Results of a Gene Panel Approach in a Cohort of Patients with Incomplete Distal Renal Tubular Acidosis and Nephrolithiasis.

BACKGROUND: Distal renal tubular acidosis (dRTA) is characterized by an impairment of urinary acidification resulting in metabolic acidosis, hypokalemia, and inappropriately elevated urine pH. If not treated, this chronic condition eventually leads to nephrocalcinosis, nephrolithiasis, impaired renal function, and bone demineralization. dRTA is a well-defined entity that can be diagnosed by genetic testing of 5 genes known to be disease-causative. Incomplete dRTA (idRTA) is defined as impaired urinary acidification that does not lead to overt metabolic acidosis and therefore can be diagnosed if patients fail to adequately acidify urine after an ammonium chloride (NH4Cl) challenge or furosemide and fludrocortisone test. It is still uncertain whether idRTA represents a distinct entity or is part of the dRTA spectrum and whether it is caused by mutations in the same genes of overt dRTA. METHODS: In this cross-sectional study, we investigated a group of 22 stone formers whose clinical features were suspicious of idRTA. They underwent an NH4Cl challenge and were found to have impaired urinary acidification ability. These patients were then analyzed by genetic testing with sequencing of 5 genes: SLC4A1, ATP6V1B1, ATP6V0A4, FOXI1, and WDR72. RESULTS: Two unrelated individuals were found to have two different variants in SLC4A1 that had never been described before. CONCLUSIONS: Our results suggest the involvement of other genes or nongenetic tubular dysfunction in the pathogenesis of idRTA in stone formers. However, genetic testing may represent a cost-effective tool to recognize, treat, and prevent complications in these patients.

Identification of new candidate genes for spina bifida through exome sequencing.

PURPOSE: Neural tube defects are a group of birth defects caused by failure of neural tube closure during development. The etiology of NTD, requiring a complex interaction between environmental and genetic factors, is not well understood. METHODS: We performed whole-exome sequencing (WES) in six trios, with a single affected proband with spina bifida, to identify rare/novel variants as potential causes of the NTD. RESULTS: Our analysis identified four de novo and ten X-linked recessive variants in four of the six probands, all of them in genes previously never implicated in NTD. Among the 14 variants, we ruled out six of them, based on different criteria and pursued the evaluation of eight potential candidates in the following genes: RXRγ, DTX1, COL15A1, ARHGAP36, TKTL1, AMOT, GPR50, and NKRF. The de novo variants where located in the RXRγ, DTX1, and COL15A1 genes while ARHGAP36, TKTL1, AMOT, GPR50, and NKRF carry X-linked recessive variants. This analysis also revealed that four patients presented multiple variants, while we were unable to identify any significant variant in two patients. CONCLUSIONS: Our preliminary conclusion support a major role for the de novo variants with respect to the X-linked recessive variants where the X-linked could represent a contribution to the phenotype in an oligogenic model.

Phenotypic effects of chronic and acute use of methiopropamine in a mouse model.

Methiopropamine (MPA) is a structural analogue of methamphetamine and belongs to the category of the novel psychoactive substances. To the best of our knowledge, no experimental study has been performed to evaluate the organ damage evoked by MPA administration in an animal model. Therefore, the main purpose of the present study was to investigate the histological changes in CD-1 male mice following the chronic administration of MPA. MPA-chronically treated mice showed myocardial damage with features consistent with repeated episodes of ischemia and a pattern of kidney damage and gastrointestinal ischemia, with ischemic-necrotic lesions of variable extent. In agreement with the analogies between MPA and methamphetamine, we link organ damage secondary to MPA administration to the vasoconstrictive effect exhibited by both compounds. Chronically MPA-treated mice did not show changes in body weight, food intake, thermoregulation, muscular strength and motor coordination in the accelerod test. However, acute MPA administration significantly increased their heart rate and promoted vasoconstriction, which were associated with the sudden death of a subset of animals (40% of all chronically treated mice). In conclusion, the present study demonstrates that MPA consumption could induce health hazards, highlighting the risk of sudden catastrophic events; therefore, clinicians should be aware of these data and consider MPA screening when no other drug is identified by a urine drug screen.

The intestinal stem cell markers Bmi1 and Lgr5 identify two functionally distinct populations.

