Genetic landscape of congenital insensitivity to pain and hereditary sensory and autonomic neuropathies.

Congenital insensitivity to pain (CIP) and hereditary sensory and autonomic neuropathies (HSAN) are clinically and genetically heterogeneous disorders exclusively or predominantly affecting the sensory and autonomic neurons. Due to the rarity of the diseases and findings based mainly on single case reports or small case series, knowledge about these disorders is limited. Here, we describe the molecular workup of a large international cohort of CIP/HSAN patients including patients from normally under-represented countries. We identify 80 previously unreported pathogenic or likely pathogenic variants in a total of 73 families in the >20 known CIP/HSAN-associated genes. The data expand the spectrum of disease-relevant alterations in CIP/HSAN, including novel variants in previously rarely recognized entities such as ATL3-, FLVCR1- and NGF-associated neuropathies and previously under-recognized mutation types such as larger deletions. In silico predictions, heterologous expression studies, segregation analyses and metabolic tests helped to overcome limitations of current variant classification schemes that often fail to categorize a variant as disease-related or benign. The study sheds light on the genetic causes and disease-relevant changes within individual genes in CIP/HSAN. This is becoming increasingly important with emerging clinical trials investigating subtype or gene-specific treatment strategies.

[Sequence variants of unknown significance in small fiber neuropathy : Characterization of a heterogeneous patient population]. / Sequenzvarianten unklarer Signifikanz bei Small-Fiber-Neuropathie : Charakterisierung einer heterogenen PatientInnenpopulation.

BACKGROUND: In almost half of patients suffering from small fiber neuropathies (SFN), the etiology remains elusive. For these patients with "idiopathic SFN", symptomatic analgesic therapy is the only option. Reports on a potential genetic background of neuropathic pain syndromes are increasing and particularly in SFN patients, several genetic variants were found mainly located in genes encoding voltage-gated sodium channels. Although up to 30% of SFN patients show genetic alterations, most of these remain of "unknown pathogenic significance" and little is known about "genetic SFN". OBJECTIVES: The study aimed to determine clinical characteristics of SFN patients carrying a rare genetic variant of unknown significance in pain-associated genes. MATERIALS AND METHODS: From 2015 to 2020, 66 patients with primarily idiopathic SFN were examined and rare gene variants of unknown significance detected in 13/66 (20%) of these. A detailed medical history with focus on pain was recorded and patients filled in standardized questionnaires to assess physical and emotional burden due to pain. RESULTS: The authors found 13/66 (20%) patients with rare variants of unknown significance located in pain-associated genes who reported pain refractory to analgesic treatment, a higher number of external factors influencing clinical symptoms, and a higher level of physical impairment and emotional stress due to pain compared with patients without such genetic variants. CONCLUSIONS: Early genetic assessment is recommended to optimize the management of patients with potentially hereditary SFN. Early access to rehabilitation and mental support as well as a consequent elimination of external triggering factors should be granted.

Peripheral temperature dysregulation associated with functionally altered NaV1.8 channels.

The voltage-gated sodium channel NaV1.8 is prominently expressed in the soma and axons of small-caliber sensory neurons, and pathogenic variants of the corresponding gene SCN10A are associated with peripheral pain and autonomic dysfunction. While most disease-associated SCN10A variants confer gain-of-function properties to NaV1.8, resulting in hyperexcitability of sensory neurons, a few affect afferent excitability through a loss-of-function mechanism. Using whole-exome sequencing, we here identify a rare heterozygous SCN10A missense variant resulting in alteration p.V1287I in NaV1.8 in a patient with a 15-year history of progressively worsening temperature dysregulation in the distal extremities, particularly in the feet. Further symptoms include increasingly intensifying tingling and numbness in the fingers and increased sweating. To assess the impact of p.V1287I on channel function, we performed voltage-clamp recordings demonstrating that the alteration confers loss- and gain-of-function characteristics to NaV1.8 characterized by a right-shifted voltage dependence of channel activation and inactivation. Current-clamp recordings from transfected mouse dorsal root ganglion neurons further revealed that NaV1.8-V1287I channels broaden the action potentials of sensory neurons and increase their firing rates in response to depolarizing current stimulations, indicating a gain-of-function mechanism of the variant at the cellular level in a heterozygous setting. The data support the hypothesis that the properties of NaV1.8 p.V1287I are causative for the patient's symptoms and that nonpainful peripheral paresthesias should be considered part of the clinical spectrum of NaV1.8-associated disorders.

