Affected Family Members Research Articles

Exonic trinucleotide repeat expansions in ZFHX3 cause spinocerebellar ataxia type 4: A poly-glycine disease

Autosomal-dominant ataxia with sensory and autonomic neuropathy is a highly specific combined phenotype that we described in two Swedish kindreds in 2014; its genetic cause had remained unknown. Here, we report the discovery of exonic GGC trinucleotide repeat expansions, encoding poly-glycine, in zinc finger homeobox 3 (ZFHX3) in these families. The expansions were identified in whole-genome datasets within genomic segments that all affected family members shared. Non-expanded alleles carried one or more interruptions within the repeat. We also found ZFHX3 repeat expansions in three additional families, all from the region of Skåne in southern Sweden. Individuals with expanded repeats developed balance and gait disturbances at 15 to 60 years of age and had sensory neuropathy and slow saccades. Anticipation was observed in all families and correlated with different repeat lengths determined through long-read sequencing in two family members. The most severely affected individuals had marked autonomic dysfunction, with severe orthostatism as the most disabling clinical feature. Neuropathology revealed p62-positive intracytoplasmic and intranuclear inclusions in neurons of the central and enteric nervous system, as well as alpha-synuclein positivity. ZFHX3 is located within the 16q22 locus, to which spinocerebellar ataxia type 4 (SCA4) repeatedly had been mapped; the clinical phenotype in our families corresponded well with the unique phenotype described in SCA4, and the original SCA4 kindred originated from Sweden. ZFHX3 has known functions in neuronal development and differentiation n both the central and peripheral nervous system. Our findings demonstrate that SCA4 is caused by repeat expansions in ZFHX3.

Modeling TCIRG1 Neutropenia By Utilizing Patient Derived Induced Pluripotent Stem Cells

Background: Severe congenital neutropenia (SCN) is a hereditary hematopoietic disorder characterized by recurrent infections and leukemic transformation. We previously identified a novel heterozygous missense mutation (R736S) in TCIRG1 causing SCN in a large multigenerational family (PMID: 24753205). We also identified a statistically significant correlation in individuals with lower ANC with rare missense variants in TCIRG1 in a large cohort of population-based genome screening (PMID: 27229898). Recently, we have discovered another TCIRG1 heterozygousvariant: R736C in two unrelated families with congenital neutropenia. Independently, Shinwari et al identified a novel TCIRG1 mutation, V52L, which was correlated with the neutropenic phenotype (PMID: 35573728). Homozygous mutations of TCIRG1 are causing autosomal recessive osteopetrosis (ARO), which is characterized by osteopetrosis and secondary hematological and neurological abnormalities. The Genome Aggregation Database (gnomAD) has no data about TCIRG1 R736S and R736C variants accruing in the healthy population. This is in contrast of numerous healthy individuals with a various heterozygous variants throughout the TCIRG1 gene. Aims: We aim to generate in-vitro model of TCIRG1 neutropenia by utilizing patient derived induced pluripotent stem cells (iPSCs) and characterize the hematological abnormalities caused by mutant TCIRG1 expression. We also aim to characterize the incidence of TCIRG1-associated neutropenia and the prevalence of TCIRG1 variants by sharing this new information within the scientific community through identifying more cases/families with this disease. Methods: Medical history, differential WBC counts and whole exome sequencing (WES) was used to identify the genetic basis of neutropenia in the family with R736S mutation. The R736C mutation in the new families also were identified by WES of the index cases. Medical history, blood counts and Sanger sequencing identified their affected relatives. iPSC lines of 4 affected individuals from 2 families harboring TCIRG1 R736S and R736C mutations and healthy volunteer were generated using standard methodology. S732F benign heterozygous TCIRG1 variant was generated by utilizing CRISPR/Cas9 knock-In gene editing using a healthy volunteer iPSC line. iPSCs hematopoietic differentiation was achieved by using STEMdiff Hematopoietic Kit. Resultant CD34+ cells were differentiated towards neutrophils using a published protocol (PMID: 35795780). Cell proliferation, survival and differentiation characteristics were measured by automated cell counter and flow cytometry, cell viability staining and surface markers analysis for granulocytic differentiation. Results: The discovery of these families came through the reputation and work of the Severe Congenital Neutropenia International Registry (SCNIR) and the expanding use of WES to identify the causes of hereditary disorders. With the identification of the index cases, we have identified several affected family members in each family. In all three families the severity of neutropenia is variable and correlates with the incidence of recurrent bacterial infections. Myeloid differentiation of CD34+ cells derived from iPSCs of 4 patients from two families with R736S and R736C mutations revealed maturation, survival and proliferation abnormalities compared to volunteer cells and cell line harboring benign S732F heterozygous TCIRG1 mutation. All patient cells had from 2 to 3-fold drop in cell proliferation and cell viability drop by over 70% for the patients with R736S and up to 90% with R736C mutation compared to the controls. The myeloid differentiation in the patients' cells, measured by flow cytometry and assessed by CD66b+/CD14+ and CD11b+/CD15+ markers was impaired by 38% and 23% respectively for the patients with R736S and 43% and 24% for the patients harboring R736C mutation. Conclusions: The pathogenesis of TCIRG1-associated neutropenia is currently unknown. Carriers of pathogenic variants from these families appear to have neutropenia with variable severity. Our preliminary studies show that patient iPSC derived hematopoietic cells closely recapitulate the neutropenic phenotype. Population based genomic studies; careful family histories and WES will identify new patients with this disorder.

