Hereditary spastic paraplegias proteome: common pathways and pathogenetic mechanisms.

INTRODUCTION: Hereditary spastic paraplegias (HSPs) are a group of inherited neurodegenerative disorders characterized by progressive spasticity and weakness of the lower limbs. These conditions are caused by lesions in the neuronal pyramidal tract and exhibit clinical and genetic variability. Ongoing research focuses on understanding the underlying mechanisms of HSP onset, which ultimately lead to neuronal degeneration. Key molecular mechanisms involved include axonal transport, cytoskeleton dynamics, myelination abnormalities, membrane trafficking, organelle morphogenesis, ER homeostasis, mitochondrial dysfunction, and autophagy deregulation. AREAS COVERED: This review aims to provide an overview of the shared pathogenetic mechanisms in various forms of HSPs. By examining disease-causing gene products and their associated functional pathways, this understanding could lead to the discovery of new therapeutic targets and the development of treatments to modify the progression of the disease. EXPERT OPINION: Investigating gene functionality is crucial for identifying shared pathogenetic pathways underlying different HSP subtypes. Categorizing protein function and identifying pathways aids in finding biomarkers, predicting early onset, and guiding treatment for a better quality of life. Targeting shared mechanisms enables efficient and cost-effective therapies. Prospects involve identifying new disease-causing genes, refining molecular processes, and implementing findings in diagnosis, key for advancing HSP understanding and developing effective treatments.

Δ1 -Pyrroline-5-carboxylate synthetase deficiency: An emergent multifaceted urea cycle-related disorder.

The bifunctional homooligomeric enzyme Δ1 -pyrroline-5-carboxylate synthetase (P5CS) and its encoding gene ALDH18A1 were associated with disease in 1998. Two siblings who presented paradoxical hyperammonemia (alleviated by protein), mental disability, short stature, cataracts, cutis laxa, and joint laxity, were found to carry biallelic ALDH18A1 mutations. They showed biochemical indications of decreased ornithine/proline synthesis, agreeing with the role of P5CS in the biosynthesis of these amino acids. Of 32 patients reported with this neurocutaneous syndrome, 21 familial ones hosted homozygous or compound heterozygous ALDH18A1 mutations, while 11 sporadic ones carried de novo heterozygous ALDH18A1 mutations. In 2015 to 2016, an upper motor neuron syndrome (spastic paraparesis/paraplegia SPG9) complicated with some traits of the neurocutaneous syndrome, although without report of cutis laxa, joint laxity, or herniae, was associated with monoallelic or biallelic ALDH18A1 mutations with, respectively, dominant and recessive inheritance. Of 50 SPG9 patients reported, 14 and 36 (34/2 familial/sporadic) carried, respectively, biallelic and monoallelic mutations. Thus, two neurocutaneous syndromes (recessive and dominant cutis laxa 3, abbreviated ARCL3A and ADCL3, respectively) and two SPG9 syndromes (recessive SPG9B and dominant SPG9A) are caused by essentially different spectra of ALDH18A1 mutations. On the bases of the clinical data (including our own prior patients' reports), the ALDH18A1 mutations spectra, and our knowledge on the P5CS protein, we conclude that the four syndromes share the same pathogenic mechanisms based on decreased P5CS function. Thus, these syndromes represent a continuum of increasing severity (SPG9A < SPG9B < ADCL3 ≤ ARCL3A) of the same disease, P5CS deficiency, in which the dominant mutations cause loss-of-function by dominant-negative mechanisms.

Delineation of the 9q31 deletion syndrome: Genomic microarray characterization of two patients with overlapping deletions.

Interstitial deletions of chromosome 9q31 are very rare. The deletions in most reported patients have been detected by conventional cytogenetics, with reported breakpoints ranging between 9q21 and 9q34. Therefore, an accurate description of a "9q31 deletion syndrome" could not be established. However, based on microarray studies, a small region of overlap has recently been proposed. We report clinical features of two unrelated individuals with overlapping 9q deletions identified by SNP microarray analysis. Patient 1 has a 9 Mb deletion, while Patient 2's deletion was 21.6 Mb. The clinical features common to our patients and those in the literature include developmental delay and short stature. Patient 2 shows additional features not reported in other 9q31 deletions, such as hearing loss, ventriculomegaly, cleft lip and palate, and small kidneys, which could be due to the larger size of the deletion, hence the influence of the genes in the region beyond the smallest region of overlap. Based on the comparison of these patients with the previously reported patients, we redefine the smallest region of overlap and characterize the clinical features of the 9q31 deletion syndrome.