High Levels of Chromosomal Copy Number Alterations and TP53 Mutations Correlate with Poor Outcome in Younger Breast Cancer Patients.

Prognosis in young patients with breast cancer is generally poor, yet considerable differences in clinical outcomes between individual patients exist. To understand the genetic basis of the disparate clinical courses, tumors were collected from 34 younger women, 17 with good and 17 with poor outcomes, as determined by disease-specific survival during a follow-up period of 17 years. The clinicopathologic parameters of the tumors were complemented with DNA image cytometry profiles, enumeration of copy numbers of eight breast cancer genes by multicolor fluorescence in situ hybridization, and targeted sequence analysis of 563 cancer genes. Both groups included diploid and aneuploid tumors. The degree of intratumor heterogeneity was significantly higher in aneuploid versus diploid cases, and so were gains of the oncogenes MYC and ZNF217. Significantly more copy number alterations were observed in the group with poor outcome. Almost all tumors in the group with long survival were classified as luminal A, whereas triple-negative tumors predominantly occurred in the short survival group. Mutations in PIK3CA were more common in the group with good outcome, whereas TP53 mutations were more frequent in patients with poor outcomes. This study shows that TP53 mutations and the extent of genomic imbalances are associated with poor outcome in younger breast cancer patients and thus emphasize the central role of genomic instability vis-a-vis tumor aggressiveness.

Spatial UBE2N protein expression indicates genomic instability in colorectal cancers.

BACKGROUND: One major hallmark of colorectal cancers (CRC) is genomic instability with its contribution to tumor heterogeneity and therapy resistance. To facilitate the investigation of intra-sample phenotypes and the de novo identification of tumor sub-populations, imaging mass spectrometry (IMS) provides a powerful technique to elucidate the spatial distribution patterns of peptides and proteins in tissue sections. METHODS: In the present study, we analyzed an in-house compiled tissue microarray (n = 60) comprising CRCs and control tissues by IMS. After obtaining protein profiles through direct analysis of tissue sections, two validation sets were used for immunohistochemical evaluation. RESULTS: A total of 28 m/z values in the mass range 800-3500 Da distinguished euploid from aneuploid CRCs (p < 0.001, ROC AUC values < 0.385 or > 0.635). After liquid chromatograph-mass spectrometry identification, UBE2N could be successfully validated by immunohistochemistry in the initial sample cohort (p = 0.0274, ROC AUC = 0.7937) and in an independent sample set of 90 clinical specimens (p = 0.0070, ROC AUC = 0.6957). CONCLUSIONS: The results showed that FFPE protein expression profiling of surgically resected CRC tissue extracts by MALDI-TOF MS has potential value for improved molecular classification. Particularly, the protein expression of UBE2N was validated in an independent clinical cohort to distinguish euploid from aneuploid CRCs.

Reduced Gray Matter Volume and Cortical Thickness in Patients With Small-Fiber Neuropathy.

Small-fiber neuropathy (SFN) is defined by degeneration or dysfunction of peripheral sensory nerve endings. Central correlates have been identified on the level of gray matter volume (GMV) and cortical thickness (CT) changes. However, across SFN etiologies knowledge about a common structural brain signature is still lacking. Therefore, we recruited 26 SFN patients and 25 age- and sex-matched healthy controls to conduct voxel-based- and surface-based morphometry. Across all patients, we found reduced GMV in widespread frontal regions, left caudate, insula and superior parietal lobule. Surface-based morphometry analysis revealed reduced CT in the right precentral gyrus of SFN patients. In a region-based approach, patients had reduced GMV in the left caudate. Since pathogenic gain-of-function variants in voltage-gated sodium channels (Nav) have been associated with SFN pathophysiology, we explored brain morphological patterns in a homogenous subsample of patients carrying rare heterozygous missense variants. Whole brain- and region-based approaches revealed GMV reductions in the bilateral caudate for Nav variant carriers. Further research is needed to analyze the specific role of Nav variants for structural brain alterations. Together, we conclude that SFN patients have specific GMV and CT alterations, potentially forming potential new central biomarkers for this condition. Our results might help to better understand underlying or compensatory mechanisms of chronic pain perception in the future. PERSPECTIVE: This study reveals structural brain changes in small-fiber neuropathy (SFN) patients, particularly in frontal regions, caudate, insula, and parietal lobule. Notably, individuals with SFN and specific Nav variants exhibit bilateral caudate abnormalities. These findings may serve as potential central biomarkers for SFN and provide insights into chronic pain perception mechanisms.

