HDAC9 structural variants disrupting TWIST1 transcriptional regulation lead to craniofacial and limb malformations.

Structural variants (SVs) can affect protein-coding sequences as well as gene regulatory elements. However, SVs disrupting protein-coding sequences that also function as cis-regulatory elements remain largely uncharacterized. Here, we show that craniosynostosis patients with SVs containing the histone deacetylase 9 (HDAC9) protein-coding sequence are associated with disruption of TWIST1 regulatory elements that reside within the HDAC9 sequence. Based on SVs within the HDAC9-TWIST1 locus, we defined the 3'-HDAC9 sequence as a critical TWIST1 regulatory region, encompassing craniofacial TWIST1 enhancers and CTCF sites. Deletions of either Twist1 enhancers (eTw5-7Δ/Δ) or CTCF site (CTCF-5Δ/Δ) within the Hdac9 protein-coding sequence led to decreased Twist1 expression and altered anterior/posterior limb expression patterns of SHH pathway genes. This decreased Twist1 expression results in a smaller sized and asymmetric skull and polydactyly that resembles Twist1+/- mouse phenotype. Chromatin conformation analysis revealed that the Twist1 promoter interacts with Hdac9 sequences that encompass Twist1 enhancers and a CTCF site, and that interactions depended on the presence of both regulatory regions. Finally, a large inversion of the entire Hdac9 sequence (Hdac9 INV/+) in mice that does not disrupt Hdac9 expression but repositions Twist1 regulatory elements showed decreased Twist1 expression and led to a craniosynostosis-like phenotype and polydactyly. Thus, our study elucidates essential components of TWIST1 transcriptional machinery that reside within the HDAC9 sequence. It suggests that SVs encompassing protein-coding sequences could lead to a phenotype that is not attributed to its protein function but rather to a disruption of the transcriptional regulation of a nearby gene.

SRPK3 Is Essential for Cognitive and Ocular Development in Humans and Zebrafish, Explaining X-Linked Intellectual Disability.

OBJECTIVE: Intellectual disability is often the outcome of neurodevelopmental disorders and is characterized by significant impairments in intellectual and adaptive functioning. X-linked intellectual disability (XLID) is a subset of these disorders caused by genetic defects on the X chromosome, affecting about 2 out of 1,000 males. In syndromic form, it leads to a broad range of cognitive, behavioral, ocular, and physical disabilities. METHODS: Employing exome or genome sequencing, here we identified 4 missense variants (c.475C > G; p.H159D, c.1373C > A; p.T458N, and c.1585G > A; p.E529K, c.953C > T; p.S318L) and a putative truncating variant (c.1413_1414del; p.Y471*) in the SRPK3 gene in 9 XLID patients from 5 unrelated families. To validate SRPK3 as a novel XLID gene, we established a knockout (KO) model of the SRPK3 orthologue in zebrafish. RESULTS: The 8 patients ascertained postnatally shared common clinical features including intellectual disability, agenesis of the corpus callosum, abnormal eye movement, and ataxia. A ninth case, ascertained prenatally, had a complex structural brain phenotype. Together, these data indicate a pathological role of SRPK3 in neurodevelopmental disorders. In post-fertilization day 5 larvae (free swimming stage), KO zebrafish exhibited severe deficits in eye movement and swim bladder inflation, mimicking uncontrolled ocular movement and physical clumsiness observed in human patients. In adult KO zebrafish, cerebellar agenesis and behavioral abnormalities were observed, recapitulating human phenotypes of cerebellar atrophy and intellectual disability. INTERPRETATION: Overall, these results suggest a crucial role of SRPK3 in the pathogenesis of syndromic X-linked intellectual disability and provide new insights into brain development, cognitive and ocular dysfunction in both humans and zebrafish. ANN NEUROL 2024;96:914-931.

The c.-265G>A GLA gene promoter variant causes Fabry disease: The hidden culprit identified.

Here, we report the identification and functional characterization of a novel GLA variant, not detectable by routine molecular tests, in a family with FD suspicion.

Magnetic resonance imaging scoring system of the lower limbs in adult patients with suspected idiopathic inflammatory myopathy.