The small intestine epithelium undergoes rapid and continuous regeneration supported by crypt intestinal stem cells (ISCs). Bmi1 and Lgr5 have been independently identified to mark long-lived multipotent ISCs by lineage tracing in mice; however, the functional distinctions between these two populations remain undefined. Here, we demonstrate that Bmi1 and Lgr5 mark two functionally distinct ISCs in vivo. Lgr5 marks mitotically active ISCs that exhibit exquisite sensitivity to canonical Wnt modulation, contribute robustly to homeostatic regeneration, and are quantitatively ablated by irradiation. In contrast, Bmi1 marks quiescent ISCs that are insensitive to Wnt perturbations, contribute weakly to homeostatic regeneration, and are resistant to high-dose radiation injury. After irradiation, however, the normally quiescent Bmi1(+) ISCs dramatically proliferate to clonally repopulate multiple contiguous crypts and villi. Clonogenic culture of isolated single Bmi1(+) ISCs yields long-lived self-renewing spheroids of intestinal epithelium that produce Lgr5-expressing cells, thereby establishing a lineage relationship between these two populations in vitro. Taken together, these data provide direct evidence that Bmi1 marks quiescent, injury-inducible reserve ISCs that exhibit striking functional distinctions from Lgr5(+) ISCs and support a model whereby distinct ISC populations facilitate homeostatic vs. injury-induced regeneration.

From Churchill to Elephants: The Role of Protective Genes against Cancer.

Richard Peto's paradox, first described in 1975 from an epidemiological perspective, established an inverse correlation between the probability of developing cancer in multicellular organisms and the number of cells. Larger animals exhibit fewer tumors compared to smaller ones, though exceptions exist. Mice are more susceptible to cancer than humans, while elephants and whales demonstrate significantly lower cancer prevalence rates than humans. How nature and evolution have addressed the issue of cancer in the animal kingdom remains largely unexplored. In the field of medicine, much attention has been devoted to cancer-predisposing genes, as they offer avenues for intervention, including blocking, downregulating, early diagnosis, and targeted treatment. Predisposing genes also tend to manifest clinically earlier and more aggressively, making them easier to identify. However, despite significant strides in modern medicine, the role of protective genes lags behind. Identifying genes with a mild predisposing effect poses a significant challenge. Consequently, comprehending the protective function conferred by genes becomes even more elusive, and their very existence is subject to questioning. While the role of variable expressivity and penetrance defects of the same variant in a family is well-documented for many hereditary cancer syndromes, attempts to delineate the function of protective/modifier alleles have been restricted to a few instances. In this review, we endeavor to elucidate the role of protective genes observed in the animal kingdom, within certain genetic syndromes that appear to act as cancer-resistant/repressor alleles. Additionally, we explore the role of protective alleles in conditions predisposing to cancer. The ultimate goal is to discern why individuals, like Winston Churchill, managed to live up to 91 years of age, despite engaging in minimal physical activity, consuming large quantities of alcohol daily, and not abstaining from smoking.

RADX Gene Variant May Predispose to Familial Asperger Syndrome.

Asperger syndrome (AS) is a pervasive developmental disorder characterized by general impairment in socialization, stereotypical behavior, defective adaptation to the social context usually without intellectual disability, and some high functioning areas related to memory and mathematics. Clinical criteria are not well defined and the etiology is heterogeneous and mostly unknown. Like in typical autism spectrum disorders (ASD), the genetic background plays a crucial role in AS, and often an almost mendelian segregation can be observed in some families. We performed a whole exome sequencing (WES) in three relatives of a family with vertical transmission of AS-ASD to identify variants in candidate genes segregating with the phenotype. Variant p.(Cys834Ser) in the RADX gene was the only one segregating among all the affected family members. This gene encodes a single-strand DNA binding factor, which mediates the recruitment of genome maintenance proteins to sites of replication stress. Replication stress and genome instability have been reported recently in neural progenitor cells derived from ASD patients, leading to a disruption of long neural genes involved in cell-cell adhesion and migration. We propose RADX as a new gene that when mutated could represent a predisposing factor to AS-ASD.

Identification by Exome Sequencing of Predisposing Variants in Familial Cases of Autoinflammatory Recurrent Fevers.