Painful stimulation increases functional connectivity between supplementary motor area and thalamus in patients with small fibre neuropathy.

BACKGROUND: The lead symptom of small fibre neuropathy (SFN) is neuropathic pain. Recent functional magnetic resonance imaging (fMRI) studies have indicated central changes in SFN patients of different etiologies. However, less is known about brain functional connectivity during acute pain processing in idiopathic SFN. METHODS: We conducted fMRI with thermal heat pain application (left volar forearm) in 32 idiopathic SFN patients and 31 healthy controls. We performed functional connectivity analyses with right supplementary motor area (SMA), left insula, and left caudate nucleus (CN) as seed regions, respectively. Since pathogenic gain-of-function variants in voltage gated sodium channels (Nav) have been linked to SFN pathophysiology, explorative connectivity analyses were performed in a homogenous subsample of patients carrying rare heterozygous missense variants. RESULTS: For right SMA, we found significantly higher connectivity with the right thalamus in SFN patients compared to controls. This connectivity correlated significantly with intraepidermal nerve fibre density, suggesting a link between peripheral and central pain processing. We found significantly reduced connections between right SMA and right middle frontal gyrus in patients with Nav variants. Likewise, connectivity between left CN and right frontal pole was decreased. CONCLUSIONS: Aberrant functional connectivity in SFN is in line with previous research on other chronic pain syndromes. Functional connectivity changes may be linked to SFN, highlighting the need to determine if they result from peripheral changes causing abnormal somatosensory processing. This understanding may be crucial for assessing their impact on painful symptoms and therapy response. SIGNIFICANCE STATEMENT: We found increased functional connectivity between SMA and thalamus during painful stimulation in patients with idiopathic SFN. Connectivity correlated significantly with intraepidermal nerve fibre density, suggesting a link between peripheral and central pain processing. Our findings emphasize the importance of investigating functional connectivity changes as a potential feature of SFN.

Genetic pain loss disorders.

Genetic pain loss includes congenital insensitivity to pain (CIP), hereditary sensory neuropathies and, if autonomic nerves are involved, hereditary sensory and autonomic neuropathy (HSAN). This heterogeneous group of disorders highlights the essential role of nociception in protecting against tissue damage. Patients with genetic pain loss have recurrent injuries, burns and poorly healing wounds as disease hallmarks. CIP and HSAN are caused by pathogenic genetic variants in >20 genes that lead to developmental defects, neurodegeneration or altered neuronal excitability of peripheral damage-sensing neurons. These genetic variants lead to hyperactivity of sodium channels, disturbed haem metabolism, altered clathrin-mediated transport and impaired gene regulatory mechanisms affecting epigenetic marks, long non-coding RNAs and repetitive elements. Therapies for pain loss disorders are mainly symptomatic but the first targeted therapies are being tested. Conversely, chronic pain remains one of the greatest unresolved medical challenges, and the genes and mechanisms associated with pain loss offer new targets for analgesics. Given the progress that has been made, the coming years are promising both in terms of targeted treatments for pain loss disorders and the development of innovative pain medicines based on knowledge of these genetic diseases.

The impact of CBP expression in estrogen receptor-positive breast cancer.

BACKGROUND: The development of new biomarkers with diagnostic, prognostic and therapeutic prominence will greatly enhance the management of breast cancer (BC). Several reports suggest the involvement of the histone acetyltransferases CREB-binding protein (CBP) and general control non-depressible 5 (GCN5) in tumor formation; however, their clinical significance in BC remains poorly understood. This study aims to investigate the value of CBP and GCN5 as markers and/or targets for BC prognosis and therapy. Expression of CBP, GCN5, estrogen receptor α (ERα), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) in BC was analyzed in cell lines by western blot and in patients' tissues by immunohistochemistry. The gene amplification data were also analyzed for CBP and GCN5 using the publicly available data from BC patients. RESULTS: Elevated expression of CBP and GCN5 was detected in BC tissues from patients and cell lines more than normal ones. In particular, CBP was more expressed in luminal A and B subtypes. Using chemical and biological inhibitors for CBP, ERα and HER2 showed a strong association between CBP and the expression of ERα and HER2. Moreover, analysis of the CREBBP (for CBP) and KAT2A (for GCN5) genes in a larger number of patients in publicly available databases showed amplification of both genes in BC patients. Amplification of CREBBP gene was observed in luminal A, luminal B and triple-negative but not in HER2 overexpressing subtypes. Furthermore, patients with high CREBBP or KAT2A gene expression had better 5-year disease-free survival than the low gene expression group (p = 0.0018 and p < 0.00001, respectively). CONCLUSIONS: We conclude that the persistent amplification and overexpression of CBP in ERα- and PR-positive BC highlights the significance of CBP as a new diagnostic marker and therapeutic target in hormone-positive BC.

