 months. In both probands, zoledronic acid treatment significantly improved the clinical symptoms, bone lesions, ambulation, and body habitus. Before treatment, both probands showed a marked increase in serum levels of osteocalcin, procollagen type I N-terminal propeptide, and cross-linked carboxyterminal telopeptide of type I collagen, reflecting an increased bone turnover. Bone marker levels returned to their normal values during treatment. Zoledronic acid treatment may be an important therapeutic option in patients with severe CED. Biochemical markers of bone turnover could be considered as surrogate indexes of CED activity.

Short Stature in Isodicentric Y Chromosome and Three Copies of the SHOX Gene: Clinical Report and Review of Literature.

Short stature homeobox gene (SHOX) mutations and pseudoautosomal region 1 (PAR1) deletions encompassing SHOX are known causes of Léri-Weill dyschondrosteosis and isolated short stature, while 3 copies of SHOX in cases with triple sex chromosome constitution are responsible for tall stature. Duplications involving SHOX have been rarely reported, and they were found in individuals with short, normal and tall stature. An adopted boy with short stature, isodicentric Y chromosome and 3 copies of SHOX is described. Normal growth hormone (GH) secretion and insulin-like growth factor 1 (IGF1) increase during an IGF1 generation test were found, ruling out impaired GH-IGF1 axis. No other organic or psychiatric causes of impaired growth were found. GH treatment improved linear growth, as reported in children with SHOX haploinsufficiency. This new report and the review of literature support that SHOX duplication may cause short stature, especially in those children with duplications of the 5'SHOX regulatory elements. Chromosome analysis and detailed molecular characterization of the duplicated region should be warranted in individuals with SHOX duplications in order to investigate the presence of occult chromosome imbalance. Additional reports and follow-up till adult height are needed to give conclusions on long-term efficacy and safety of GH treatment in short children with SHOX duplication.

Growth in Boys with 45,X/46,XY Mosaicism: Effect of Growth Hormone Treatment on Statural Growth.

45,X/46,XY mosaicism is a rare sex chromosome disorder of sex development. Short stature is a main feature of boys with this condition. Different causes likely contribute to growth impairment. Growth hormone (GH) has been administered to treat short stature in boys with 45,X/46,XY mosaicism, but conflicting data are available. Here, spontaneous growth patterns as well as short- and long-term follow-up studies during GH therapy in these patients are reviewed. Short- and mid-term data showed an improvement of the growth pattern in GH-treated boys, mainly when hormonal therapy was started early, while long-term follow-up demonstrated similar adult heights in GH-treated and untreated patients. Individual biological factors (e.g. different chromosome constitution, different mosaicism among various tissues, impaired pubertal growth spurt), non-homogeneous GH doses and different ages at start of therapy may contribute to the variable results. Thus, early GH therapy at pharmacological doses may improve the growth pattern of short boys with 45,X/46,XY mosaicism, but data on adult height are disappointing. Evaluation of larger patient samples treated by homogeneous doses and long-term follow-up studies assessing adult height and safety are needed to reach definitive conclusions on GH therapy in boys with 45,X/46,XY mosaicism.

625 kb microduplication at Xp22.12 including RPS6KA3 in a child with mild intellectual disability.

Here, we report on a patient with a 625 kb duplication in Xp22.12, detected by array comparative genomic hybridization (CGH). The duplicated region contains only one gene, RPS6KA3, that results in partial duplication. The same duplication was present in his mother and his maternal uncle. This partial duplication inhibits the RPS6KA3 expression, mimicking the effect of loss-of-function mutations associated with Coffin-Lowry syndrome (CLS). The phenotype of the patient here presented is not fully evocative of this syndrome because he does not present some of the facial, digital and skeletal abnormalities that are considered the main diagnostic features of CLS. This case is one of the few examples where RPS6KA3 mutations are associated with a non-specific X-linked mental retardation.

Clinical Phenotype of DiGeorge Syndrome with Negative Genetic Tests: A Case of DiGeorge-Like Syndrome?