Hemoglobin Monza: A New Variant of Unstable Hemoglobin

Introduction: Unstable hemoglobins are variants with structural abnormalities; while not hindering protein formation, they lead to protein destabilization and can cause hemolytic crises. Most unstable hemoglobins are caused by point mutations that result in the premature dissociation of heme from the globin chain and usually involve His residues (Weatherall et al. 2001 Nat.Rev.Genetics; Scheps et al. 2018 EJH). Here a new unstable hemoglobin, named Hb-Monza is presented, exhibiting a long duplication in the HBB gene without apparent alterations in α-β subunit interaction or involvement of specific His residues. The variant was identified in a family, with four out of six members involved and is associated with mild chronic hemolysis and acute severe hemolytic crises during febrile infections. Methods: The presence of erythrocyte membrane abnormalities was tested by glycerol lysis, Pink, osmotic resistance and by EMA binding test. G6PD and PK activity levels were tested both during the hemolytic crisis and at resolution. Abnormal variants were screened by HPLC and electrophoresis. Stability of the Hb variant was assessed by the Isopropanol Precipitation Test. DNA was extracted from peripheral blood leukocytes, followed by PCR amplification of HBB, HBA1 and HBA2 genes. Sequencing was performed using Genetic Analyzer 3500. A 3D model of the variants was produced by SWISS-MODEL using the crystal structure of the deoxy human hemoglobin (PDB 1A3N) as template and refined by Maestro Suite. Results: The index patient (pt) was a 13-year-old girl of Chinese origin; she was the firstborn of 4 children (2 sisters and 1 brother) and came to ER with fever and hyperchromic urine. Blood tests revealed severe anemia (Hb 5.2 g/dL) and signs of hemolysis. Direct Coombs test was negative both for warm and cold antibodies. She tested positive for Mycoplasma Pneumoniae, Epstein Barr Virus (EBV) and Parvovirus B19 (IgG and IgM) antibodies. The child's infection cleared during hospitalization, and hemoglobin levels increased without transfusion. Her hemoglobin further rose to 11 g/dL in outpatient follow-up although signs of low-level chronic hemolysis persisted. All affected family members exhibit mild chronic hemolytic anemia, with all, except the mother, experiencing at least one acute severe hemolytic state triggered by infection. HPLC showed the presence of an abnormal fraction of hemoglobin with slow migration speed in alkaline electrophoresis and extremely slow elution in HPLC (4.8 min). The abnormal Hb fraction in the proband, mother, brother and sister was 20.8%, 17.7%, 28.1%, and 25.6% respectively. The father and the youngest sibling did not show this band. Sequencing of HBB demonstrated an in-frame duplication of 69 bases corresponding to 23 amino acids located in exon 2 (c.176_244dup; p.Pro59_Asn81dup); this was present in all pts exhibiting the abnormal fraction at HPLC. A functional validation of Hb Monza utilizing the isopropanol test confirmed the unstable nature of the variant (Figure 1). The 3D-model analysis (Figure 2) showed that the duplication is situated amidst helix E and F of HBB resulting in a disordered conformation protruding from the Hb structure The binding of the heme group remains unaffected and the coordination of the heme group with His residues of both the E and F helix is preserved. The tertiary structure of the Hb beta chain is scarcely affected. Conclusions: This report introduces a novel HBB gene variant, Hb Monza (HBB: c.176_244dup), associated with subclinical chronic hemolysis and acute hemolytic crises during fever-inducing infections. Only a few HBB duplications exceeding 20 bp have been documented, all leading to Beta-Thalassemia clinical features through production of a truncated beta globin chain or failure of HBB to bind the alpha chain (e.g. Frischknecht et al. 2007 Haematologica). However, 3D-modeling of Hb Monza indicates minimal tertiary structure alteration. The inclusion of 23 additional amino acids results in a disordered conformation protruding from the Hb structure possibly facilitates direct bonding between the iron molecule in the heme group and internal amino acid side chains due to increased structural flexibility. This could lead during thermal or metabolic stress, to Hb precipitation. Further data from molecular dynamic experiments will be presented. Dr. Civettini and Dr. Corti contributed equally to this article

Women's Knowledge and Attitudes Regarding the Risk Factors and Warning Signs of Breast Cancer in the Eastern Province, Saudi Arabia: A Cross-Sectional Study.

Introduction Breast cancer is the leading type of cancer in women globally, contributing significantly to cancer-related deaths. In Saudi Arabia, it ranks as the second most common cancer among women. Studies have highlighted a lack of awareness about breast cancer, negatively impacting breast self-examination practices. The Eastern Province has particularly high incidence rates. Knowledge of risk and protective factors can aid in prevention and early detection. While some studies show good awareness, this research aims to assess women's knowledge and attitudes about breast cancer risk factors and warning signs in the Eastern Province. Methodology This is a cross-sectional study conducted in Saudi Arabia's Eastern Provincefrom June to October 2023 to assessbreast cancer awareness among adult females. Data were collected via an online survey. Data were cleaned in MS Excel(Microsoft Corporation, Redmond, Washington) and analyzed in IBM SPSS Statistics for Windows, Version 23(Released 2015; IBM Corp., Armonk, New York). Results Our study assessed breast cancer awareness in 697 participants in Saudi Arabia's Eastern Province. Most were aged 20-24 years (30.3%), married (49.2%), and Saudi nationals (97.6%), with higher education (59.4%). Significantly, 69.2% received education on breast cancer risk factors/signs. Some had personal breast cancer diagnoses (5.0%), and 31.7% had affected family members. Participants showed good awareness of various risk factors and warning signs. Significant associations were found between nationality, personal breast cancer diagnosis, and knowledge and attitudes about risk factors. Age, marital status, occupation, education on breast cancer, and personal breast cancer diagnosis influenced knowledge and attitudes about warning signs. Conclusion Our study findings indicate generally good awareness of breast cancer risk factors and warning signs among participants. Age, marital status, occupation, education on breast cancer, and personal breast cancer diagnosis influenced knowledge and attitudes about risk factors and warning signs. Educational efforts should target lesser-known risk factors to enhance prevention and early detection.