Periodic fever syndromes include autoinflammatory disorders (AID) that involve innate immunity. These disorders are characterized by recurrent fevers and aberrant multi-organ inflammation, without any involvement of T or B cells or the presence of autoantibodies. A complex genetic architecture has been recognized for many AID. However, this complexity has only been partially uncovered for familial Mediterranean fever and other conditions that have a classical monogenic origin and Mendelian transmission. Several gene panels are currently available for molecular diagnosis in patients suspected of having AID. However, even when an extensive number of genes (up to 50-100) are tested in a cohort of clinically selected patients, the diagnostic yield of AID ranges between 15% and 25%, depending on the clinical criteria used for patient selection. In the remaining 75-85% of cases, it is conceivable that the causative gene or genes responsible for a specific condition are still elusive. In these cases, the disease could be explained by variants, either recessive or dominant, that have a major effect on unknown genes, or by the cumulative impact of different variants in more than one gene, each with minor additive effects. In this study, we focused our attention on five familial cases of AID presenting with classical autosomal dominant transmission. To identify the probable monogenic cause, we performed exome sequencing. Through prioritization, filtering, and segregation analysis, we identified a few variants for each family. Subsequent bioinformatics evaluation and pathway analysis helped to narrow down the best candidate genes for each family to FCRL6, PKN1, STAB1, PTDGR, and VCAM1. Future studies on larger cohorts of familial cases will help confirm the pathogenic role of these genes in the pathogenesis of these complex disorders.

Base-Excision Repair Mutational Signature in Two Sebaceous Carcinomas of the Eyelid.

Personalized medicine aims to develop tailored treatments for individual patients based on specific mutations present in the affected organ. This approach has proven paramount in cancer treatment, as each tumor carries distinct driver mutations that respond to targeted drugs and, in some cases, may confer resistance to other therapies. Particularly for rare conditions, personalized medicine has the potential to revolutionize treatment strategies. Rare cancers often lack extensive datasets of molecular and pathological information, large-scale trials for novel therapies, and established treatment guidelines. Consequently, surgery is frequently the only viable option for many rare tumors, when feasible, as traditional multimodal approaches employed for more common cancers often play a limited role. Sebaceous carcinoma of the eyelid is an exceptionally rare cancer affecting the eye's adnexal tissues, most frequently reported in Asia, but whose prevalence is significantly increasing even in Europe and the US. The sole established curative treatment is surgical excision, which can lead to significant disfigurement. In cases of metastatic sebaceous carcinoma, validated drug options are currently lacking. In this project, we set out to characterize the mutational landscape of two sebaceous carcinomas of the eyelid following surgical excision. Utilizing available bioinformatics tools, we demonstrated our ability to identify common features promptly and accurately in both tumors. These features included a Base-Excision Repair mutational signature, a notably high tumor mutational burden, and key driver mutations in somatic tissues. These findings had not been previously reported in similar studies. This report underscores how, in the case of rare tumors, it is possible to comprehensively characterize the mutational landscape of each individual case, potentially opening doors to targeted therapeutic options.

Bmi1 lineage tracing identifies a self-renewing pancreatic acinar cell subpopulation capable of maintaining pancreatic organ homeostasis.

A central question in stem cell biology is whether organ homeostasis is maintained in adult organs through undifferentiated stem cells or self-duplication of specialized cell populations. To address this issue in the exocrine pancreas we analyzed the Bmi1-labeled cell lineage of pancreatic acinar cells. Previously, we had shown that inducible linage tracing with Bmi1-Cre-estrogen receptor (ER) in the small intestine specifically, labels "classical" undifferentiated intestinal stem cells. In this article we demonstrate that the Bmi1-Cre-ER system labels a subpopulation of differentiated acinar cells in the exocrine pancreas whose derivatives are still present, at a steady-state level, 1 year after a single TM pulse. This study suggests that Bmi1 is a marker for a subpopulation of self-renewing acinar cells, indicating that self-renewal is not an exclusive feature of adult undifferentiated stem cells. Further, the extended period that Bmi1-labeled acinar cells retain a pulse of BrdU suggests that some of this subpopulation of cells are not continuously replicating, but rather are set aside until needed. This cellular behavior is again reminiscent of behavior normally associated with more classical adult stem cells. Setting aside cells capable of self-renewal until needed retains the advantage of protecting this subpopulation of cells from DNA damage induced during replication.

The Clinical Chameleon of Autoinflammatory Diseases in Children.

