Kari Lima

Exome Sequencing and Genetic Testing for MODY

Context Genetic testing for monogenic diabetes is important for patient care. Given the extensive genetic and clinical heterogeneity of diabetes, exome sequencing might provide additional diagnostic potential when standard Sanger sequencing-based diagnostics is inconclusive. Objective The aim of the study was to examine the performance of exome sequencing for a molecular diagnosis of MODY in patients who have undergone conventional diagnostic sequencing of candidate genes with negative results. Research Design and Methods We performed exome enrichment followed by high-throughput sequencing in nine patients with suspected MODY. They were Sanger sequencing-negative for mutations in the HNF1A, HNF4A, GCK, HNF1B and INS genes. We excluded common, non-coding and synonymous gene variants, and performed in-depth analysis on filtered sequence variants in a pre-defined set of 111 genes implicated in glucose metabolism. Results On average, we obtained 45 X median coverage of the entire targeted exome and found 199 rare coding variants per individual. We identified 0–4 rare non-synonymous and nonsense variants per individual in our a priori list of 111 candidate genes. Three of the variants were considered pathogenic (in ABCC8, HNF4A and PPARG, respectively), thus exome sequencing led to a genetic diagnosis in at least three of the nine patients. Approximately 91% of known heterozygous SNPs in the target exomes were detected, but we also found low coverage in some key diabetes genes using our current exome sequencing approach. Novel variants in the genes ARAP1, GLIS3, MADD, NOTCH2 and WFS1 need further investigation to reveal their possible role in diabetes. Conclusion Our results demonstrate that exome sequencing can improve molecular diagnostics of MODY when used as a complement to Sanger sequencing. However, improvements will be needed, especially concerning coverage, before the full potential of exome sequencing can be realized.

Flow cytometry study of blood cell subtypes reflects autoimmune and inflammatory processes in autoimmune polyendocrine syndrome type I.

Autoimmune polyendocrine syndrome type I (APS I) is a recessive disorder caused by mutations in the autoimmune regulator (AIRE) gene. AIRE is expressed in medullary epithelial cells where it activates transcription of organ‐specific proteins in thymus, thereby regulating autoimmunity. Patients with APS I have, in addition to autoimmune manifestations in endocrine organs, also often ectodermal dystrophies and chronic mucocutaneous candidiasis. The aim of this study was to characterize immune cell subpopulations in patients with APS I and their close relatives. Extensive blood mononuclear cell immunophenotyping was carried out on 19 patients with APS I, 18 first grade relatives and corresponding sex‐ and age‐matched healthy controls using flow cytometry. We found a significant relative reduction in T helper cells coexpressing CCR6 and CXCR3 in patients with APS I compared to controls (mean = 4.10% versus 5.94% respectively, P = 0.035). The pools of CD16+ monocytes and regulatory T cells (Tregs) were also lower in patients compared with healthy individuals (mean = 15.75% versus 26.78%, P = 0.028 and mean = 4.12% versus 6.73%, P = 0.029, respectively). This is the first report describing reduced numbers of CCR6+CXCR3+ T helper cells and CD16+ monocytes in patients with APS I We further confirm previous findings of reduced numbers of Tregs in these patients.

