clin chem lab med 2021; 59, special suppl, pp s94 s835

Genetic diseases

T063

GENETIC TESTING OF MATURITY-ONSET DIABETES OF THE YOUNG USING NEXT-GENERATION SEQUENCING.

M. Exposito Garcia 1, L. Rosado Jimenez 1, Y. Mestre Terkemani 1, M.A. Moreno Locubiche 1, M.D. Sarabia Meseguer1, A.M. Hernandez Martinez 1, J.A. Noguera Velasco 1, F. Ruiz Espejo 11GENOMICS LABORATORY, DEPARTMENT OF CLINICAL ANALYSIS, VIRGEN DE LA ARRIXACA UNIVERSITY CLINICAL HOSPITAL,MURCIA.

BACKGROUND-AIM

MODY (Maturity Onset Diabetes of the Young) presents autosomal dominant inheritance, dysfunction of pancreaticbeta cells and onset before 25 years.

METHODS

Fifty-two index cases with suspected MODY were selected. A clinical exome was made using the Gen Exome Panel v1.0kit on the NexSeq500 platform (Illumina). Data Genomics software (Imegen) was used for the bioinformatics analysis.The clinical characterization of the gene variants was carried out according to the ACMG recommendations. Thedatabases used for the variant analysis were ClinVar and HGMD. The Selene software was used to obtain the medicalrecords.The specific variants were verified by Sanger methodology and the large rearrangements by MLPA (Multiplex LigationProbe Amplification).The SPSS v27 program was used to compare the phenotypic characteristics between the group 1 (patients carrying apathogenic variant) and the group 2 (non-carriers patients). Quantitative variables (age of debut, HbA1c, BMI, serumlevels of insulin and C-peptide) were compared using the Mann-Whitney U test, and qualitative variables (patients withpancreatic antibodies, insulin treatment and family history) with Fisher's exact test. A value of p <0.05 was consideredstatistically significant.

RESULTS

The diagnostic yield was 18%. The subtypes of MODY diagnosed were: MODY 2 (25%), MODY 3 (18.75%), MODY 1(12.5%), MODY 12 (12.5%), MODY 8 (6.25%), MODY 5 (6.25%), MODY 10 (6.25%), MODY 4 (6.25%) and MODY 13 (6.25%).At the molecular level, 16 pathogenic variants were found, the type changes being: 68.75% missense, 12.5% frameshift,12.5% large rearrangements, and 6.25% nonsense. Excluding the benign variants detected, the percentage of the restof the variants was: 75% pathogenic, 6.25% probably pathogenic, and 18.75% unknown clinical significance.Regarding the phenotypic characteristics, significant differences were observed between both groups only in seruminsulin levels, higher in group 2, and in patients with insulin treatment, higher in group 1.

CONCLUSIONS

Massive sequencing analysis is an essential tool to increase the diagnostic performance of MODY.The genetic study has been beneficial in diagnosing MODY even though both study groups presented similarphenotypic characteristics in most parameters.

Poster Abstracts – EuroMedLab Munich 2021 – Munich, Nov 28-Dec 02, 2021 • DOI 10.1515/cclm-2021-5020 Clin Chem Lab Med 2021; 59, Special Suppl, pp S94 – S998, Nov/Dec 2021 • Copyright © by Walter de Gruyter • Berlin • Boston S462

Genetic diseases

T064

EARLY DETECTION OF GENETIC HYPERTRIGLYCERIDEMIAS BY LABORATORY TELEMEDICINE IN PRIMARY CARE.

T. Arrobas Velilla 2, S. Martin 2, M.J. Ariza 1, J. Rioja Villodres 1, P. Valdivieso Felices 1, A. Leon-Justel 21Medical and Health Research Centre (CIMES) University of Malaga2Virgen Macarena University Hospital

BACKGROUND-AIM

Familial hypertriglyceridaemia is a disorder of lipid metabolism with autosomal dominant inheritance occurring in thepopulation with an incidence around 0.5-1%. Deficiency of lipoprotein lipase enzyme activity leads to an increase intriglycerides that may be genetically caused. The clinical laboratory by applying cardiovascular risk laboratory (CVR)telemedicine can make an early diagnosis of this type of underdiagnosed dyslipidaemia in primary care. The obtectiveof the study is to identify potential patients with genetic hypertriglyceridemia in routine analyses by applying CVRlaboratory telemedicine for priority referral to genetic dyslipidemia consultation.