The very first line of defense in humans is innate immunity, serving as a critical strongpoint in the regulation of inflammation. Abnormalities of the innate immunity machinery make up a motley group of rare diseases, named 'autoinflammatory', which are caused by mutations in genes involved in different immune pathways. Self-limited inflammatory bouts involving skin, serosal membranes, joints, gut and other districts of the human body burst and recur with variable periodicity in most autoinflammatory diseases (ADs), often leading to secondary amyloidosis as a long-term complication. Dysregulated inflammasome activity, overproduction of interleukin (IL)-1 or other IL-1-related cytokines and delayed shutdown of inflammation are pivotal keys in the majority of ADs. The recent progress of cellular biology has clarified many molecular mechanisms behind monogenic ADs, such as familial Mediterranean fever, tumor necrosis factor receptor-associated periodic syndrome (or 'autosomal dominant familial periodic fever'), cryopyrin-associated periodic syndrome, mevalonate kinase deficiency, hereditary pyogenic diseases, idiopathic granulomatous diseases and defects of the ubiquitin-proteasome pathway. A long-lasting history of recurrent fevers should require the ruling out of chronic infections and malignancies before considering ADs in children. Little is known about the potential origin of polygenic ADs, in which sterile cytokine-mediated inflammation results from the activation of the innate immunity network, without familial recurrency, such as periodic fever/aphthous stomatitis/pharyngitis/cervical adenopathy (PFAPA) syndrome. The puzzle of febrile attacks recurring over time with chameleonic multi-inflammatory symptoms in children demands the inspection of the mixture of clinical data, inflammation parameters in the different disease phases, assessment of therapeutic efficacy of a handful of drugs such as corticosteroids, colchicine or IL-1 antagonists, and genotype analysis to exclude or confirm a monogenic origin.

Morphology and immunophenotyping of a monolateral ovotestis in a 46,XderY/45,X mosaic individual with ambiguous genitalia.

Disorders of sexual development represent a pathologic and clinical challenge. Many different clinical syndromes exist, and several classifications have been proposed in relation to different risks for malignant degeneration. The morphology, cytogenetics, and immunophenotype of a monolateral ovotestis in a 3-month-old individual with ambiguous genitalia and right inguinal mass are reported. The inguinal mass consisted of a tiny female genital tract with a hermaphroditic gonad with focal placental-like alkaline phosphatase-stained gonocytes; chromosome analysis disclosed a mosaic constitution: 46,XderY/45,X with a rearranged Y chromosome. A sharp morphologic distinction between true hermaphroditism and mixed gonadal dysgenesis probably does not exist, and cytogenetic characterization is mandatory. The presence of placental-like alkaline phosphatase-stained gonocytes indicates a high risk of malignant transformation, and justifies the surgical removal of the dysgenetic gonad. Fertility is unlikely.

In vivo evaluation of PhiC31 recombinase activity using a self-excision cassette.

Gene targeting allows precise tailoring of the mouse genome such that desired modifications can be introduced under precise temporal and spatial control. This can be achieved through the use of site-specific recombinases, which mediate deletion or inversion of genomic DNA flanked by recombinase-specific recognition sites, coupled with gene targeting to introduce the recombinase recognition sites at the desired genomic locations within the mouse genome. The introduction of multiple modifications at the same locus often requires use of multiple recombination systems. The most commonly used recombination system is Cre/lox. We here evaluated in vivo the ability of PhiC31 phage integrase to induce a genomic deletion in mouse. We engineered a self-excision cassette, modeled after one previously designed for Cre, containing a positive selection marker and PhiC31 driven by a testis-specific promoter, all flanked by PhiC31 specific attP/B sites. We found in vivo PhiC31 mediated self-excision in 38% of transmitted alleles, although 18% of these showed evidence of imprecise deletion. Furthermore, in the 69% of un-recombined cassettes, sequence analysis revealed that PhiC31 mediated an intra-molecular deletion of the attB site preventing any subsequent recombination. This study demonstrates that PhiC31 can be used to automatically remove Neo, in the male chimera germline, although it is not as efficient or as accurate as Cre.

DNA Methylation in the Diagnosis of Monogenic Diseases.

DNA methylation in the human genome is largely programmed and shaped by transcription factor binding and interaction between DNA methyltransferases and histone marks during gamete and embryo development. Normal methylation profiles can be modified at single or multiple loci, more frequently as consequences of genetic variants acting in cis or in trans, or in some cases stochastically or through interaction with environmental factors. For many developmental disorders, specific methylation patterns or signatures can be detected in blood DNA. The recent use of high-throughput assays investigating the whole genome has largely increased the number of diseases for which DNA methylation analysis provides information for their diagnosis. Here, we review the methylation abnormalities that have been associated with mono/oligogenic diseases, their relationship with genotype and phenotype and relevance for diagnosis, as well as the limitations in their use and interpretation of results.

Rare missense variants in the ALPK1 gene may predispose to periodic fever, aphthous stomatitis, pharyngitis and adenitis (PFAPA) syndrome.