Phenotypic Description of A Patient with ODLURO Syndrome and Functional Characterization of the Pathogenetic Role of A Synonymous Variant c.186G>A in KMT2E Gene.

O'Donnell-Luria-Rodan (ODLURO) syndrome is an autosomal dominant disorder caused by mutations in the KMT2E gene. The clinical phonotype of the affected individuals is typically characterized by global developmental delay, autism, epilepsy, hypotonia, macrocephaly, and very mild dysmorphic facial features. In this report, we describe the case of a 6-year-old boy with ODLURO syndrome who is a carrier of the synonymous mutation c.186G>A (p.Ala62=) in the KMT2E gene, predicted to alter splicing by in silico tools. Given the lack of functional studies on the c.186G>A variant, in order to assess its potential functional effect, we sequenced the patient's cDNA demonstrating its impact on the mechanism of splicing. To the best of our knowledge, our patient is the second to date reported carrying this synonymous mutation, but he is the first whose functional investigation has confirmed the deleterious consequence of the variant, resulting in exon 4 skipping. Additionally, we suggest a potential etiological mechanism that could be responsible for the aberrant splicing mechanism in KMT2E.

Case report: Functional characterization of a novel CHD7 intronic variant in patients with CHARGE syndrome.

Background: Because CHARGE syndrome is characterized by high clinical variability, molecular confirmation of the clinical diagnosis is of pivotal importance. Most patients have a pathogenic variant in the CHD7 gene; however, variants are distributed throughout the gene and most cases are due to de novo mutations. Often, assessing the pathogenetic effect of a variant can be challenging, requiring the design of a unique assay for each specific case. Method: Here we describe a new CHD7 intronic variant, c.5607+17A>G, identified in two unrelated patients. In order to characterize the molecular effect of the variant, minigenes were constructed using exon trapping vectors. Results: The experimental approach pinpoints the pathogenetic effect of the variant on CHD7 gene splicing, subsequently confirmed using cDNA synthetized from RNA extracted from patient lymphocytes. Our results were further corroborated by the introduction of other substitutions at the same nucleotide position, showing that c.5607+17A>G specifically alters splicing possibly due to the generation of a recognition motif for the recruitment of a splicing effector. Conclusion: Here we identify a novel pathogenetic variant affecting splicing, and we provide a detailed molecular characterization and possible functional explanation.

Hereditary spastic paraplegia: Genetic heterogeneity and common pathways.

Hereditary Spastic Paraplegias (HSPs) are a heterogeneous group of disease, mainly characterized by progressive spasticity and weakness of the lower limbs resulting from distal degeneration of corticospinal tract axons. Although HSPs represent rare or ultra-rare conditions, with reported cases of mutated genes found in single families, overall, with 87 forms described, they are an important health and economic problem for society and patients. In fact, they are chronic and life-hindering conditions, still lacking a specific therapy. Notwithstanding the number of forms described, and 73 causative genes identified, overall, the molecular diagnostic rate varies among 29% to 61.8%, based on recent published analysis, suggesting that more genes are involved in HSP and/or that different molecular diagnostic approaches are necessary. The accumulating data in this field highlight several peculiar features of HSPs, such as genetic heterogeneity, the discovery that different mutations in a single gene can be transmitted in dominant and recessive trait in families and allelic heterogeneity, resulting in the involvement of HSP-genes in other conditions. Based on the observation of protein functions, the activity of many different proteins encoded by HSP-related genes converges into some distinct pathophysiological mechanisms. This suggests that common pathways could be a potential target for a therapy, possibly addressing several forms at once. Furthermore, the overlap of HSP genes with other neurological conditions can further expand this concept.

Association of hereditary thrombocythemia and distal limb defects with a thrombopoietin gene mutation.