METHODS

In the analytical validation procedure, patients with severe hypertriglyceridemia >880 mg/dl on at least two occasionswere selected, and their clinical history was reviewed in order to rule out secondary causes. A genetic study is carriedout after informed consent with the following candidate genes: USF1, GALNT2, LPL GPIHBP1 LMF1 APOC2 APOA5 APOEAPOE APOC3 GALNT2 GCKR GPD1 LIPC. Patients with a positive diagnosis are referred as a priority to the geneticdyslipidaemia clinic for evaluation according to standard clinical practice.

RESULTS

We selected 30 potential candidates who met the inclusion criteria, obtaining genetic cause in heterozygosis in90% of the cases, and family segregation of the variations of uncertain significance was carried out. In one case,familial segregation of the variant C.427delC, p.Arg143AlafsTer57 was carried out in two children, one of them beinga heterozygous carrier. The most frequent variations were:APOA5 het rs3135506 1 c.56C>G p.Ser19TrpGPIHBP1 het rs11538389 1 c.41G>T p.Cys14PheAPOA5 het rs651821 1 c.-3G>AGCKR het rs1260326 1 c.1337T>C p.Leu446ProAPOE het rs7412 1 c.526C>T p.Arg176CysAPOE het rs429358 1 c.388T>C p.Cys130ArgAPOA5 het rs651821 1 c.-3G>AAPOB het rs375936466 2 c.9293A>G p.Tyr3098CysANGPTL8 het rs2278426 2 c.175C>T p.Arg59TrpAPOB het rs676210 2 c.8216C>T p.Pro2739LeuAPOB het rs679899 2 c.1853C>T p.Ala618ValLMF1 het rs4984948 2 c.1685C>G p.Pro562ArgAPOB hom rs676210 2 c.8216C>T p.Pro2739LeuAPOB het rs1042034 2 c.13013G>A p.Ser4338AsnCETP het rs5880 2 c.1168G>C p.Ala390ProLMF1 het rs35168378 2 c.1091G>A p.Arg364GlnLPL het rs328 3 c.1421C>G p.Ser474Ter

CONCLUSIONS

Early detection from the clinical laboratory by applying laboratory telemedicine is cost-effective, promotes quality ofcare and multidisciplinary work for the benefit of the patient.


Genetic diseases

T065

HIGH INCIDENCE OF SMN1 EXON 8/9 DELETIONS IN SPINAL MUSCULAR ATROPHY (SMA)

P.K. Manuprasad 1, C.K. Reshma 1, K. Vaidyanathan 11Amrita Institute of Medical Science, Kochi, Kerala, India

BACKGROUND-AIM

Spinal muscular atrophy (SMA) is an autosomal recessive genetic disorder characterised by motor neuron degeneration.The global prevalence of SMA is 1-2 in 100,000 and incidence is 1 in 10,000. About 96% of SMA cases are due to thepathogenic variants in SMN1 gene, mainly due to the deletions in SMN1 exon 7 and 8. There are few studies from Indiafor this disorder in which prevention is justified through carrier screening. Our aim was to study the frequency of SMApatients lacking SMN1 gene exon 7 and 8 in South Indian population.

METHODS

Sixty patients with symptoms of SMA or having family history of SMA has been included for the genetic analysis usingMLPA technique. All patients are from the Department of Pediatric Genetics, Amrita Institute of Medical Sciences(AIMS), Kochi, Kerala during the period of 2018-2019. Genetic analysis was done in Molecular Genetics Laboratory,AIMS, Kochi. DNA extracted from the Blood samples collected in EDTA tube. Copy number status of both SMN1 andSMN2 were determined using the SALSA MLPA Probemix P021 SMA at MRC Holland specific for the SMN1 exon 7 and8 deletions.

RESULTS

In the sixty samples analysed, we have identified pathogenic homozygous SMN1 exon 7 and 8 deletions in 27/60samples (45%), while 23 /60 were carriers for SMN1 exon 7 or 8 deletions. All the patients screened with symptomsof SMA identified had homozygous deletion in SMN1 exon 7 and 8 (100%). Parents of thirty-three patients (withfamily history of SMA) were genetically tested, in which twenty-three were carriers (23/33) for any of these SMN1 exondeletions.