Stuttering associated with a pathogenic variant in the chaperone protein cyclophilin 40.

Stuttering is a common speech disorder that interrupts speech fluency and tends to cluster in families. Typically, stuttering is characterized by speech sounds, words or syllables which may be repeated or prolonged and speech that may be further interrupted by hesitations or 'blocks'. Rare variants in a small number of genes encoding lysosomal pathway proteins have been linked to stuttering. We studied a large four-generation family in which persistent stuttering was inherited in an autosomal dominant manner with disruption of the cortico-basal-ganglia-thalamo-cortical network found on imaging. Exome sequencing of three affected family members revealed the PPID c.808C>T (p.Pro270Ser) variant that segregated with stuttering in the family. We generated a Ppid p.Pro270Ser knock-in mouse model and performed ex vivo imaging to assess for brain changes. Diffusion-weighted MRI in the mouse revealed significant microstructural changes in the left corticospinal tract, as previously implicated in stuttering. Quantitative susceptibility mapping also detected changes in cortico-striatal-thalamo-cortical loop tissue composition, consistent with findings in affected family members. This is the first report to implicate a chaperone protein in the pathogenesis of stuttering. The humanized Ppid murine model recapitulates network findings observed in affected family members.

Hereditary thrombocythemia due to splicing donor site mutation of THPO in a Japanese family.

Thrombopoietin (THPO) is an essential factor for platelet production. Hereditary thrombocythemia (HT) is caused by a germline mutation of THPO, MPL, or JAK2 and is inherited in an autosomal-dominant manner. We identified a Japanese family with HT due to a point mutation of the splicing donor site of the THPO gene (THPO c.13 + 1G > A). Bone marrow biopsy showed increased megakaryocytes mimicking essential thrombocythemia. One affected family member developed chronic myeloid leukemia. We cloned the mutation and developed mutated and wild type THPO expression vectors. Molecular analysis showed that the mutation causes an exon 3 skipping transcript of THPO that abrogates a suppressive untranslated upstream open reading frame. Although the transcript levels of THPO mRNA were comparable, mutated transcripts were more efficiently translated and THPO protein expression was significantly higher than that of the wild type.

Mutation in PDGFRbeta: a potential new pathogenic variant for mitral valve prolapse

Abstract Background Mitral valve prolapse (MVP) due to myxomatous degeneration is characterized by familial clustering, but few pathogenic genes have been identified. In a genetic screening program for MVP patients, we identified a platelet-derived growth factor receptor β (PDGFRβ)-E162K missense variant in a family with a bi-leaflet MVP phenotype in four affected family members. PDGFRβ is a receptor tyrosine kinase, which plays an important role in vascular development. Purpose In this study, we investigated whether the E162K substitution in PDGFRβ could lead to mitral valve abnormalities. Methods To assess the functional effects of the missense variant, proliferation and migration assays were performed using HeLa cells overexpressing wild-type (WT) or mutant PDGFRβ. Immunostainings were performed to determine PDGFRβ expression in the diseased valves from a family member undergoing surgical operation and from additional patients operated for MVP. Mouse models were created with the mouse equivalent of the human PDGFRβ-E162K variant (PDGFRβ-E161K) using CRISPR/CAS9. The variant was not lethal and a total of 12 homozygous PDGFRβ-E161K-/- mice, 11 heterozygous PDGFRβ-E161K+/- mice, and 7 WT mice were subjected to transthoracic echocardiography. In addition, histology was performed in all 30 mice to detect potential valvular defects. Results Stimulated HeLa cells expressing PDGFRβ-E162K showed reduced proliferation and migration, suggesting a loss of function of the mutant receptor. PDGFRβ was expressed in human diseased mitral valves. Echocardiographic evaluation revealed a significant larger mitral valve annulus diameter in both PDGFRβ-E161K+/- and PDGFRβ-E161K-/- compared to WT (WT: 1.97 mm; PDGFRβ-E161K+/-:2.13, p=0.031; PDGFRβ-E161K-/-: 2.42 mm, p<0.0001). Histological data in PDGFRβ-E161K-/- mice showed significant leaflet thickening in the free edge (FE) of the posterior mitral valve leaflet (PMVL) compared to WT (WT: 72.34 ɥm; PDGFRβ-E161K-/-: 109.6 ɥm, p=0.040) and a larger leaflet area of the PMVL compared to WT (WT: 25280 ɥm²; PDGFRβ-E161K-/-: 44494 ɥm², p=0.020). Moreover, an altered organization of the extracellular matrix reminiscent of the myxomatous degeneration in MVP was observed. Conclusions The PDGFRβ-E162K variant is associated with familial MVP and alters the function of PDGFRβ. Mice harboring this mutation display mitral valve defects and can be used as a model for mitral valve disease.