We report a case of DiGeorge-like syndrome in which immunodeficiency coexisting with juvenile idiopathic arthritis, congenital heart disease, delay in emergence of language and in motor milestones, feeding and growing problems, enamel hypoplasia, mild skeletal anomalies, and facial dysmorphisms are associated with no abnormalities found on genetic tests.

Molecular analysis, pathogenic mechanisms, and readthrough therapy on a large cohort of Kabuki syndrome patients.

Kabuki syndrome (KS) is a multiple congenital anomalies syndrome characterized by characteristic facial features and varying degrees of mental retardation, caused by mutations in KMT2D/MLL2 and KDM6A/UTX genes. In this study, we performed a mutational screening on 303 Kabuki patients by direct sequencing, MLPA, and quantitative PCR identifying 133 KMT2D, 62 never described before, and four KDM6A mutations, three of them are novel. We found that a number of KMT2D truncating mutations result in mRNA degradation through the nonsense-mediated mRNA decay, contributing to protein haploinsufficiency. Furthermore, we demonstrated that the reduction of KMT2D protein level in patients' lymphoblastoid and skin fibroblast cell lines carrying KMT2D-truncating mutations affects the expression levels of known KMT2D target genes. Finally, we hypothesized that the KS patients may benefit from a readthrough therapy to restore physiological levels of KMT2D and KDM6A proteins. To assess this, we performed a proof-of-principle study on 14 KMT2D and two KDM6A nonsense mutations using specific compounds that mediate translational readthrough and thereby stimulate the re-expression of full-length functional proteins. Our experimental data showed that both KMT2D and KDM6A nonsense mutations displayed high levels of readthrough in response to gentamicin treatment, paving the way to further studies aimed at eventually treating some Kabuki patients with readthrough inducers.

NR5A1 gene mutations: clinical, endocrine and genetic features in two girls with 46,XY disorder of sex development.

BACKGROUND: Steroidogenic factor 1, encoded by the NR5A1 gene, is a key regulator of endocrine function within the hypothalamic-pituitary-steroidogenic axis. Both homozygous, compound heterozygous and heterozygous mutations in the NR5A1 gene may determine 46,XY disorders of sex development (DSD). PATIENTS AND METHODS: NR5A1 gene sequencing was performed in a cohort of 6 patients with 46,XY DSD without specific diagnosis. RESULTS: Heterozygous NR5A1 gene mutations were found in 2 girls, aged 0.5 years and 14 years. The older girl harbored the c.250C>T transition in exon 4 (p.Arg84Cys), previously reported in a Japanese girl. The younger girl presented a de novo novel exon 6 heterozygous frameshift mutation (c.1074dupG) in codon 359 associated with the p.Gly146Ala polymorphism the latter inherited from her father. This baby showed severe impairment of androgen secretion from the first months of life. Overt adrenal insufficiency did not occur, but the older girl showed subnormal cortisol peak after ACTH stimulation. CONCLUSIONS: NR5A1 gene mutations are a relatively frequent cause of 46,XY DSD in humans. Clear indications for management of these individuals remain elusive, mainly when diagnosis is made in infancy. Long-term monitoring of adrenal function should be recommended.

IGF-I generation test in prepubertal children with Noonan syndrome due to mutations in the PTPN11 gene.

BACKGROUND: Short stature represents one of the main features of children with Noonan syndrome. The reason for impaired growth remains largely unknown. OBJECTIVE: To assess GH and IGF1 secretion in children with Noonan syndrome. PATIENTS: 12 prepubertal children with Noonan syndrome due to mutations in the PTPN11 gene [7 males, 6 females; median age, years: 8.6 (range 5.1-13.4)] were studied; 12 prepubertal children with short stature (SS) [7 males, 5 females; median age, years: 8.1 (range 4.8-13.1)] served as the control group. MEASUREMENTS: GH secretion after arginine stimulation test; IGF1 generation test by measurement of IGF1 levels before and after recombinant GH (rGH) administration (0.05 mg/kg/day for 4 days). RESULTS: Baseline and stimulated peak values of GH were not significantly different between the two groups. At +120 minutes, GH levels remained significantly higher (p = 0.0121) in comparison with baseline values in children with Noonan syndrome. Baseline IGFI levels in patients and in SS controls were not significantly different, in contrast to values after the rGH generation test [205 ng/mL (interquartiles 138.2-252.5 ng/mL) and 284.5 ng/mL (interquartiles 172-476 ng/mL), respectively; p = 0.0248]. IGF1 values were significantly related to height (baseline: r = 773, p = 0.0320; peak: r = 0.591, p = 0.0428) in children with Noonan syndrome. CONCLUSIONS: Blunted increase of IGF1 after the rGH generation test was present in children with Noonan syndrome due to mutations in the PTPN11 gene in comparison with SS children. This finding may be due to partial GH resistance in the former likely related to altered Ras-MAPK signaling pathway.