Hereditary thrombocythemia is a rare autosomal dominant disorder caused by mutations in either the thrombopoietin gene (TPO) or its receptor c-MPL. TPO mutations described so far lead to thrombopoietin overproduction through increased translation of m-RNA. Unilateral transverse reduction limb defects are usually sporadic and generally thought to be caused by vascular disruptions. Reports of inherited unilateral limb defects are extremely rare. In the present study, we describe a family with segregation of G185T TPO mutation in the 5' UTR region in 4 subjects with thrombocythemia. Three of these patients also present congenital transverse limb defects. Association of these events gives a strong hint of the in vivo involvement of thrombopoietin in vasculogenesis, confirming the role of TPO in human development of the hemangioblast, the embryonic progenitor of the hematopoietic and endothelial lineages. This is the first report showing that vascular disruptions could be secondary to specific gene derangements.

The clear cell sarcoma functional genomic landscape.

Clear cell sarcoma (CCS) is a deadly malignancy affecting adolescents and young adults. It is characterized by reciprocal translocations resulting in expression of the chimeric EWSR1-ATF1 or EWSR1-CREB1 fusion proteins, driving sarcomagenesis. Besides these characteristics, CCS has remained genomically uncharacterized. Copy number analysis of human CCSs showed frequent amplifications of the MITF locus and chromosomes 7 and 8. Few alterations were shared with Ewing sarcoma or desmoplastic, small round cell tumors, which are other EWSR1-rearranged tumors. Exome sequencing in mouse tumors generated by expression of EWSR1-ATF1 from the Rosa26 locus demonstrated no other repeated pathogenic variants. Additionally, we generated a new CCS mouse by Cre-loxP-induced chromosomal translocation between Ewsr1 and Atf1, resulting in copy number loss of chromosome 6 and chromosome 15 instability, including amplification of a portion syntenic to human chromosome 8, surrounding Myc. Additional experiments in the Rosa26 conditional model demonstrated that Mitf or Myc can contribute to sarcomagenesis. Copy number observations in human tumors and genetic experiments in mice rendered, for the first time to our knowledge, a functional landscape of the CCS genome. These data advance efforts to understand the biology of CCS using innovative models that will eventually allow us to validate preclinical therapies necessary to achieve longer and better survival for young patients with this disease.

MYH9 related disease: four novel mutations of the tail domain of myosin-9 correlating with a mild clinical phenotype.

MYH9-related disease (MYH9-RD) is a rare autosomal dominant disorder caused by mutations in MYH9, the gene encoding the heavy chain of non-muscle myosin IIA. All patients present congenital macrothrombocytopenia and inclusion bodies in neutrophils. Some of them can also develop sensorineural deafness, presenile cataract, and/or progressive nephropathy leading to end-stage renal failure. We report four families, each with a novel mutation: two missense mutations, in exons 31 and 32, and two out of frame deletions in exon 40. They were associated with no bleeding diathesis, normal, or only slightly reduced platelet count and no extra-hematological manifestations, confirming that alterations of the tail domain cause a mild form of MYH9-RD with no clinically relevant defects.

A heritable cause of cleft lip and palate--Van der Woude syndrome caused by a novel IRF6 mutation. Review of the literature and of the differential diagnosis.

BACKGROUND: Orofacial clefts are common congenital malformations usually characterized by a multifactorial etiology. These heterogeneous defects comprise cleft lip (CL), CL with cleft palate (CL/P), and cleft palate, sometimes observed in recognizable syndromes, with mendelian, chromosomal, or environmental pathogenesis. The Van der Woude syndrome is a mendelian CL/P, accounting for about 2% of all cases and caused by mutations in the interferon regulatory factor 6 (IRF6) gene, located on 1q32.2 chromosome. OBJECTIVE: Here, we describe a familial case with a novel IRF6 mutation segregating in the maternal line, displaying a highly intrafamilial variable clinical expression. CONCLUSION: This report emphasizes the role of the clinician in recognizing the clinical variability and the genetic heterogeneity of CL/P.

Seizures and Cardiomyopathy in a Patient with Pallister-Killian Syndrome due to Hexasomy 12p Mosaicism.