PURPOSE: We aim to propose a visual quantitative score for muscle edema in lower limb MRI to contribute to the diagnosis of idiopathic inflammatory myopathy (IIM). MATERIAL AND METHODS: We retrospectively evaluated 85 consecutive patients (mean age 57.4 ± 13.9 years; 56.5% female) with suspected IIM (muscle weakness and/or persistent hyper-CPK-emia with/without myalgia) who underwent MRI of lower limbs using T2-weighted fast recovery-fast spin echo images and fat-sat T2 echo planar images. Muscle inflammation was evaluated bilaterally in 11 muscles of the thigh and eight muscles of the leg. Edema in each muscle was graded according to a four-point Likert-type scale adding up to 114 points ([11 + 8)] × 3 × 2). Diagnostic accuracy of the total edema score was explored by assessing sensitivity and specificity using the area under the ROC curve. Final diagnoses were made by a multidisciplinary Expert Consensus Panel applying the Bohan and Peter diagnostic criteria whenever possible. RESULTS: Of the 85 included patients, 34 (40%) received a final diagnosis of IIM (IIM group) while 51 (60%) received an alternative diagnosis (non-IIM group). A cutoff score ≥ 18 was able to correctly classify patients having an IIM with an area under the curve of 0.85, specificity of 96%, and sensitivity of 52.9%. CONCLUSION: Our study demonstrates that a quantitative MRI score for muscle edema in the lower limbs (thighs and legs) aids in distinguishing IIM from conditions that mimic it.

Adult phenotype in Koolen-de Vries/KANSL1 haploinsufficiency syndrome.

BACKGROUND: Koolen-de Vries syndrome (KdVS) is a multisystem neurodevelopmental disorder caused by 17q21.31 deletions or mutations in KANSL1. It was mainly described in children. METHODS: A retrospective study on 9 subjects aged 19-45 years and revision of 18 literature patients, with the purpose to get insights into the phenotypic evolution with time, and into the clinical manifestations in adulthood. RESULTS: Seven patients had a 17q21.31 deletion and two a point mutation in KANSL1. All had intellectual disability, which was mild in five (56%) and moderate in four (44%). Epilepsy was diagnosed in four subjects (44%), with onset from 1 to 7 years and full remission before 9 years in 3/4 patients. Scoliosis affected seven individuals (77.7%) and it was substantially stable with age in 5/7 patients, allowing for simple daily activities. Two subjects had severely progressive scoliosis, which was surgically corrected. Overweight or true obesity did occur after puberty in six patients (67%). Behaviour abnormalities were recorded in six patients (67%). The facial phenotype slightly evolved with time to include thick eyebrows, elongated nose and pronounced pointed chin. Despite behaviour abnormalities, happy disposition and sociable attitudes were common. Half of patients had fluent language and were good at writing and reading. Rich language, although limited to single words or short sentences, and very limited or absent skills in writing and reading were observed in the remaining patients. Autonomy in daily activities and personal care was usually limited. CONCLUSIONS: Distinctive features in adult KdVS subjects include intellectual disability, overweight/obesity, behaviour abnormalities with preserved social interest, ability in language, slight worsening of the facial phenotype and no seizures.

Refining the mutational spectrum and gene-phenotype correlates in pontocerebellar hypoplasia: results of a multicentric study.

BACKGROUND: Pontocerebellar hypoplasias (PCH) comprise a group of genetically heterogeneous disorders characterised by concurrent hypoplasia of the pons and the cerebellum and variable clinical and imaging features. The current classification includes 13 subtypes, with ~20 known causative genes. Attempts have been made to delineate the phenotypic spectrum associated to specific PCH genes, yet clinical and neuroradiological features are not consistent across studies, making it difficult to define gene-specific outcomes. METHODS: We performed deep clinical and imaging phenotyping in 56 probands with a neuroradiological diagnosis of PCH, who underwent NGS-based panel sequencing of PCH genes and MLPA for CASK rearrangements. Next, we conducted a phenotype-based unsupervised hierarchical cluster analysis to investigate associations between genes and specific phenotypic clusters. RESULTS: A genetic diagnosis was obtained in 43 probands (77%). The most common causative gene was CASK, which accounted for nearly half cases (45%) and was mutated in females and occasionally in males. The European founder mutation p.Ala307Ser in TSEN54 and pathogenic variants in EXOSC3 accounted for 18% and 9% of cases, respectively. VLDLR, TOE1 and RARS2 were mutated in single patients. We were able to confirm only few previously reported associations, including jitteriness and clonus with TSEN54 and lower motor neuron signs with EXOSC3. When considering multiple features simultaneously, a clear association with a phenotypic cluster only emerged for EXOSC3. CONCLUSION: CASK represents the major PCH causative gene in Italy. Phenotypic variability associated with the most common genetic causes of PCH is wider than previously thought, with marked overlap between CASK and TSEN54-associated disorders.