Hypophosphatemic rickets.

PURPOSE OF REVIEW: Description of the recent advances on the regulation of phosphate metabolism, gene mutations, and new approaches to treatment in patients with hypophosphatemic rickets. RECENT FINDINGS: Fibroblast growth factor 23 (FGF23) overproduction may be a primary cause of hypophosphatemic rickets. Inactivating mutations of phosphate-regulating gene with homologies to endopeptidases on the X chromosome, dentin matrix acidic phosphoprotein 1, and ectonucleotide pyrophosphatase/phosphodiesterase 1 are associated with X-linked hypophosphatemic rickets, autosomal recessive hypophosphatemic rickets 1, and autosomal recessive hypophosphatemic rickets 2, respectively. Activating mutations of FGF23 gene is the cause of autosomal dominant hypophosphatemic rickets. Iron deficiency may affect autosomal dominant hypophosphatemic rickets phenotype by regulating FGF23 production.Current treatment with activated vitamin D metabolites and oral inorganic phosphate salts may partially correct skeletal lesions and linear growth in patients with hypophosphatemic rickets. However, some patients have poor improvement by the current treatment. SUMMARY: Identification of the causative mutation in patients with hypophosphatemic rickets may be useful to confirm the diagnosis and probably for prognosis. Inhibition of FGF23 overproduction by anti-FGF23 neutralizing antibodies could be a future approach for treatment of patients with FGF23-dependent hypophosphatemic rickets.

Acro-cardio-facial syndrome: a microdeletion syndrome?

Acro-cardio-facial syndrome (ACFS) is an infrequently reported, variable condition characterized by split-hand and split-foot malformation and congenital heart defect (CHD), along with cleft lip and palate, genital anomalies, unusual face and intellectual disability. An autosomal recessive pattern of inheritance has been suggested because of affected sibs born to unaffected parents and parental consanguinity; the cause is unknown. We describe a newborn with the clinical manifestations of ACFS in whom a deletion of the region 6q21-q22.3 was detected by array CGH. We compare the clinical features of the present patient with earlier reported patients with similar 6q deletions and patients diagnosed with ACFS. The similarities between these patient groups suggest that ACFS may be a microdeletion syndrome caused by loss of the 6q21-22.3 region. The recurrence in families may be explained by prenatal germline mosaicism. Alternatively, ACFS may be a genetically heterogeneous disorder which can also be caused by biallelic mutations of an autosomal recessive gene.

Molecular cytogenetic characterization of an interstitial deletion of chromosome 21 (21q22.13q22.3) in a patient with dysmorphic features, intellectual disability and severe generalized epilepsy.

We report on a de novo interstitial deletion of chromosome 21q in a patient presenting with characteristic facial features, intellectual disability, and epilepsy. The deletion extent was about 4.9 Mb from position 37713441 bp (21q22.13) to position 42665162 bp (21q22.3) (NCBI36/hg18 map). Patients with partial monosomy 21 are quite rare; this anomaly has been associated with a wide spectrum of clinical signs, ranging from very mild to quite severe phenotypes. This variability results from variability in the deleted regions, thus accurate molecular definition of the chromosomal breakpoints is necessary to make better genotype-phenotype correlations. We compared our patient's phenotype with the few other patients reported in the literature and found to have similar deletion when analyzed by array CGH. The minimal overlapping region contains only two genes, DYRK1A and KCNJ6, which may play a major role in these patients' phenotype.