CONCLUSIONS

A high incidence of deletions in exons 7 and 8 in SMN1 gene was identified in patients with SMA in the South Indianpopulation studied. A higher incidence of deletion mutations in SMN1 gene were also identified in parents of childrenwith proven family history.


Genetic diseases

T066

MODULAR AND FLEXIBLE GENETIC NEWBORN SCREENING FROM DRIED BLOOD SPOTS

J. Bzdok 1, L. Czibere 1, S. Burggraf 1, O. Landt 2, W. Röschinger 1, M. Becker 1, J. Durner 11Labor Becker & Kollegen2TIBMolbiol Berlin

BACKGROUND-AIM

Some diseases in newborns cannot be detected using traditional methods like fluorescence immuno assays ortandem mass spectrometry, because there are neither metabolite, hormone nor enzyme activity markers in the blood.Molecular techniques are an alternative tool for diagnosis of diseases in newborns. Early treatment is often crucial fora successful therapy. Genetic testing has become in many countries part of the regular newborn screening.

METHODS

Nucleic acid can be easily extracted from a single dried blood spot with a yield allowing lots of genetic assays. Multicolorreal-time PCR testing allows to test for several genetic markers in one assay.Here we demonstrate a flexible system which can be easily adapted to different screening programs and allows anefficient high-throughput newborn screening.

RESULTS

Based on a simple and rapid DNA extraction procedure from dried blood spots we conducted from January 2018 untilMarch 2019 a pilot study for three mutations in the cystinosin (CTNS) gene, responsible for nephropathic cystinosis,and a deletion of exon 7 of the survival motor neuron 1 (SMN1) gene, responsible for spinal muscular atrophy (SMA).TREC-based severe-combined-immunodeficiency (SCID) testing has been moved to the same extraction method. Early2019 we combined SCID testing with SMA. Recently we evaluated an additional assay for screening for the two beta-globin gene mutations HbS and HbC, causing sickle cell disease. Adding a new target can be achieved within a coupleof weeks.At least 20 independent PCR reactions can be performed using the DNA extracted from one dried blood spot.Theoretically up to 6 different parameters can be analysed in one PCR reaction e.g. using a Roche 480 instrument. Thisflexibility opens a wide field for genetic screening assays. In addition the DNA can also be used for next generationsequencing applications.

CONCLUSIONS

We show results from our different genetic screening projects, which include data from >500,000 newborns.


Genetic diseases

T067

NEONATAL DIAGNOSIS OF A PATIENT WITH KLINELFELTER SYNDROME AND A CLASSICAL FORM OF CONGENITALADRENAL HYPERPLASIA

M.C. Morante Palacios 2, M. Arriba Domènech 2, B. Ezquieta 2, M. Orera 11Clinical Genetics, Clinical Biochemistry, Hospital General Universitario Gregorio Marañon, Madrid2Molecular Diagnostics, Clinical Biochemistry, Hospital General Universitario Gregorio Marañon, Madrid

BACKGROUND-AIM

Congenital adrenal hyperplasia (CAH) is a recessive disease mostly due to steroid 21-hydroxylase deficiency. It isencoded by the CYP21A2 gene and exhibits a good genotype-phenotype correlation with a wide range of clinicalmanifestations depending on the degree of enzymatic activity. Classical salt wasting forms (CSWF) with a completelack of enzymatic activity can debut with a life-threating hyponatremic dehydration and variable signs of androgenexcess. Klinelfelter Syndrome (KS) –also known as 47,XXY– is a sex chromosome aneuploidy that causes infertility andhypogonadism in males.

METHODS

A 39-year-old pregnant woman with a combined first trimester screening test had a risk value of 1/146 (cut off 1/270)for trisomy 21. Both, noninvasive prenatal testing (NIPT) and amniocentesis showed a 47,XXY karyotype.The full-term newborn showed scrotal hyperpigmentation and macrogenitosomy, starting with vomiting, lethargyand signs of severe dehydration during his first days of life. Neonatal screening resulted in 17-hydroxyprogesteronelevels >800mg/L. Abdominal ultrasound revealed bilateral adrenal hyperplasia. Gluco-mineralocorticoids replacementtherapies started immediately and CYP21A2 gene analyses were performed.