Consolidation of the clinical and genetic definition of a SOX4-related neurodevelopmental syndrome.

BACKGROUND: A neurodevelopmental syndrome was recently reported in four patients with SOX4 heterozygous missense variants in the high-mobility-group (HMG) DNA-binding domain. The present study aimed to consolidate clinical and genetic knowledge of this syndrome. METHODS: We newly identified 17 patients with SOX4 variants, predicted variant pathogenicity using in silico tests and in vitro functional assays and analysed the patients' phenotypes. RESULTS: All variants were novel, distinct and heterozygous. Seven HMG-domain missense and five stop-gain variants were classified as pathogenic or likely pathogenic variant (L/PV) as they precluded SOX4 transcriptional activity in vitro. Five HMG-domain and non-HMG-domain missense variants were classified as of uncertain significance (VUS) due to negative results from functional tests. When known, inheritance was de novo or from a mosaic unaffected or non-mosaic affected parent for patients with L/PV, and from a non-mosaic asymptomatic or affected parent for patients with VUS. All patients had neurodevelopmental, neurological and dysmorphic features, and at least one cardiovascular, ophthalmological, musculoskeletal or other somatic anomaly. Patients with L/PV were overall more affected than patients with VUS. They resembled patients with other neurodevelopmental diseases, including the SOX11-related and Coffin-Siris (CSS) syndromes, but lacked the most specific features of CSS. CONCLUSION: These findings consolidate evidence of a fairly non-specific neurodevelopmental syndrome due to SOX4 haploinsufficiency in neurogenesis and multiple other developmental processes.

Recurrent NF1 gene variants and their genotype/phenotype correlations in patients with Neurofibromatosis type I.

Neurofibromatosis type I, a genetic condition due to pathogenic variants in the NF1 gene, is burdened by a high rate of complications, including neoplasms, which increase morbidity and mortality for the disease. We retrospectively re-evaluated the NF1 gene variants found in the period 2000-2019 and we studied for genotype/phenotype correlations of disease complications and neoplasms 34 variants, which were shared by at least two unrelated families (range 2-11) for a total 141 of probands and 21 relatives affected by Neurofibromatosis type I. Recurrent variants could be ascribed to the most common mutational mechanisms (C to T transition, microsatellite slippage, non-homologous recombination). In genotype/phenotype correlations, the variants p.Arg440*, p.Tyr489Cys, and p.Arg1947*, together with the gross gene deletions, displayed the highest rates of complications. When considering neoplasms, carriers of variants falling in the extradomain region at the 5' end of NF1 had a lower age-related cancer frequency than the rest of the gene sequence, showing a borderline significance (p = 0.045), which was not conserved after correction with covariates. We conclude that (1) hotspots in NF1 occur via different mutational mechanisms, (2) several variants are associated with high rates of complications and cancers, and (3) there is an initial evidence toward a lower cancer risk for carriers of variants in the 5' end of the NF1 gene although not significant at the multivariate analysis.

De Novo SOX4 Variants Cause a Neurodevelopmental Disease Associated with Mild Dysmorphism.

SOX4, together with SOX11 and SOX12, forms group C of SRY-related (SOX) transcription factors. They play key roles, often in redundancy, in multiple developmental pathways, including neurogenesis and skeletogenesis. De novo SOX11 heterozygous mutations have been shown to cause intellectual disability, growth deficiency, and dysmorphic features compatible with mild Coffin-Siris syndrome. Using trio-based exome sequencing, we here identify de novo SOX4 heterozygous missense variants in four children who share developmental delay, intellectual disability, and mild facial and digital morphological abnormalities. SOX4 is highly expressed in areas of active neurogenesis in human fetuses, and sox4 knockdown in Xenopus embryos diminishes brain and whole-body size. The SOX4 variants cluster in the highly conserved, SOX family-specific HMG domain, but each alters a different residue. In silico tools predict that each variant affects a distinct structural feature of this DNA-binding domain, and functional assays demonstrate that these SOX4 proteins carrying these variants are unable to bind DNA in vitro and transactivate SOX reporter genes in cultured cells. These variants are not found in the gnomAD database of individuals with presumably normal development, but 12 other SOX4 HMG-domain missense variants are recorded and all demonstrate partial to full activity in the reporter assay. Taken together, these findings point to specific SOX4 HMG-domain missense variants as the cause of a characteristic human neurodevelopmental disorder associated with mild facial and digital dysmorphism.