Digenic inheritance accounts for phenotypic variability in amelogenesis imperfecta.

Amelogenesis imperfecta (AI) represents a group of clinically and genetically heterogeneous disorders that affect enamel formation and mineralization. Although AI is commonly considered a monogenic disorder, digenic inheritance is rarely reported. In this study, we recruited two nonconsanguineous Chinese families exhibiting diverse phenotypes of enamel defects among affected family members. Digenic variants were discovered in both probands. In family 1, the proband inherited a paternal frameshift variant in LAMA3 (NM_198129.4:c.3712dup) and a maternal deletion encompassing the entire AMELX gene. This resulted in a combined hypoplastic and hypomineralized AI phenotype, which was distinct from the parents' manifestations. In family 2, whole-exome sequencing analysis revealed the proband carried a maternal heterozygous splicing variant in COL17A1 (NC_000010.11 (NM_000494.3): c.4156 + 2dup) and compound heterozygous variants in RELT (paternal: NM_032871.4:c.260A > T; maternal: NM_032871.4:c.521 T > G). These genetic changes caused the abundant irregular enamel defects observed in the proband, whereas other affected family members carrying heterozygous variants in both COL17A1 and RELT displayed only horizontal grooves as their phenotype. The pathogenicity of the novel COL17A1 splice site variant was confirmed through RT-PCR and minigene assay. This study enhances our understanding by highlighting the potential association between the co-occurrence of variants in two genes and variable phenotypes observed in AI patients.

Abstract 15579: Stem Cell Modeling of a Candidate Genetic Variant in a Family With Complex Inherited Cardiomyopathy

Introduction: Inherited cardiomyopathies, such as arrhythmogenic right (or left) ventricular cardiomyopathy (ARVC; ALVC) and dilated cardiomyopathy (DCM), can lead to sudden cardiac death. Mechanisms of arrhythmogenesis in these cardiomyopathies are not fully understood, with ~ 20% of patients carrying a pathogenic variant in a validated cardiomyopathy-associated gene. Aims: Here, we investigate a family with highly variable expression of arrhythmogenic cardiomyopathy. The two affected siblings have differential ventricle involved and arrhythmia burden: with one fulfilling criteria for ARVC, and the other for ALVC. Their father has a non-ischemic cardiomyopathy with ventricular arrhythmias and a family history of sudden death. The maternal side is unaffected. The sibling with ALVC harbors a paternally inherited rare missense variant of uncertain significance (VUS) in plakoglobin ( JUP) (c.356C>T; p.Pro119Leu), an ARVC-associated gene that does not segregate in the sibling with ARVC. Hypothesis: Study of patient-specific inducible pluripotent stem cell-derived cardiomyocytes (iPSC-CM) can define an elusive genetic etiology as well molecular pathways associated with cardiomyopathy in the affected family members. Methods: We reprogrammed iPSC lines from 3 affected family members and 2 unaffected maternal controls and differentiated them into cardiomyocytes. Results: Differentiation to iPSC-CMs resulted in transcriptionally upregulated markers of cardiac stress and contractile function in affected family members ( NPPA, NPPB, MYH7 ). iPSC-CM harboring JUP c.356C>T did not differ in plakoglobin RNA or protein expression. Hypertrophy was observed in the iPSC-CMs from affected family members after isoproterenol stimulation. Whole exome sequencing results revealed rare candidate variants (MAF<5x10 -5 , REVEL>0.7) that segregated in affected family members. Conclusions: iPSC-CMs from affected family members recapitulate cardiomyopathy phenotypes. Affected family members harbor novel candidate variants not previously associated with cardiomyopathies. Future work will utilize base editing to evaluate for compound contributions of several novel VUS on cardiomyopathy phenotypes in iPSC-CM from family members.

Challenging Stereotypes: Cases of Breast Cancer in Men

Breast cancer is the most common malignant tumor in women, internationally, and the leading cause of death from cancer. However, breast cancer in men is an uncommon pathology, corresponding to less than 1% of neoplasms in men. The lifetime risk of breast cancer in a man is approximately 1:1000, compared to 1:8 for a woman. Currently, an increase in timely diagnosis has been demonstrated thanks to the programs that are carried out for the early detection of breast cancer worldwide. Although, unfortunately, early diagnosis is still difficult due to the lack of information that a large part of the population has regarding self-examination, not only in our country, but also around the world, and in the case of breast cancer in men, being such an uncommon condition, its diagnosis is more difficult. The incidence of breast cancer is increasing in the developing world, due to longer life expectancy, increased urbanization and the adoption of Western lifestyles. Among the risk factors that have been seen to increase the risk of breast cancer in men, with scientifically proven evidence, are mainly hormonal disorders, family history and mutations of certain genes that predispose to the disease. The hormonal disorders mentioned above refer to an increase in circulating estrogen levels and a deficiency in the synthesis or action of testosterone. These alterations can be caused by disorders at the level of testicular testosterone synthesis, such as: orchitis, orchiectomy, undescended testicle, congenital inguinal hernia and others. Patients who voluntarily consume exogenous estrogens as part of the treatment of prostate cancer or transsexuals should also be taken into account. Another risk factor that has been found to be implicated in the presentation of this type of neoplasia are medications; many frequently used medications are testosterone antagonists. Alcohol is also implicated, since it decreases testosterone synthesis and the number of testicular receptors for gonadotropin. A higher prevalence has also been seen in patients with obesity, in whom there is a double risk, because as they have a higher peripheral aromatization of androgens, they synthesize a greater amount of circulating estrogens. Approximately 20% of men with breast cancer have a first-line family history, which indicates a two to three times higher risk of developing the disease at some point in their lives and that this risk doubles exponentially with the number of affected family members. Currently there are many programs and organizations that promote the fight against breast cancer, such as the World Health Organization, where there are comprehensive national cancer control programs that are integrated with non-communicable diseases and other related problems. Comprehensive cancer control encompasses prevention, early detection, diagnosis and treatment, rehabilitation and palliative care. Raising awareness of the general public about the problem of breast cancer and control mechanisms, as well as promoting appropriate policies and programs, are key strategies for population-based breast cancer control. Based on what has been mentioned above, the work will be carried out in order to have a better approach to breast cancer in men, due to the little information we have about it, as well as to raise awareness among the general population about the risk factors, prognostic factors, timely diagnosis and treatment of patients with this type of pathology.