Single Cell Genetic Profiling of Tumors of Breast Cancer Patients Aged 50 Years and Older Reveals Enormous Intratumor Heterogeneity Independent of Individual Prognosis.

PURPOSE: Older breast cancer patients are underrepresented in cancer research even though the majority (81.4%) of women dying of breast cancer are 55 years and older. Here we study a common phenomenon observed in breast cancer which is a large inter- and intratumor heterogeneity; this poses a tremendous clinical challenge, for example with respect to treatment stratification. To further elucidate genomic instability and tumor heterogeneity in older patients, we analyzed the genetic aberration profiles of 39 breast cancer patients aged 50 years and older (median 67 years) with either short (median 2.4 years) or long survival (median 19 years). The analysis was based on copy number enumeration of eight breast cancer-associated genes using multiplex interphase fluorescence in situ hybridization (miFISH) of single cells, and by targeted next-generation sequencing of 563 cancer-related genes. RESULTS: We detected enormous inter- and intratumor heterogeneity, yet maintenance of common cancer gene mutations and breast cancer specific chromosomal gains and losses. The gain of COX2 was most common (72%), followed by MYC (69%); losses were most prevalent for CDH1 (74%) and TP53 (69%). The degree of intratumor heterogeneity did not correlate with disease outcome. Comparing the miFISH results of diploid with aneuploid tumor samples significant differences were found: aneuploid tumors showed significantly higher average signal numbers, copy number alterations (CNAs) and instability indices. Mutations in PIKC3A were mostly restricted to luminal A tumors. Furthermore, a significant co-occurrence of CNAs of DBC2/MYC, HER2/DBC2 and HER2/TP53 and mutual exclusivity of CNAs of HER2 and PIK3CA mutations and CNAs of CCND1 and PIK3CA mutations were revealed. CONCLUSION: Our results provide a comprehensive picture of genome instability profiles with a large variety of inter- and intratumor heterogeneity in breast cancer patients aged 50 years and older. In most cases, the distribution of chromosomal aneuploidies was consistent with previous results; however, striking exceptions, such as tumors driven by exclusive loss of chromosomes, were identified.

Hard wiring of normal tissue-specific chromosome-wide gene expression levels is an additional factor driving cancer type-specific aneuploidies.

BACKGROUND: Many carcinomas have recurrent chromosomal aneuploidies specific to the tissue of tumor origin. The reason for this specificity is not completely understood. METHODS: In this study, we looked at the frequency of chromosomal arm gains and losses in different cancer types from the The Cancer Genome Atlas (TCGA) and compared them to the mean gene expression of each chromosome arm in corresponding normal tissues of origin from the Genotype-Tissue Expression (GTEx) database, in addition to the distribution of tissue-specific oncogenes and tumor suppressors on different chromosome arms. RESULTS: This analysis revealed a complex picture of factors driving tumor karyotype evolution in which some recurrent chromosomal copy number reflect the chromosome arm-wide gene expression levels of the their normal tissue of tumor origin. CONCLUSIONS: We conclude that the cancer type-specific distribution of chromosomal arm gains and losses is potentially "hardwiring" gene expression levels characteristic of the normal tissue of tumor origin, in addition to broadly modulating the expression of tissue-specific tumor driver genes.

Genome Instability Profiles Predict Disease Outcome in a Cohort of 4,003 Patients with Breast Cancer.