Pallister-Killian syndrome (PKS) is a rare disorder presenting with developmental delay, numerous dysmorphic features, and skin pigmentation anomalies. It is caused by mosaic tetrasomy of the short arm of chromosome 12. In most instances, tetrasomy is due to a supernumerary isochromosome i(12)(p10). Although mitotic instability is a generally accepted behavior for supernumerary chromosomes, hexasomy 12p due to a gain of an isochromosome 12p, has been hardly ever reported. We report a 10 year follow-up on a girl with 2 copies of isochromosome consisting of the short arm of chromosome 12, who has craniofacial features seen in PKS, such as sparse hair with an unusual pattern, sparse eyebrows, lacrimal duct stenosis, submucous cleft palate, Pallister lip (a relatively long philtrum continuing into the vermillion border of the upper lip), narrow palate, and wide alveolar ridges. She also has other abnormalities, including unilateral renal dysgenesis, rectovaginal fistula, pre-axial polydactyly of the right hand, severe global developmental delay, and hypotonia as well as some features suggestive of mosaicism such as bilateral asymmetry, patchy areas of rough skin, and retinal mottling. Initial cytogenetic studies from peripheral blood showed a normal female karyotype. Further cytogenetic studies on a skin biopsy showed mosaicism with 2 copies of the supernumerary isochromosome 12p.

Benign albeit glycolytic: MCT4 expression and lactate release in giant cell tumour of bone.

Giant cell tumour of bone (GCTB) is a histologically benign, locally aggressive skeletal lesion with an unpredictable propensity to relapse after surgery and a rare metastatic potential. The microscopic picture of GCTB shows different cell types, including multinucleated giant cells, mononuclear cells of the macrophage-monocyte lineage, and spindle cells. The histogenesis of GCTB is still debated, and morphologic, radiographic or molecular features are not predictive of the clinical course. Characterization of the unexplored cell metabolism of GCTB offers significant clues for the understanding of this elusive pathologic entity. In this study we aimed to characterize GCTB energetic metabolism, with a particular focus on lactate release and the expression of monocarboxylate transporters, to lie down a novel path for understanding the pathophysiology of this tumour. We measured the expression of glycolytic markers (GAPDH, PKM2, MCT4, GLUT1, HK1, LDHA, lactate release) in 25 tissue samples of GCTB by immunostaining and by mRNA and ELISA analyses. We also evaluated MCT1 and MCT4 expression and oxidative markers (JC1 staining and Bec index) in tumour-derived spindle cell cultures and CD14+ monocytic cells. Finally, we quantified the intratumoural and circulating levels of lactate in a series of 17 subjects with GCTB. In sharp contrast to the benign histological features of GCTB, we found a high expression of glycolytic markers, with particular reference to MCT4. Unexpectedly, this was mainly confined to the giant cell, not proliferating cell component. Accordingly, GCTB patients showed higher levels of blood lactate as compared to healthy subjects. In conclusion, taken together, our data indicate that GCTB is characterized by a highly glycolytic metabolism of its giant cell component, opening new perspectives on the pathogenesis, the natural history, and the treatment of this lesion.

Hereditary Spastic Paraplegia Is a Common Phenotypic Finding in ARG1 Deficiency, P5CS Deficiency and HHH Syndrome: Three Inborn Errors of Metabolism Caused by Alteration of an Interconnected Pathway of Glutamate and Urea Cycle Metabolism.

Hereditary Spastic Paraplegias (HSPs) are a clinically and genetically heterogeneous group of neurodegenerative disorders characterized by a progressive rigidity and weakness of the lower limbs, caused by pyramidal tract lesions. As of today, 80 different forms of HSP have been mapped, 64 genes have been cloned, and new forms are constantly being described. HSPs represent an intensively studied field, and the functional understanding of the biochemical and molecular pathogenetic pathways are starting to be elucidated. Recently, dominant and recessive mutations in the ALDH18A1 gene resulting in the deficiency of the encoded enzyme (delta-1-pyrroline-5-carboxylate synthase, P5CS) have been pathogenetically linked to HSP. P5CS is a critical enzyme in the conversion of glutamate to pyrroline-5-carboxylate, an intermediate that enters in the proline biosynthesis and that is connected with the urea cycle. Interestingly, two urea cycle disorders, Argininemia and Hyperornithinemia-Hyperammonemia-Homocitrullinuria syndrome, are clinically characterized by highly penetrant spastic paraplegia. These three diseases represent a peculiar group of HSPs caused by Inborn Errors of Metabolism. Here we comment on these forms, on the common features among them and on the hypotheses for possible shared pathogenetic mechanisms causing the HSP phenotype.