Abstract 13631: Desmoplakin Cardiomyopathy in Pediatric Patients - A Distinct, Underrecognized Cohort of Arrhythmogenic Cardiomyopathy

Introduction: Arrhythmogenic cardiomyopathy is a spectrum of diagnoses including the historically recognized arrhythmogenic right ventricular cardiomyopathy. More recent literature describes predominately left ventricular (LV) or biventricular (BiV) involvement. Variants in the desmoplakin ( DSP) gene commonly cause LV and BiV phenotypes, though data in children are sparse. Hypothesis: Pediatric patients (pts) with DSP mutation have predominantly LV or BiV disease and may present with early onset malignant arrhythmia. Methods: We performed a multicenter, retrospective cohort study of pts <21 years with pathogenic or likely pathogenic DSP variants from 6 tertiary referral children’s hospitals. We describe clinical variation in DSP probands (PBs), the first affected family member prompting genetic testing due to clinical history, as compared to genotype-positive family members (FMs) <21 years of age diagnosed by familial cascade screening. Results: We identified 34 pts; 12 (35%) were PBs and 22 (65%) were FMs. Median age at genetic diagnosis was 13.3 and 12.3 years for PBs and FMs, respectively. Four PBs had LV-predominant disease; 6 had BiV disease (table 1). The most common initial diagnosis for PBs was myocarditis and dilated cardiomyopathy. ECG abnormalities were common in PBs (75%) and included left axis deviation, inferolateral T-wave inversion, and low QRS complex amplitude. The LV ejection fraction (EF) on echo was 48% and 65% for PBs and FMs, respectively. Eight PBs underwent MRI and 6 had late gadolinium enhancement involving the LV only, while 2 had BiV involvement. Six PBs underwent ICD implantation and 2 received appropriate ICD therapy (LVEF 25% and 41%). Two PBs had cardiac transplantation. Conclusion: Pediatric DSP -cardiomyopathy has predominantly LV or BiV involvement and may be misdiagnosed as myocarditis or dilated cardiomyopathy. Probands in particular, may present with early malignant ventricular arrhythmias and significant LV dysfunction.

Cancer Predisposition Genetic Testing Among Children with Hematologic Malignancies

Introduction: There is growing recognition of germline cancer predisposition syndromes (CPS) that increase risk of hematologic malignancies including leukemias, lymphomas, bone marrow failure syndromes and myeloproliferative diseases. Somatic (tumor) testing can identify potential germline variants based on multiple factors including variant allele fraction, sequence characteristics, and other findings; however, subsequent germline testing is required to characterize the lesion. Paired tumor and germline testing (T/N testing) is more accurate to diagnose CPS and may mitigate loss to follow-up, reduce time to CPS diagnosis, and possibly alleviate unnecessary anxiety due to suspected but ultimately disproved germline conditions. Previously, our center performed tumor-only diagnostic testing for patients with newly diagnosed hematologic malignancies. In 2020 we transitioned to paired T/N testing for all new hematologic malignancy diagnoses with patient/caregiver consent. Here we report the frequency of CPS diagnoses suggested by tumor-only testing and subsequently confirmed with germline testing, as well as CPS diagnoses identified from the outset by T/N testing. Methods: Data was automatically abstracted from the Children's Hospital of Philadelphia (CHOP) Diagnostic Genomics Database (FileMaker and Nexus) for 1057 pediatric, adolescent, and young adult patients who underwent testing with the CHOP Comprehensive Hematologic Malignancy panel between 2016 and 2023. This panel interrogates 117 genes for single nucleotide variants, insertions, deletions, and copy number variants. Of those tested, 932 patients underwent tumor-only testing, and 125 patients underwent paired T/N testing. Characteristics of patients with possible pre-established genetic syndromes (e.g. Trisomy 21) were cross-checked via manual chart review. Results: Schematic representation of patient testing is summarized in Figure 1. Among 932 patients who underwent tumor-only testing, 98 were identified to have potential or previously established germline pathogenic variants. Of these, 9 had known genetic syndromes and 8 had non-cancer related genetic diagnoses (e.g. Usher syndrome). Of the 81 patients with a suspected CPS, 26 were referred from an outside hospital with unavailable follow-up data. Among suspected germline patients from our center (n=55), 34 (62%) did not undergo confirmatory testing. Of the 21 (38%) who underwent confirmatory testing, 2 (10%) were diagnosed with a CPS. For patients with T/N testing, 16 of 125 (7%) were identified to have germline conditions; 8 were pre-established diagnoses and 4 were non-cancer related genetic diagnoses. Thus, 4 (3%) of patients with T/N testing were newly diagnosed with a CPS. New CPS diagnoses included: ETV6 thrombocytopenia and leukemia predisposition syndrome, CBL syndrome, GATA2 MDS/AML predisposition syndrome, Noonan syndrome, and CEBPA AML predisposition syndrome. Conclusion: At our center, 62% of patients with suspected germline conditions on tumor-only testing had no follow up testing documented in the available medical record. These data suggest that paired T/N testing at the time of hematologic malignancy diagnosis assists in mitigating loss-to-follow up for patients with possible CPS. Additionally, T/N testing may streamline result interpretation, reduce time to diagnosis, aid in treatment decisions, and guide cascade testing for potentially affected family members. When possible, genetic testing decisions and results should be discussed with a trained genetic counselor. Over time, increasing utilization of paired T/N testing may also enhance understanding of CPS-associated genes that increase risk of hematologic malignancies.