De Novo ZMYND8 variants result in an autosomal dominant neurodevelopmental disorder with cardiac malformations.

PURPOSE: ZMYND8 encodes a multidomain protein that serves as a central interactive hub for coordinating critical roles in transcription regulation, chromatin remodeling, regulation of super-enhancers, DNA damage response and tumor suppression. We delineate a novel neurocognitive disorder caused by variants in the ZMYND8 gene. METHODS: An international collaboration, exome sequencing, molecular modeling, yeast two-hybrid assays, analysis of available transcriptomic data and a knockdown Drosophila model were used to characterize the ZMYND8 variants. RESULTS: ZMYND8 variants were identified in 11 unrelated individuals; 10 occurred de novo and one suspected de novo; 2 were truncating, 9 were missense, of which one was recurrent. The disorder is characterized by intellectual disability with variable cardiovascular, ophthalmologic and minor skeletal anomalies. Missense variants in the PWWP domain of ZMYND8 abolish the interaction with Drebrin and missense variants in the MYND domain disrupt the interaction with GATAD2A. ZMYND8 is broadly expressed across cell types in all brain regions and shows highest expression in the early stages of brain development. Neuronal knockdown of the DrosophilaZMYND8 ortholog results in decreased habituation learning, consistent with a role in cognitive function. CONCLUSION: We present genomic and functional evidence for disruption of ZMYND8 as a novel etiology of syndromic intellectual disability.

Preferences of Italian patients for return of secondary findings from clinical genome/exome sequencing.

Exome/genome sequencing (ES/GS) is increasingly becoming routine in clinical genetic diagnosis, yet issues regarding how to disclose and manage secondary findings (SFs) remain to be addressed, and limited evidence is available on patients' preferences. We carried out semi-structured interviews with 307 individuals undergoing clinical genetic testing to explore their preferences for return of SFs in the hypothetical scenario that their test would be performed using ES/GS. Participants were 254 females (82.7%) and 53 males (17.3%), aged 18-86 years; 73.9% (81.1% of those with lower education levels) reported no prior knowledge of ES/GS. Prior knowledge of ES/GS was more common among patients tested for Mendelian conditions (34.5%), compared to those undergoing cancer genetic testing (22.3%) or carrier screening (7.4%). Despite this reported lack of knowledge, most participants (213, 69.6%) stated they would prefer to be informed of all possible results. Reasons in favor of disclosure included wanting to be aware of any risks (168; 83.6%) and to help relatives (23; 11.4%), but also hope that preventive measures might become available in the future (10, 5%). Conversely, potential negative impact on quality of life was the commonest motivation against disclosure. Among 179 participants seen for cancer genetic counseling who were interviewed again after test disclosure, 81.9% had not heard about ES/GS in the meantime; however, the proportion of participants opting for disclosure of any variants was lower (116; 64.8%), with 36 (20.1%) changing opinion compared to the first interview. Based on these findings, we conclude that genetic counseling for ES/GS should involve enhanced education and decision-making support to enable informed consent to SFs disclosure.

An international cohort study of autosomal dominant tubulointerstitial kidney disease due to REN mutations identifies distinct clinical subtypes.