P5CS expression study in a new family with ALDH18A1-associated hereditary spastic paraplegia SPG9.

In 2015-2016, we and others reported ALDH18A1 mutations causing dominant (SPG9A) or recessive (SPG9B) spastic paraplegia. In vitro production of the ALDH18A1 product, Δ1 -pyrroline-5-carboxylate synthetase (P5CS), appeared necessary for cracking SPG9 disease-causing mechanisms. We now describe a baculovirus-insect cell system that yields mgs of pure human P5CS and that has proven highly valuable with two novel P5CS mutations reported here in new SPG9B patients. We conclude that both mutations are disease-causing, that SPG9B associates with partial P5CS deficiency and that it is clinically more severe than SPG9A, as reflected in onset age, disability, cognitive status, growth, and dysmorphic traits.

Position of nonmuscle myosin heavy chain IIA (NMMHC-IIA) mutations predicts the natural history of MYH9-related disease.

MYH9-related disease (MYH9-RD) is a rare autosomal-dominant disorder caused by mutations in MYH9, the gene for the heavy chain of nonmuscle myosin IIA (NMMHC-IIA). All patients present from birth with macrothrombocytopenia, but in infancy or adult life, some of them develop sensorineural deafness, presenile cataracts, and/or progressive nephritis leading to end-stage renal failure. No consistent correlations have been identified between the 27 different MYH9 mutations identified so far and the variable clinical evolution of the disease. We have evaluated 108 consecutive MYH9-RD patients belonging to 50 unrelated pedigrees. The risk of noncongenital manifestations associated with different genotypes was estimated over time by event-free survival analysis. We demonstrated that all subjects with mutations in the motor domain of NMMHC-IIA present with severe thrombocytopenia and develop nephritis and deafness before the age of 40 years, while those with mutations in the tail domain have a much lower risk of noncongenital complications and significantly higher platelet counts. We also evaluated the clinical course of patients with mutations in the four most frequently affected residues of NMMHC-IIA (responsible for 70% of MYH9-RD cases). We concluded that mutations at residue 1933 do not induce kidney damage or cataracts and cause deafness only in the elderly, those in position 702 result in severe thrombocytopenia and produce nephritis and deafness at a juvenile age, while alterations at residue 1424 or 1841 result in intermediate clinical pictures. These findings are relevant not only to patients' clinical management but also to the elucidation of the pathogenesis of the disease.

A novel locus for syndromic chronic idiopathic intestinal pseudo-obstruction maps to chromosome 8q23-q24.

Chronic idiopathic intestinal pseudo-obstruction (CIIP) is a rare and severe clinical syndrome characterized by symptoms and signs of intestinal occlusion, in the absence of any mechanical obstruction of the gut lumen. In the attempt to identify the genetic basis of CIIP, we analyzed a Turkish pedigree with a high degree of consanguinity in which three siblings presented with a syndromic form of CIIP. All affected family members were characterized by recurrent, self-limiting subocclusive episodes, long-segment Barrett esophagus, and a variety of minor cardiac valve or septal defects. In some patients full-thickness intestinal biopsy samples were obtained and tissues were processed for immunohistochemistry using antibodies to different markers of the intestinal neuromuscular tract. Full-thickness biopsies of the gut wall showed abnormalities of both the neural and muscular components suggesting an underlying intestinal neuro-myopathy. Blood samples were collected for DNA extraction from each available family member and DNAs were genotyped using 382 microsatellites spanning the entire genome with the aim to take advantage of the homozygosity mapping approach. Linkage analysis identified a new syndromic locus on chromosome 8q23-q24 (multipoint LOD score=5.01). Our data strongly support the presence of a new genetic locus associated with CIIP, long-segment Barrett esophagus, and cardiac involvement on chromosome 8.

The breakpoint identified in a balanced de novo translocation t(7;9)(p14.1;q31.3) disrupts the A-kinase (PRKA) anchor protein 2 gene (AKAP2) on chromosome 9 in a patient with Kallmann syndrome and bone anomalies.