RESULTS

Analyses by MLPA (CAH P050-C1-0128) revealed a compound heterozygous of two severe alterations: deletion/conversion involving exons 1 to 3 and complete deletion of exons 1 to 7. Segregation studies confirmed the completedeletion of CYP21A2 in the father and the deletion/conversion of exons 1 to 3 in the mother, both in heterozygosity.

CONCLUSIONS

To our knowledge, this is the earliest diagnosed case of coexistent KS and CAH. Males with CSWF diagnosed andtreated within their first days of life could have an unpredictable clinical response that could balanced out KS-relatedhypogonadism as puberty progresses into adulthood. In contrast, KS males with non-classical forms of CAH mayremain undiagnosed and be unnoticed until adulthood due the opposite effect of androgens. This case highlights thecontribution of molecular studies in diagnosis, the importance of including actionable not-infrequent severe diseasesin newborn screening programs and the valuable input of NIPT in increasing early diagnosis of sex chromosomesaneuploidies.


Genetic diseases

T068

PRENATAL GENETIC COUNSELING FOR PATIENT WITH FAMILY HISTORY OF FRAGILE X SYNDROME

A. Vílchez Rodríguez 1, M.L. Bellido Díaz 2, M. Perez García 2, S. Esteve Poblador 11Área de Diagnóstico Biológico, Hospital Universitario La Ribera, Alzira, Spain2Hospital Universitario Vírgen de las Nieves, Granada, Spain

BACKGROUND-AIM

Fragile X syndrome (FXS) is the most common cause of inherited intellectual disability with characteristic phenotype(elongated face, large ears, macroorchidism after puberty and in some cases autistic features). It is an X-linkedmonogenic disease of dominant inheritance. It is caused by transcriptional silencing of the FMR1 gene (Xq27.3) dueto progressive expansion and subsequent methylation of trinucleotide repeats (CGG)n in the 5' untranslated regionof the gene.

METHODS

A 22-year-old woman with a family history of Fragile X syndrome. She reports that her mother is a carrier of acomplete mutation without clinical manifestation. Her two brothers also have a complete mutation, both of them haveintellectual disability. She goes to a genetic consultation, since she is pregnant, requesting advice to know the risk oftransmission of this disease to the offspring.

RESULTS

The patient was studied, being a carrier of the complete mutation (> 200 repetitions of the CGG trinucleotide).Ultrasonography performed at week 13, revealed a male fetus, so the study was indicated due the risk associated withsex. The result of the chorionic villus biopsy indicated that the fetus was not a carrier of the expansion.

CONCLUSIONS

Any woman desiring gestation with a family history of Fragile X syndrome is a candidate for genetic counseling. First ofall, the mother must be studied to see if she has a premutation or the complete mutation of the FMR1 gene. If she hasthe mutation, prenatal diagnosis is performed according to the fetal sex. In males, the study continues, because if themother has the complete mutation, she will certainly present intellectual deficit. If the fetus is female, prenatal studyis not indicated because of the risk associated with invasive techniques, since due to the penetrance of the disease andinactivation of the X chromosome in females, the possibility of clinical manifestations is greatly reduced.


Genetic diseases

T069

CONSEQUENCES OF APPARENTLY BALANCED TRANSLOCATIONS: DELETION AT FAMILY TRANSLOCATION BREAKINGPOINT

A. Vílchez Rodríguez 1, M.L. Bellido Díaz 2, M. Ortuño Alonso 1, S. Esteve Poblador 11Área de Diagnóstico Biológico, Hospital Universitario La Ribera, Alzira, Spain2Hospital Universitario Vírgen de las Nieves, Granada, Spain

BACKGROUND-AIM

Balanced autosomal translocation is the most common structural chromosomal rearrangement. It corresponds to 0.2%of newborns, most of them being inherited and 0.02% de novo.In these translocations there is no loss or gain of genes and the individual is apparently healthy, although in theoffspring there may be an imbalance, leading to various health problems.

METHODS

A 21-year-old woman, was diagnosed with a balanced translocation 46, XX, t(14,18)(q24.3;q12.2) in 1993 due torepeated abortions. Her 3 children present the same translocation diagnosed in a prenatal karyotype study (1994,1998, 2003) respectively.