There have been few clinical or scientific reports of autosomal dominant tubulointerstitial kidney disease due to REN mutations (ADTKD-REN), limiting characterization. To further study this, we formed an international cohort characterizing 111 individuals from 30 families with both clinical and laboratory findings. Sixty-nine individuals had a REN mutation in the signal peptide region (signal group), 27 in the prosegment (prosegment group), and 15 in the mature renin peptide (mature group). Signal group patients were most severely affected, presenting at a mean age of 19.7 years, with the prosegment group presenting at 22.4 years, and the mature group at 37 years. Anemia was present in childhood in 91% in the signal group, 69% prosegment, and none of the mature group. REN signal peptide mutations reduced hydrophobicity of the signal peptide, which is necessary for recognition and translocation across the endoplasmic reticulum, leading to aberrant delivery of preprorenin into the cytoplasm. REN mutations in the prosegment led to deposition of prorenin and renin in the endoplasmic reticulum-Golgi intermediate compartment and decreased prorenin secretion. Mutations in mature renin led to deposition of the mutant prorenin in the endoplasmic reticulum, similar to patients with ADTKD-UMOD, with a rate of progression to end stage kidney disease (63.6 years) that was significantly slower vs. the signal (53.1 years) and prosegment groups (50.8 years) (significant hazard ratio 0.367). Thus, clinical and laboratory studies revealed subtypes of ADTKD-REN that are pathophysiologically, diagnostically, and clinically distinct.

Autozygosity-driven genetic diagnosis in consanguineous families from Italy and the Greater Middle East.

Autozygosity-driven exome analysis has been shown effective for identification of genes underlying recessive diseases especially in countries of the so-called Greater Middle East (GME), where high consanguinity unravels the phenotypic effects of recessive alleles and large family sizes facilitate homozygosity mapping. In Italy, as in most European countries, consanguinity is estimated low. Nonetheless, consanguineous Italian families are not uncommon in publications of genetic findings and are often key to new associations of genes with rare diseases. We collected 52 patients from 47 consanguineous families with suspected recessive diseases, 29 originated in GME countries and 18 of Italian descent. We performed autozygosity-driven exome analysis by detecting long runs of homozygosity (ROHs > 1.5 Mb) and by prioritizing candidate clinical variants within. We identified a pathogenic synonymous variant that had been previously missed in NARS2 and we increased an initial high diagnostic rate (47%) to 55% by matchmaking our candidate genes and including in the analysis shorter ROHs that may also happen to be autozygous. GME and Italian families contributed to diagnostic yield comparably. We found no significant difference either in the extension of the autozygous genome, or in the distribution of candidate clinical variants between GME and Italian families, while we showed that the average autozygous genome was larger and the mean number of candidate clinical variants was significantly higher (p = 0.003) in mutation-positive than in mutation-negative individuals, suggesting that these features influence the likelihood that the disease is autozygosity-related. We highlight the utility of autozygosity-driven genomic analysis also in countries and/or communities, where consanguinity is not widespread cultural tradition.

Aortic arch geometry predicts outcome in patients with Loeys-Dietz syndrome independent of the causative gene.

This study aimed to investigate the potential association between imaging features and cardiovascular outcomes in patients with Loeys­Dietz syndrome (LDS). We performed a retrospective cohort study of 36 patients with LDS and described cardiovascular events and imaging data. We observed different clinical courses in patients with LDS, irrespective of the causative gene. Angular or elongated aortic arch geometry correlated with aortic dissection (R = .39, p = .02), occurrence of the first cardiovascular event before 45 years of age (R = .36, p = .03), and the number of operations (R = 0.47, p = .004), but not with age (R = −.05, p = .79) or the causative gene (R = −0.04, p = .79). Relative absences of cardiovascular events at ages 20, 40, and 60 were 100, 75, and 56%, respectively, in patients with "romanesque" aortic arches, and 74, 39, and 21%, respectively, in patients with "gothic" and "elongated" aortic arches (p = .03). Angular or elongated aortic arch geometry is associated with early­onset of disease and a worse cardiovascular outcome in LDS patients. Large multicenter studies are warranted to elucidate the impact of aortic arch morphology evaluation in clinical practice.

Kidney Transplant in Fabry Disease: A Revision of the Literature.