How affected family members communicate about their loved one’s non-medical opioid use

Family members of people who misuse opioids frequently experience stigma due to their association with non-medical opioid use (NMOU). Stigma may affect how family members communicate about NMOU and seek social support. Guided by communication privacy management theory, this study sought to understand how affected family members (AFMs) communicate about NMOU within and outside of the immediate family. In-depth interviews were conducted with 34 adults with an immediate relative with NMOU. Qualitative analyses utilized a common iterative approach. Findings identified complex dialectical tensions that families traverse in both wanting to conceal stigmatizing information while simultaneously wanting to disclose information to engage support for themselves during stressful experiences. Strategies to resolve this tension included focusing on the good, functional updates, and moving from closed to open communication boundaries. Treatment for individuals with NMOU should engage families and assist with the resolution of communication privacy management tensions.

The Family of the City, the Family of the Country

Abstract In this Article, we argue that the state supports different familial practices in urban and rural areas. Spatial laws and policies are affecting family members’ ability, availability, and commitment to care for one another based on their geographical location. We support the argument using a bottom-up approach that works with three case studies from three jurisdictions: China, Israel, and the United States to uncover and understand the phenomenon. In all these instances, state policies and laws effectively create gaps in familial practices between the city and the country. The analysis identifies and compares two categories of state involvement: differential treatment and spatially blind policies. Collectively, the case studies introduce to family law scholarship the rich, contextual phenomenon of state involvement in spatially shaping families.

Physical illness and changes in spousal depression: a register study on Finnish middle-aged couples

Abstract Background Physical health decline may affect family members’ mental health by causing distress and, in case of functional impairments, need to provide care. We examined how diseases common in midlife but with different disease progression and level of impairment are associated with short-term and long-term changes in spousal depression. Methods We used Finnish total population register data from 2000-2019 to identify 91,849 men and 70,836 women aged 30-70 that experienced onset of severe diseases without cognitive impairment (myocardial infarction, prostate cancer, breast cancer), conditions with sudden onset and immediate impairment (stroke, traumatic brain injury) or progressive conditions with increasing impairment (Parkinson's disease, multiple sclerosis, dementia) while living with a spouse. We measured depression with antidepressant purchases and used fixed-effects linear probability models to examine spouses’ depression trajectories before and after onset of spousal disease Results Preliminary findings show that the onset of any type of spousal illness is associated with an increase in depression. Among men this increase is similar in magnitude irrespective of the type of spouse's illness, whereas among women the increase is smaller if the spouse is diagnosed with a disease without cognitive impairment. When the spouse is diagnosed with a progressive condition, female spouses’ depression increases more gradually, whereas a large immediate increase in depression follows sudden onset conditions. One year after diagnosis there are little changes in spousal depression. Conclusions Diseases common in midlife increase spouse's risk of depression around the time of diagnosis, and the effect appears persistent. Adequate support should thus be available to family members from the start of physical illness. When planning support to female spouses, the male spouse's cognitive impairment and associated care needs should be carefully evaluated. Key messages • Diseases common in midlife increase spouse's depression risk around the time of diagnosis, and the effect appears persistent. Support to spouses should be available from the start of illness. • Cognitive impairment of the male spouse seems to increase female spouse's depression particularly strongly, suggesting that caregiving help could be especially beneficial for women.

Alport Syndrome: A Comprehensive Review.

Alport syndrome is an genetic disorder that distresses the basement membrane of the kidneys and can also impact other organs, such as the cochlea of theinner ear and eyes. It is characterized by mutation causing abnormalities in the collagen within the basement membrane, which has a crucial role in the filtration process of the kidneys. These abnormalities lead to progressive kidney damage and often result in chronic kidney disease. In some cases of Alport syndrome, the abnormal collagen can also affect the cochlea in the inner ear, leading to sensorineural hearing loss. Additionally, changes in the ocular lens, named anterior lenticonus, can occur, causing vision problems. Alport syndrome can manifest differently among individuals, and its severity can vary. Some people may experience mild symptoms, while others may develop more severe kidney problems, including end-stage renal disease, which may need dialysis or kidney transplant. Treatment for Alport syndrome primarily focuses on managing its symptoms and complications. Regular monitoring of kidney function and blood pressure, along with medications to control hypertension, are crucial aspects of the management plan. In cases of severe kidney damage, kidney transplantation may be necessary. As with any medical condition, early detection and intervention can improve results and quality of life for persons with Alport syndrome. Therefore, if there is a family history of the disorder or any concerning symptoms, it is essential to seek medical attention promptly. Genetic testing can help confirm the diagnosis and identify affected family members, allowing for appropriate monitoring and management.