Hypoparathyroidism and autoimmunity in the 22q11.2 deletion syndrome

OBJECTIVE To characterize the endocrine and autoimmune disturbances with emphasis on parathyroid dysfunction in patients with 22q11.2 deletion syndrome (22q11.2 DS). Design In this nationwide survey; 59 patients (age 1-54 years) out of 86 invited with a 22q11.2 DS were recruited through all the genetic institutes in Norway. METHODS Data was collected from blood tests, medical records, a physical examination and a semi-structured interview. We registered autoimmune diseases and measured autoantibodies, hormone levels and HLA types. RESULTS Twenty-eight (47%) patients had hypoparathyroidism or a history of neonatal or transient hypocalcemia. Fifteen patients had neonatal hypocalcemia. Fourteen patients had permanent hypoparathyroidism including seven (54%) of those above age 15 years. A history of neonatal hypocalcemia did not predict later occurring hypoparathyroidism. Parathyroid hormone levels were generally low indicating a low reserve capacity. Twenty-eight patients were positive for autoantibodies. Six (10%) persons had developed an autoimmune disease, and all were females (P<0.02). Hypoparathyroidism correlated with autoimmune diseases (P<0.05), however, no antibodies were detected against the parathyroid glands. CONCLUSIONS Hypoparathyroidism and autoimmunity occur frequently in the 22q11.2 DS. Neonatal hypocalcemia is not associated with later development of permanent hypoparathyroidism. Hypoparathyroidism may present at any age, also in adults, and warrants regular measurement of calcium levels. Hypoparathyroidism and autoimmunity occur frequently together. Our findings of autoimmune diseases in 10% of the patients highlight the importance of stringent screening and follow-up routines.

A longitudinal follow-up of autoimmune polyendocrine syndrome type 1

Context: Autoimmune polyendocrine syndrome type 1 (APS1) is a childhood-onset monogenic disease defined by the presence of two of the three major components: hypoparathyroidism, primary adrenocortical insufficiency, and chronic mucocutaneous candidiasis (CMC). Information on longitudinal follow-up of APS1 is sparse. Objective: To describe the phenotypes of APS1 and correlate the clinical features with autoantibody profiles and autoimmune regulator (AIRE) mutations during extended follow-up (1996–2016). Patients: All known Norwegian patients with APS1. Results: Fifty-two patients from 34 families were identified. The majority presented with one of the major disease components during childhood. Enamel hypoplasia, hypoparathyroidism, and CMC were the most frequent components. With age, most patients presented three to five disease manifestations, although some had milder phenotypes diagnosed in adulthood. Fifteen of the patients died during follow-up (median age at death, 34 years) or were deceased siblings with a high probability of undisclosed APS1. All except three had interferon-ω) autoantibodies, and all had organ-specific autoantibodies. The most common AIRE mutation was c.967_979del13, found in homozygosity in 15 patients. A mild phenotype was associated with the splice mutation c.879+1G>A. Primary adrenocortical insufficiency and type 1 diabetes were associated with protective human leucocyte antigen genotypes. Conclusions: Multiple presumable autoimmune manifestations, in particular hypoparathyroidism, CMC, and enamel hypoplasia, should prompt further diagnostic workup using autoantibody analyses (eg, interferon-ω) and AIRE sequencing to reveal APS1, even in adults. Treatment is complicated, and mortality is high. Structured follow-up should be performed in a specialized center.

Epidemiology and Health-Related Quality of Life in Hypoparathyroidism in Norway

Objective: The epidemiology of hypoparathyroidism (HP) is largely unknown. We aimed to determine prevalence, etiologies, health related quality of life (HRQOL) and treatment pattern of HP. Methods: Patients with HP and 22q11 deletion syndrome (DiGeorge syndrome) were identified in electronic hospital registries. All identified patients were invited to participate in a survey. Among patients who responded, HRQOL was determined by Short Form 36 and Hospital Anxiety and Depression scale. Autoantibodies were measured and candidate genes (CaSR, AIRE, GATA3, and 22q11-deletion) were sequenced for classification of etiology. Results: We identified 522 patients (511 alive) and estimated overall prevalence at 102 per million divided among postsurgical HP (64 per million), nonsurgical HP (30 per million), and pseudo-HP (8 per million). Nonsurgical HP comprised autosomal dominant hypocalcemia (21%), autoimmune polyendocrine syndrome type 1 (17%), DiGeorge/22q11 deletion syndrome (15%), idiopathic HP (44%), and others (4%). Among the 283 respondents (median age, 53 years [range, 9–89], 75% females), seven formerly classified as idiopathic were reclassified after genetic and immunological analyses, whereas 26 (37% of nonsurgical HP) remained idiopathic. Most were treated with vitamin D (94%) and calcium (70%), and 10 received PTH. HP patients scored significantly worse than the normative population on Short Form 36 and Hospital Anxiety and Depression scale; patients with postsurgical scored worse than those with nonsurgical HP and pseudo-HP, especially on physical health. Conclusions: We found higher prevalence of nonsurgical HP in Norway than reported elsewhere. Genetic testing and autoimmunity screening of idiopathic HP identified a specific cause in 21%. Further research is necessary to unravel the causes of idiopathic HP and to improve the reduced HRQOL reported by HP patients.