PFAPA is an autoinflammatory syndrome characterized by periodic fever, aphthous stomatitis, sterile pharingitis, and adenitis, with an onset usually before the age of five. While the condition is most commonly sporadic, a few cases are familial and are usually compatible with an autosomal dominant (AD) transmission pattern, with reduced penetrance in some pedigrees. We performed exome analysis in a family where PFAPA was present in three relatives in two generations showing apparent AD segregation, identifying several rare and/or novel heterozygous variants in genes involved in the autoinflammatory pathway. Following segregation analysis of candidate variants, only one, c. 2770T>C p.(S924P) in the ALPK1 gene, was found to be consistently present in affected family members. ALPK1 is broadly expressed in different tissues and its protein is the intracellular kinase activated by the bacterial ADP-heptose bisphosphate that phosphorylates and activates TRAF-Interacting protein with Forkhead-Associated domain (TIFA) and triggers the immediate response to Gram-negative bacterial invasion. Sequencing analysis of 13 additional sporadic cases and 10 familial PFAPA cases identified two additional heterozygous missense variants c.1024G>C p.(D342H) and c.710C>T p.(T237M) in two sporadic patients, suggesting that rare variants in ALPK1 may represent a predisposing factor for recurrent periodic fever in a pediatric population.

Defective activation of the MAPK/ERK pathway, leading to PARP1 and DNMT1 dysregulation, is a common defect in IgA nephropathy and Henoch-Schönlein purpura.

Studies on IgA nephropathy (IgAN) have identified, through GWAS, linkage analysis, and pathway scanning, molecular defects in familial and sporadic IgAN patients. In our previous study, we identified a novel variant in the SPRY2 gene that segregates with the disease in one large family. The functional characterization of this variant led us to discover that the MAPK/ERK pathway was defective not only in this family, but also in two sporadic IgAN patients wild type for SPRY2. In the present study, we have deepened the molecular analysis of the MAPK/ERK pathway and extended our evaluation to a larger cohort of sporadic patients and to one additional family. We found that the ERK pathway is defective in IgAN patients and in patients affected by another IgA-mediated disorder, Henoch-Schönlein purpura (HSP). Furthermore, we found that two other proteins, PARP1 and DNMT1, respectively involved in DNA repair and in antibody class switching and methylation maintenance duties, were critically downregulated in IgAN and HSP patients. This study opens up the possibility that defective ERK activation, in some patients, leads to PARP1 and DNMT1 downregulation suggesting that IgAN could be the consequence of a dysregulated epigenetic maintenance leading to the upregulation of several genes. In particular, PARP1 could be used as a potential biomarker for the disease.

Two brothers with 22q13 deletion syndrome and features suggestive of the Clark-Baraitser syndrome.

We report on two brothers with moderate-to-severe mental retardation, severe macrocephaly, obesity, characteristic face, big hands and feet, advanced bone age and brain abnormalities, including frontal cortical atrophy. These two boys resembled the two brothers described by , two maternal cousins subsequently reported by and a Brazilian boy described by . Upon further investigation, we detected a cryptic subtelomeric deletion of chromosome region 22q13, not present in either parent and probably due to a maternal germinal mosaicism. Thus, we describe the first familial case of 22q13 deletion and recommend that patients with a phenotype suggestive of the so-called Clark-Baraitser syndrome be tested for submicroscopic 22qter deletion.

A SPRY2 mutation leading to MAPK/ERK pathway inhibition is associated with an autosomal dominant form of IgA nephropathy.

IgA nephropathy (IgAN) represents the most common primary glomerulonephritis worldwide with a prevalence of 25-50% among patients with primary glomerulopathies. In ~5-10% of the patients the disease segregates with an autosomal dominant (AD) pattern. Association studies identified loci on chromosomes 1q32, 6p21, 8p23, 17p13, 22q12, whereas classical linkage studies on AD families identified loci on chromosomes 2q36, 4q26-31, 6q22, 17q12-22. We have studied a large Sicilian family where IgAN segregates with an AD transmission. To identify the causal gene, the exomes of two affected and one unaffected individual have been sequenced. From the bioinformatics analysis a p.(Arg119Trp) variant in the SPRY2 gene was identified as the probable disease-causing mutation. Moreover, functional characterization of this variant showed that it is responsible for the inhibition of the MAPK/ERK1/2 pathway. The same effect was observed in two sporadic IgAN patients carriers of wild-type SPRY2, suggesting that downregulation of the MAPK/ERK1/2 pathway represents a common mechanism leading to IgAN.