FRI496 A Rare Case Of Thyroid Hormone Resistance Beta

Abstract Disclosure: H.K. Rai: None. R.M. Pradhan: None. A. Chaudhuri: None. Background: Genetic mutation in the thyroid hormone receptor leads to the reduced affinity of hormone binding; thus, causing thyroid hormone resistance. It is characterized by a wide range of phenotypic variations and subsequent diagnostic and treatment challenges. Here, we present a case of resistance to the thyroid hormone Beta, with a rare genetic abnormality, that was misdiagnosed as hyperthyroidism. Case: A 46-year-old woman diagnosed with hyperthyroidism s/p RAIA resulting in hypothyroidism on levothyroxine therapy (112mcg daily, weight: 55kg), presented with ten pounds of weight gain, constipation, and cold intolerance for the past six months. TFTs were as follows: TSH: 6.493 mcUnits/ml (normal: 0.400-5.00 mcUnits/ml), and free T4: 1.83 ng/dL (normal: 0.80-1.80 ng/dL). The patient was clinically hypothyroid despite having an elevated FT4 and TSH level. She was also on metoprolol succinate 25 mg daily for sinus tachycardia for the past 1 year. Upon inquiring about further details, the patient informed that at the age of 22, she had significant weight loss, palpitations, and anxiety, but also reported dry skin. The patient was seen by multiple endocrinologists and was provided with contradictory diagnoses. She had RAI scan which was significant for increased uptake and subsequently underwent RAIA. The patient was seen by multiple physicians in the past, levothyroxine dose was adjusted on various occasions but her TFTs never normalized. Post-ablation, she continued to require a higher than the weight-based dose of levothyroxine. Upon review of family history, the patient reported that her father, sister, and daughter had a similar pattern of TFTs with elevated FT4 and TSH values. All of them had palpitations and anxiety that were not severe enough to seek medical care. Family history was also significant for ADHD and hyperlipidemia present in the daughter and father. All affected family members have short stature. The levothyroxine dose was slowly titrated based on the clinical presentation. She was euthyroid with 150 mcg daily of levothyroxine with TSH of 4.869 mcUnits/ml and free T4: 1.85 ng/dl. Given her presentation and suspicion of thyroid hormone resistance, we decided to proceed with genetic testing. The result was positive for the THRB (Resistance to thyroid hormone beta) variant c.904A>G.p.(IIe302Val), which is classified as a variant of uncertain significance, as there is currently insufficient evidence to support its disease-causing role. However, there is a strong association between the gene and the patient's phenotype. Conclusion: The distinctive feature of RTH beta is high levels of free T4 while TSH concentration is normal or elevated, with a combination of hypothyroid and hyperthyroid symptoms. Such patients are often misdiagnosed. We herein describe a novel pathogenic RTH beta gene variant causing thyroid hormone resistance that needs to be further explored. Presentation: Friday, June 16, 2023

THU432 Severe Osteoporosis Associated With A Novel Plastin 3 (PLS3) Mutation

Abstract Disclosure: A. Ibrahim: None. L. Esper: None. S.M. Gandhi: None. E.S. Nylen: None. Early Onset Osteoporosis (EOOP) affects children and young adults and can be caused by endocrinopathies, chronic medical conditions, nutritional deficiencies, and/or genetic mutations. The most common inherited cause is osteogenesis imperfecta(OI), and a rare variant linked to OI includes a defect in the X chromosomal gene encoding Plastin 3 (PLS3). In our case, a 34-year-old male was referred for severe osteoporosis. He had been diagnosed with osteopenia at age 21. At age 24, imaging revealed multiple vertebral compression fractures and DXA scan confirmed severe osteoporosis. He required L3-L5 kyphoplasty and started on oral bisphosphonates for presumed OI despite negative gene mutation (i.e., COL1A1/COL1A2). He continued treatment for only 2 years. 8 years later, he had persistent osteoporosis (DXA L2-4 Z-score -3.6, LFN Z-score -3.2). He had no evidence of kyphosis or scoliosis and no extraskeletal manifestations such as blue-grey sclera or any dysmorphic features. The workup was negative for secondary causes of EOOP and he denied a family history of osteoporosis. Labs showed normal 24-hour urine calcium, bone markers, and bone Alkaline Phosphatase. Karyotyping showed normal XY and gene analysis was again negative for OI. He started treatment with intravenous Zoledronic acid, however, had a 2-year gap between doses initially causing BMD to drop by 6.5% in L2-L4 (Z -4.1) and by 5.7% in LFN (Z -3.1). Subsequently, he received a total of 5 doses of Zoledronic acid to date and had no further fractures. DXA significantly improved by 10.4% in L2-4 (Z -2.6), and 16.3% in LFN (Z -2.0). Repeated expanded gene testing showed a novel PLS3 hemizygous mutation (deletion at site C.234_237+10del of Exon 3). This case demonstrates a novel PLS3 mutation variant to be a plausible cause of adulthood EOOP which was responsive to bisphosphonate treatment. PLS3 is an F-actin binding and bundling protein involved in cytoskeletal remodeling and may have roles in calcium homeostasis, cell migration, endocytosis, DNA repair, and membrane trafficking. Currently, less than 30 loss of function variants of PLS3 have been identified and all lead to severe osteoporosis- particularly in children. However, our patient’s known onset of bone loss occurred much later- in his twenties and he had no evidence of extra-skeletal manifestations. The cause for such late presentation is unclear but may be attributed to this specific variant of PLS3. This case highlights that PLS3 mutation should be on the differential for EOOP even in early adulthood. Our patient’s BMD has markedly improved with bisphosphonates and he sustained no further fractures after starting therapy. Timely diagnosis and earlier detection will allow for appropriate genetic counseling, earlier screening, and treatment of affected family members. Presentation: Thursday, June 15, 2023