Dental developmental disturbances in 50 individuals with the 22q11.2 deletion syndrome; relation to medical conditions?

Abstract Objective. The aims of the study were to examine tooth and enamel disturbances in individuals with 22q11.2 deletion syndrome and to analyze associations with medical conditions, birth characteristics and blood values of calcium and PTH. Materials and methods. Fifty individuals participated in the study (27 females, median age 10 years, range 1.5–44). Congenital absence of teeth was studied on orthopantomograms; 1148 teeth were examined, both clinically and radiologically, and enamel hypomineralizations and hypoplasias were recorded. Medical history and findings were recorded as part of a larger study on the manifestations of 22q11.2-deletion syndrome in Norway. Results. Tooth agenesis was observed in 15% of study participants. Sixty-six percent of the participants and 26.0% of teeth presented with enamel disturbances. Of these, 12 individuals (24.0%) and 215 teeth (18.7%) had hypomineralizations and four individuals (8.0%) and 86 teeth (7.5%) had hypoplasias. Seventeen participants (34.0%) presented with both types of disturbance, but rarely in the same tooth. Only two teeth (0.17%) had both types of disturbance. Hypomineralizations were twice as frequent in permanent as in primary teeth. No correlations were found to medical conditions, except that participants with congenital cardiac anomalies presented with fewer total enamel disturbances and hypomineralizations in permanent teeth than those without. Conclusions. Enamel disturbances were frequently seen. There were more hypomineralizations than hypoplasias. Hypoparathyroidism and/or hypocalcemia are not clear etiological factors for enamel disturbances and there were no major correlations between medical conditions and enamel disturbances.

Neonatal Levels of T-cell Receptor Excision Circles (TREC) in Patients with 22q11.2 Deletion Syndrome and Later Disease Features

PurposeNewborns with severe T-cell lymphopenia, including those with 22q11.2 deletion syndrome (DS), have low numbers of T-cell receptor excision circles (TRECs). The aim of this study was to determine a possible correlation between neonatal TRECs in 22q11.2DS and the development of different phenotypes to elucidate the prognostic value of TREC in this disease.MethodsIn this national survey including 46 patients with 22q11.2DS born after 2005, TREC levels were determined using stored newborn screening blood spots on filter cards. Patients were grouped into quartiles according to their TREC values, except the two infants with thymus aplasia.ResultsThe two patients with thymic aplasia had no detectable TREC. The rest had no severe clinical immunodeficiency. There was a significant correlation between low TRECs and the proportion of patients with CD3+CD4+T-cells below the 5th percentile of healthy infants (p = 0.027) as well as the proportion with an abnormal thymus feature either no thymus or remnant thymus as observed during heart surgery (p = 0.022). Significantly lower TRECs (p = 0.019) were found in patients with cardiac defects compared to no such defects. Patients within the lowest quartile of TREC values (<71 TRECs/μL, n = 11) had more frequent severe cardiac defects than the other quartiles (p = 0.010). Eight of these patients in the lowest quartile needed an operation/intervention within two weeks after birth or died because of a cardiac defect.ConclusionThe low TREC values not only correlate with decreased T-cell immunity, but also with the occurrence of heart defects in the patients.