Fabry disease is classified as a rare X-linked disease caused by a complete or partial defect of enzyme alpha-galactosidase, due to GLA gene mutations. This disorder leads to intracellular globotriaosylceramide (Gb3) deposition associated with increased Gb3 plasma levels. Most of the symptoms of the disease, involving kidneys, heart and nervous system, result from this progressive Gb3 deposition. The incidence is estimated in 1/50,000 to 1/117,000 in males. Fabry nephropathy begins with microalbuminuria and/or proteinuria, which, in the classic form, appear from childhood. Thus, a progressive decline of renal function can start at a young age, and evolve to kidney failure, requiring dialysis or renal transplantation. Enzyme replacement therapy (ERT), available since 2001 for Fabry disease, has been increasingly introduced into the clinical practice, with overall positive short-term and long-term effects in terms of ventricular hypertrophy and renal function. Kidney transplantation represents a relevant therapeutic option for Fabry nephropathy management, for patients reaching end-stage renal disease, but little is known about long-term outcomes, overall patient survival or the possible role of ERT after transplant. The purpose of this review is to analyze the literature on every aspect related to kidney transplantation in patients with Fabry nephropathy: from the analysis of transplant outcomes, to the likelihood of disease recurrence, up to the effects of ERT and its possible interference with immunosuppression.

Recurrent De Novo and Biallelic Variation of ATAD3A, Encoding a Mitochondrial Membrane Protein, Results in Distinct Neurological Syndromes.

ATPase family AAA-domain containing protein 3A (ATAD3A) is a nuclear-encoded mitochondrial membrane protein implicated in mitochondrial dynamics, nucleoid organization, protein translation, cell growth, and cholesterol metabolism. We identified a recurrent de novo ATAD3A c.1582C>T (p.Arg528Trp) variant by whole-exome sequencing (WES) in five unrelated individuals with a core phenotype of global developmental delay, hypotonia, optic atrophy, axonal neuropathy, and hypertrophic cardiomyopathy. We also describe two families with biallelic variants in ATAD3A, including a homozygous variant in two siblings, and biallelic ATAD3A deletions mediated by nonallelic homologous recombination (NAHR) between ATAD3A and gene family members ATAD3B and ATAD3C. Tissue-specific overexpression of borR534W, the Drosophila mutation homologous to the human c.1582C>T (p.Arg528Trp) variant, resulted in a dramatic decrease in mitochondrial content, aberrant mitochondrial morphology, and increased autophagy. Homozygous null bor larvae showed a significant decrease of mitochondria, while overexpression of borWT resulted in larger, elongated mitochondria. Finally, fibroblasts of an affected individual exhibited increased mitophagy. We conclude that the p.Arg528Trp variant functions through a dominant-negative mechanism that results in small mitochondria that trigger mitophagy, resulting in a reduction in mitochondrial content. ATAD3A variation represents an additional link between mitochondrial dynamics and recognizable neurological syndromes, as seen with MFN2, OPA1, DNM1L, and STAT2 mutations.

HDAC8 Loss of Function and SHOX Haploinsufficiency: Two Independent Genetic Defects Responsible for a Complex Phenotype.

We report a patient with developmental delay, brachydactyly type E, short stature, and tetralogy of Fallot. Brachydactyly-mental retardation syndrome (BDMR) was suspected based on the phenotype; however, array CGH excluded a 2q37 deletion, but identified a deletion encompassing the SHOX gene. BDMR is characterized by cognitive impairment, skeletal abnormalities involving hands and feet, short stature, and overweight. Most affected individuals carry relatively large 2q37 deletions encompassing HDAC4. This gene encodes a histone deacetylase involved in epigenetic regulation of cell growth and differentiation, specifically during endochondral bone formation in chondrocyte hypertrophy. Since SHOX haploinsufficiency can cause skeletal defects and short stature but would not fully explain the clinical picture of this patient, exome sequencing was performed, and a heterozygous HDAC8 frameshift mutation was identified. HDAC8 is a distinct histone deacetylase involved in cohesin recycling and is responsible for an X-linked dominant Cornelia de Lange-like phenotype. A new blended clinical phenotype may be explained by the result of a dual molecular diagnosis, which represents a combination of 2 independent genetic defects, with relevant implications for genetic counseling, clinical management, and prognosis.

Bi-allelic mutations in TRAPPC2L result in a neurodevelopmental disorder and have an impact on RAB11 in fibroblasts.