SAT231 Next-generation Sequencing For Detection Of Underlying Genetic Causes Of Nonsurgical Hypoparathyroidism: Preliminary Results From A Sponsored Testing Program

Abstract Disclosure: M. Mannstadt: Advisory Board Member; Self; Takeda, Calcilytix, Amolyt. Research Investigator; Self; Takeda, Calcilytix, Amolyt. A. Mathew: Employee; Self; BridgeBio Pharma. A. Sridhar: Employee; Self; BridgeBio Pharma. L.S. Smith: Employee; Self; BridgeBio Pharma. M. Roberts: Employee; Self; BridgeBio Pharma. S. Adler: Employee; Self; BridgeBio Pharma. Hypoparathyroidism is a rare endocrine disorder characterized by inadequate production of parathyroid hormone to maintain normal blood calcium levels. Hypoparathyroidism is most frequently caused by damage to or removal of the parathyroid glands but can also be associated with genetic etiologies. Genetic forms of hypoparathyroidism can present as isolated or as part of a syndrome and include disorders of parathyroid gland formation, parathyroid hormone secretion, and damage to the parathyroid gland through autoimmunity. Genetic testing may uncover the underlying etiology of nonsurgical hypoparathyroidism and can help confirm clinical diagnosis, guide medical treatment, and identify affected family members. A sponsored genetic testing program was made available for patients with suspected genetic hypoparathyroidism who met program eligibility criteria. The next-generation sequencing panel has evolved to include 26 genes known to be associated with hypoparathyroidism: ACADM, AIRE, ATP1A1, CASR, CHD7, CLDN16, CLDN19, CNNM2, DHCR7, EGF, FAM111A, FXYD2, GATA3, GCM2, GNA11, HADHA, HADHB, KCNA1, NEBL, PTH, SEMA3E, SLC12A3, SOX3, TBCE, TBX1 and TRPM6. A total of 169 samples between December 2020 and December 2022 were tested from participants with a mean±SD age of 23.4±20.4 (range 0-81) who were diagnosed with nonsurgical/idiopathic hypoparathyroidism (73.9%), hypocalcemia suspected to be of genetic cause (23.7%) or had a relative with a confirmed diagnosis of genetic hypoparathyroidism (2.4%). Pathogenic or likely pathogenic variants, and variants of uncertain significance were identified in 64 individuals (37.9%). Amongst these 64 participants, 77 variants were detected with 46.9% of variant harboring individuals documented as having unknown or no family history. In order of frequency, the relative number of individuals with detected variants are as follows: CASR 56.3% (36/64), AIRE 12.5% (8/64), TBX1 9.38% (6/64), GATA3 7.8% (5/64), GNA11 6.25% (4/64), CHD7 3.13% (2/64), FAM111A 3.13% (2/64), PTH 3.13% (2/64), ACADM 1.6% (1/64), GCM2 1.6% (1/64), HADHB 1.6% (1/64) and TBCE 1.6% (1/64). Of note, 5 individuals had variants identified in more than 1 gene. Notably, these data demonstrate that genetic testing may identify clinically-relevant variants in approximately 2 of 5 individuals with nonsurgical hypoparathyroidism. The most common genetic form of hypoparathyroidism was found to be autosomal dominant hypocalcemia type 1 (21.3% of individuals tested; 36/169), caused by gain-of-function variants in the CASR gene. Overall, this ongoing sponsored testing program provides an efficient path to aid in the diagnosis of genetic causes and may ultimately improve the understanding and management of nonsurgical hypoparathyroidism. Presentation: Saturday, June 17, 2023

Clinical and genetic screening in a large Iranian family with Marfan syndrome: A case study.

Marfan syndrome (MFS) is an autosomal dominant genetic disorder caused by pathogenic variants of the fibrillin-1-encoding FBN1 gene that commonly affects the cardiovascular, skeletal, and ocular systems. This study aimed to evaluate the clinical features and genetic causes of the MFS phenotype in a large Iranian family. Seventeen affected family members were examined clinically by cardiologists and ophthalmologists. The proband, a 48-year-old woman with obvious signs of MFS, her DNA sample subjected to whole-exome sequencing (WES). The candidate variant was validated by bidirectional sequencing of proband and other available family members. In silico analysis and molecular modeling were conducted to determine the pathogenic effects of the candidate variants. The most frequent cardiac complications are mitral valve prolapse and regurgitation. Ophthalmic examination revealed iridodonesis and ectopic lentis. A heterozygous missense variant (c.2179T>C/p.C727R) in exon 19 of FBN1 gene was identified and found to cosegregate with affected family members. Its pathogenicity has been predicted using several in silico predictive algorithms. Molecular docking analysis indicated that the variant might affect the binding affinity between FBN1 and LTBP1 proteins by impairing disulfide bond formation. Our report expands the spectrum of the Marfan phenotype by providing details of its clinical manifestations and disease-associated molecular changes. It also highlights the value of WES in genetic diagnosis and contributes to genetic counseling in families with MFS.