Adults with 22q11.2 deletion syndrome have a different velopharyngeal anatomy with predisposition to velopharyngeal insufficiency

AIM To find out if subjects with 22q11.2 deletion syndrome (DS) have a different velopharyngeal anatomy which could cause velopharyngeal insufficiency (VPI). METHODS A prospective study of 16 subjects >16 years of age with 22q11.2 DS, without overt cleft palate and without previous VPI surgery, and 48 healthy controls >18 years of age were included in the study. Speech was recorded and scored blindly by two independent senior speech therapists. All 64 individuals had MRI scans, which were analyzed blindly by a consultant radiologist. RESULTS Subjects with 22q11.2 DS had a mild degree of weak pressure consonants (mean score); borderline to mild degree of hypernasality and audible nasal emission (mean score). All controls had normal speech. When comparing subjects (22q11.2 DS) to controls, we found the subjects to have the following: A shorter distance between left and right points of origin of the levator veli palatini muscle (LVP) (p < 0.0001); a more obtuse angle of origin of the LVP (bilaterally) (p < 0.009); a thinner LVP bilaterally and in the midline (p < 0.0001); a shorter LVP bilaterally (p < 0.0001); a shorter velum (p = 0.007); a larger osseous pharyngeal depth:velar length ratio (p = 0.01); a more obtuse anterior cranial base angle (nasion to sella to basion) (p < 0.0001) and posterior cranial base angle (sella to basion to foramen magnum) (p < 0.0001); a wider velopharyngeal width (p = 0.002) and a larger pharyngeal airway volume (p = 0.0007). CONCLUSION Compared with healthy controls, adults with 22q11.2 DS showed a different velopharyngeal anatomy, which will make these individuals more prone to VPI.

Increased Levels of Interferon-Inducible Protein 10 (IP-10) in 22q11.2 Deletion Syndrome.

The 22q11.2 deletion syndrome (22q11.2 DS), also known as DiGeorge syndrome, is a genetic disorder with an estimated incidence of 1:4000 births. These patients may suffer from affection of many organ systems with cardiac malformations, thymic hypoplasia or aplasia, hypoparathyroidism, palate anomalies and psychiatric disorders being the most frequent. The incidence of autoimmune diseases is increased in older patients. The aim of the present study was to examine a cytokine profile in patients with 22q11.2 DS by measuring a broad spectrum of serum cytokines. Patients with a proven deletion of chromosome 22q11.2 (n = 55) and healthy individuals (n = 54) recruited from an age‐ and sex‐comparable group were included in the study. Serum levels of 27 cytokines, including chemokines and growth factors, were analysed using multiplex technology. Interferon‐inducible protein 10 (IP‐10) was also measured by ELISA to confirm the multiplex results. The 22q11.2 DS patients had distinctly and significantly raised levels of pro‐inflammatory and angiostatic chemokine IP‐10 (P < 0.001) compared to controls. The patients with congenital heart defects (n = 31) had significantly (P = 0.018) raised serum levels of IP‐10 compared to patients born without heart defects (n = 24). The other cytokines investigated were either not detectable or did not differ between patients and controls.

PO-0996 Elevated Levels Of Interferon-inducible Protein 10 (ip-10) In Patients With 22q11.2 Deletion (digeorge) Syndrome

Background and aim The 22q11.2 deletion syndrome (DS), also known as DiGeorge syndrome, is a genetic disorder with an estimated incidence of 1 in 4000 births. These patients may suffer from disorders of many organ systems, but cardiac malformations, thymic hypoplasia/aplasia, hypoparathyroidism, cleft palate and psychiatric disorders are most frequent. In addition, the incidence of autoimmune diseases is increased in older patients. The reason for the latter is not known. Methods Only patients with a proven deletion of chromosome 22q11.2 by in situ hybridization (FISH) test or multiplex ligation-dependent probe amplification assay (MLPA) was included in the study. Age and sex matched healthy controls were included. Serum levels of 27 cytokines, chemokines and growth factors were analysed using a multiplex cytokine assay. For comparison of two groups, the Mann-Whitney U test was used. Results 22q11.2 DS patients had significantly raised serum levels of interferon-inducible protein 10 (IP-10) compared with healthy controls. In contrast, there was no significant difference in the serum levels of the other cytokines, chemokines and growth factors between 22q11.2 DS and healthy controls. Conclusion Our finding suggest that IP-10, which has been shown to contribute to the development of autoimmune diseases, might play a significant role in the pathogenesis of 22q11.2 DS.