BACKGROUND: The combination of febrile illness-induced encephalopathy and rhabdomyolysis has thus far only been described in disorders that affect cellular energy status. In the absence of specific metabolic abnormalities, diagnosis can be challenging. OBJECTIVE: The objective of this study was to identify and characterise pathogenic variants in two individuals from unrelated families, both of whom presented clinically with a similar phenotype that included neurodevelopmental delay, febrile illness-induced encephalopathy and episodes of rhabdomyolysis, followed by developmental arrest, epilepsy and tetraplegia. METHODS: Whole exome sequencing was used to identify pathogenic variants in the two individuals. Biochemical and cell biological analyses were performed on fibroblasts from these individuals and a yeast two-hybrid analysis was used to assess protein-protein interactions. RESULTS: Probands shared a homozygous TRAPPC2L variant (c.109G>T) resulting in a p.Asp37Tyr missense variant. TRAPPC2L is a component of transport protein particle (TRAPP), a group of multisubunit complexes that function in membrane traffic and autophagy. Studies in patient fibroblasts as well as in a yeast system showed that the p.Asp37Tyr protein was present but not functional and resulted in specific membrane trafficking delays. The human missense mutation and the analogous mutation in the yeast homologue Tca17 ablated the interaction between TRAPPC2L and TRAPPC10/Trs130, a component of the TRAPP II complex. Since TRAPP II activates the GTPase RAB11, we examined the activation state of this protein and found increased levels of the active RAB, correlating with changes in its cellular morphology. CONCLUSIONS: Our study implicates a RAB11 pathway in the aetiology of the TRAPPC2L disorder and has implications for other TRAPP-related disorders with similar phenotypes.

Insights into Mutation Effect in Three Poikiloderma with Neutropenia Patients by Transcript Analysis and Disease Evolution of Reported Patients with the Same Pathogenic Variants.

PURPOSE: Poikiloderma with neutropenia (PN) is a genodermatosis currently described in 77 patients, all presenting with early-onset poikiloderma, neutropenia, and several additional signs. Biallelic loss-of-function mutations in USB1 gene are detected in all molecularly tested patients but genotype-phenotype correlation remains elusive. Cancer predisposition is recognized among PN features and pathogenic variants found in patients who developed early in life myelodysplasia (n = 12), acute myeloid leukemia (n = 2), and squamous cell carcinoma (n = 2) should be kept into account in management and follow-up of novel patients. This will hopefully allow achieving data clustered on specific mutations relevant to oncological surveillance of the carrier patients. METHODS: We describe the clinical features of three unreported PN patients and characterize their USB1 pathogenic variants by transcript analysis to get insights into the effect on the overall phenotype and disease evolution. RESULTS: A Turkish boy is homozygous for the c.531delA deletion, a recurrent mutation in Turkey; an adult Italian male is compound heterozygous for two nonsense mutations, c.243G>A and c.541C>T, while an Italian boy is homozygous for the splicing c.683_693+1del variant. The identified mutations have already been reported in PN patients who developed hematologic or skin cancer. Aberrant mRNAs of all four mutated alleles could be identified confirming that transcripts of USB1 main isoform either carrying stop codons or mis-spliced may at least partially escape nonsense-mediated decay. CONCLUSIONS: Our study addresses the need of gathering insights on genotype-phenotype correlations in newly described PN patients, by transcript analysis and information on disease evolution of reported patients with the same pathogenic variants.

A mosaic intragenic microduplication of LAMA1 and a constitutional 18p11.32 microduplication in a patient with keratosis pilaris and intellectual disability.

The application of array-based comparative genomic hybridization and next-generation sequencing has identified many chromosomal microdeletions and microduplications in patients with different pathological phenotypes. Different copy number variations are described within the short arm of chromosome 18 in patients with skin diseases. In particular, full or partial monosomy 18p has also been associated with keratosis pilaris. Here, for the first time, we report a young male patient with intellectual disability, diabetes mellitus (type I), and keratosis pilaris, who exhibited a de novo 45-kb microduplication of exons 4-22 of LAMA1, located at 18p11.31, and a 432-kb 18p11.32 microduplication of paternal origin containing the genes METTL4, NDC80, and CBX3P2 and exons 1-15 of the SMCHD1 gene. The microduplication of LAMA1 was identified in skin fibroblasts but not in lymphocytes, whereas the larger microduplication was present in both tissues. We propose LAMA1 as a novel candidate gene for keratosis pilaris. Although inherited from a healthy father, the 18p11.32 microduplication, which included relevant genes, could also contribute to phenotype manifestation.