We report the molecular characterization of a patient with Kallmann syndrome and bone anomalies bearing a balanced de novo translocation t(7;9)(p14.1;q31.3) which completely disrupts the A-kinase anchor protein 2 gene (AKAP2) on chromosome 9. In order to investigate the role of AKAP2 in the pathogenesis of the disease, we analyzed the expression of Akap2 in mouse embryos. The expression pattern was consistent with the phenotype observed and mAkap2 was actually found in the olfactory bulb and in the cartilagineous structures of the embryo. Since AKAP2 is supposed to bind and compartmentalize the PKA, we also analyzed the distribution and quantity of PKA in limphoblastoid cell lines of the patient compared with a control; these experiments did not demonstrate any differences between the cell lines. Furthermore a collection of 98 DNA samples from sporadic Kallmann patients was screened for mutations in this gene. The analysis revealed two different sequence variations observed in two patients but not in 200 control chromosomes: since they have been detected also in the unaffected mother of one of the two patients we can assume that they are rare polymorphisms, although we cannot exclude that they represent mutations with incomplete penetrance. Our findings suggest that the complex phenotype with Kallmann syndrome and bone anomalies observed in our patient could be the result of the interruption of the AKAP2 gene. However, a position effect mediated by the translocation could not be excluded. The screening of AKAP2 in other Kallmann patients will be necessary to elucidate its role in the pathogenesis of the disease.

Application of a fluorescent PCR method for molecular diagnosis of posttransplant lymphoproliferative disorders on routine tissue sections.

Molecular detection of monoclonality can play an important role in the diagnosis of posttransplantation lymphoproliferative disorders (PTLD). To permit accurate molecular diagnosis of PTLD even on very small amounts of DNA extracted from routinely embedded histologic material, we adapted a commercially available PCR protocol (for FR-1, -2 and -3 regions), originally designed for use on fresh/frozen samples. We applied this approach on routine biopsy/surgical material of 10 PTLD (from nine patients). All three FR regions were always amplified, indicating that the extracted DNA was of medium quality. All five PTLD morphologically classified as lymphomas were monoclonal in at least one FR region. Thus, using the WHO histologic, immunohistochemical, and clinical criteria as the reference standard, the approach provided 100% sensitivity for detection of monoclonal malignancies, supporting the validity of the method. Of five specimens classified morphologically as polymorphic PTLD, three displayed a solitary IgH gene rearrangement peak, consistent with the presence of a monoclonal B-cell population (ie, monoclonal polymorphic PTLD). This rapid and straightforward procedure, which allows identification of a wide range of IgH rearrangements, could facilitate molecular analysis of PTLD in routine practice, while limiting consumption of valuable diagnostic material.

MYH9-related disease: May-Hegglin anomaly, Sebastian syndrome, Fechtner syndrome, and Epstein syndrome are not distinct entities but represent a variable expression of a single illness.

May-Hegglin anomaly, Sebastian syndrome, Fechtner syndrome, and Epstein syndrome are autosomal dominant macrothrombocytopenias distinguished by different combinations of clinical and laboratory signs, such as sensorineural hearing loss, cataract, nephritis, and polymorphonuclear Döhle-like bodies. Mutations in the MYH9 gene encoding for the nonmuscle myosin heavy chain IIA (NMMHC-IIA) have been identified in all these syndromes. To understand the role of the MYH9 mutations, we report the molecular defects in 12 new cases, which together with our previous works represent a cohort of 19 families. Since no genotype-phenotype correlation was established, we performed an accurate clinical and biochemical re-evaluation of patients. In addition to macrothrombocytopenia, an abnormal distribution of NMMHC-IIA within leukocytes was observed in all individuals, including those without Döhle-like bodies. Selective, high-tone hearing deficiency and cataract was diagnosed in 83% and 23%, respectively, of patients initially referred as having May-Hegglin anomaly or Sebastian syndrome. Kidney abnormalities, such as hematuria and proteinuria, affected not only patients referred as Fechtner syndrome and Epstein syndrome but also those referred as May-Hegglin anomaly and Sebastian syndrome. These findings allowed us to conclude that May-Hegglin anomaly, Sebastian syndrome, Fechtner syndrome, and Epstein syndrome are not distinct entities but rather a single disorder with a continuous clinical spectrum varying from mild macrothrombocytopenia with leukocyte inclusions to a severe form complicated by hearing loss, cataracts, and renal failure. For this new nosologic entity, we propose the term "MHY9-related disease," which better interprets the recent knowledge in this field and identifies all patients at risk of developing renal, hearing, or visual defects.