PURPOSE: The choice of therapy for patients with breast cancer is often based on clinicopathologic parameters, hormone receptor status, and HER2 amplification. To improve individual prognostication and tailored treatment decisions, we combined clinicopathologic prognostic data with genome instabilty profiles established by quantitative measurements of the DNA content. EXPERIMENTAL DESIGN: We retrospectively assessed clinical data of 4,003 patients with breast cancer with a minimum postoperative follow-up period of 10 years. For the entire cohort, we established genome instability profiles. We applied statistical methods, including correlation matrices, Kaplan-Meier curves, and multivariable Cox proportional hazard models, to ascertain the potential of standard clinicopathologic data and genome instability profiles as independent predictors of disease-specific survival in distinct subgroups, defined clinically or with respect to treatment. RESULTS: In Cox regression analyses, two parameters of the genome instability profiles, the S-phase fraction and the stemline scatter index, emerged as independent predictors in premenopausal women, outperforming all clinicopathologic parameters. In postmenopausal women, age and hormone receptor status were the predominant prognostic factors. However, by including S-phase fraction and 2.5c exceeding rate, we could improve disease outcome prediction in pT1 tumors irrespective of the lymph node status. In pT3-pT4 tumors, a higher S-phase fraction led to poorer prognosis. In patients who received adjuvant endocrine therapy, chemotherapy or radiotherapy, or a combination, the ploidy profiles improved prognostication. CONCLUSIONS: Genome instability profiles predict disease outcome in patients with breast cancer independent of clinicopathologic parameters. This applies especially to premenopausal patients. In patients receiving adjuvant therapy, the profiles improve identification of high-risk patients.

Complement-Activating Capacity of Autoantibodies Correlates With Disease Activity in Bullous Pemphigoid Patients.

Background: Bullous pemphigoid is a subepidermal blistering skin disease, associated with autoantibodies to hemidesmosomal proteins, complement activation at the dermal-epidermal junction, and dermal granulocyte infiltration. Clinical and experimental laboratory findings support conflicting hypotheses regarding the role of complement activation for the skin blistering induced by pemphigoid autoantibodies. In-depth studies on the pathogenic relevance of autoimmune complement activation in patients are largely lacking. Therefore, the aim of this study was to investigate the pathogenic relevance of complement activation in patients with bullous pemphigoid. Complement activation by autoantibodies in vivo as measured by the intensity of complement C3 deposits in the patients' skin and ex vivo by the complement-fixation assay in serum was correlated with the clinical disease activity, evaluated by Autoimmune Bullous Skin Disorder Intensity Score (ABSIS) and Bullous Pemphigoid Disease Area Index (BPDAI), as well as, with further immunopathological findings in patients with bullous pemphigoid. Results: Complement-activation capacity of autoantibodies ex vivo, but not deposition of complement in the perilesional skin of patients, correlates with the extent of skin disease (measured by ABSIS and BPDAI) and with levels of autoantibodies. Conclusions: Our study provides for the first time evidence in patients for a pathogenic role of complement activation in bullous pemphigoid and should greatly facilitate the development of novel diagnostic tools and of more specific therapies for complement-dependent autoimmune injury.

Molecular diagnosis of anti-laminin 332 (epiligrin) mucous membrane pemphigoid.

BACKGROUND: Mucous membrane pemphigoid is a group of chronic subepithelial autoimmune blistering diseases that mainly affect mucous membranes. Laminin 332-specific autoantibodies are present in approximately 1/3 of the patients, being associated with an increased risk of malignancy. Because of the severe complications, an early recognition of the disease allowing a timely therapy is essential. The gold standard methods for detection of laminin 332-specific autoantibodies, including the immunoprecipitation and immunoblotting are non-quantitative, laborious and restricted to a few specialized laboratories worldwide. In addition, the use of radioimmunoassays, although highly sensitive and specific, are laborious, expensive and tightly regulated. Therefore, there is a stringent need for a quantitative immunoassay for the routine detection of laminin 332-specific autoantibodies more broadly available to diagnostic laboratories. The aim of this study was to compare different antigenic substrates, including native, recombinant laminin 332 and laminin 332-rich keratinocyte extracellular matrix, for development of an ELISA to detect autoantibodies in mucous membrane pemphigoid. RESULTS: Using a relatively large number of sera from MMP patients with well-characterized autoantibody reactivity we show the suitability of ELISA systems using laminin 332 preparations as adjunct diagnostic tools in MMP. While glycosylation of laminin 332 does not appear to influence its recognition by MMP autoantibodies, ELISA systems using both purified, native and recombinant laminin 332 demonstrated a high sensitivity and good correlation with the detection of autoantibodies by immunoblotting. ELISA systems using different laminin 332 preparations represent a feasible and more accessible alternative for a broad range of laboratories. CONCLUSIONS: Our findings qualify the use of immunoassays with the laminin 332-rich preparations as an ancillary diagnostic tool in mucous membrane pemphigoid.