RESULTS

Her first born, at the age of 23 years, is referred from neurology to genetic consultation for mild intellectual deficitand Parkinsonian type tremor in the upper limbs. Array CGH shows a deletion at the cleavage point of the familialtranslocation probably responsible for the patient's symptoms. Arr[hg19]18q12.1(29745723_36540552)x1. Study ofhis siblings reveals the same deletion.

CONCLUSIONS

The possibility of breakpoint imbalance must be considered, since deletions or duplications can occur at breakpointsduring chromatid exchange in prophase I. The risk for these new rearrangements is extremely low, less than 0.5%.However, thanks to aCGH technology, implemented in the clinic diagnosis in 2010, imbalances have been determinedin patients with inherited balanced translocations. In a de novo translocation the risk is somewhat higher, due to thedisruption of an existing gene sequence at the breakpoint.This case shows an imbalance probably confined to germ cells since all the offspring have the deletion at the q12.2band of chromosome 18 breakpoint.


Genetic diseases

T070

DETECTION OF A RARE MUTATION C.579+1G>T IN HETEROZYGOSIS IN CYSTIC FIBROSIS CARRIER PROGENITORS

A. Vílchez Rodríguez 1, S. García Linarez 2, M. Martínez Atienza 2, S. Pedrinaci Rodríguez 21Área de Diagnóstico Biológico, Hospital Universitario La Ribera, Alzira, Spain2Hospital Universitario Vírgen de las Nieves, Granada, Spain

BACKGROUND-AIM

Cystic fibrosis (CF) is a severe disease of autosomal recessive inheritance very frequent in caucasian race, with anestimated incidence between 0.05-0.01%. It is caused by mutations of the CFTR gene, with chromosomal location7q31.2. More of 2.000 mutations have been described.

METHODS

We present the case of consanguineous couple from North Africa with 12-week gestation fetus. The couple had a sonwho died of cystic fibrosis at the age of 9 years.

RESULTS

Molecular study was performed by allele-specific multiplex PCR amplifying 65 mutations identified by fluorescentcapillary electrophoresis, which allowed us to identify this mutation for the first time in our laboratory. Both parentswere heterozygous for the 711+1G>T mutation (NM_000492.4:c.579+1G>T), which affects mRNA splicing giving analtered protein due to truncation, shortening or inclusion of intronic material. This variant is classified as pathogenicin CLINVAR. Given that both parents are carriers, there is a 25% chance of having an affected child.Chorionic villus biopsy was realized, presenting in heterozygosis the same intronic variant as his parents, confirmingthe heterozygous carrier status of cystic fibrosis.

CONCLUSIONS

The laboratory is fundamental in the diagnosis of CF, a disease that requires early detection to improve the patient'squality of life. In many cases genetic testing is needed to diagnose the pathology because there are cases describedin which the value of the sweat test may be doubtful. On the other hand, other causes of false negatives due to edemaof the stimulated area, hyponatremia, systemic steroids or malnutrition should be taken into account.Genetic counseling should be offered to couples with heterozygous mutations (identified after the birth of a firstchild with cystic fibrosis, by family history of the disease or after detection at birth of a heterozygous mutation in thenewborn) to help them make accepted medical and personal decisions.


Genetic diseases

T071

GENETIC DIAGNOSIS OF AUTOSOMAL RECESSIVE CONGENITAL ICHTHYOSIS TYPE 1

I. Gámez Gómez 1, C. González Oller 1, J.G. Martínez Fernández 1, A.M. Jiménez Gila 1, C. Avivar Oyonarte 11Biotechnology Area, Hospital de Poniente, El Ejido, Almería, Spain

BACKGROUND-AIM

Two year old patient who come to the pediatric consultation for suspected non-syndromic congenital ichthyosis,probably ichthyosis “in a bathing suit”, due to the current clinical picture.He was born as a collodion baby, which improved in the first months of life and, since then, has shown clinical ichthyosis.On examination, pseudotinea amiantacea is observed on the scalp, xerosis with cracked dry skin and excoriations dueto scratching in the genital and groin area as a bathing suit. He has no known family history.Autosomal recessive congenital ichthyosis (ARCI) are rare keratinization disorders that are included in thenon-syndromic forms of ichthyosis. Classically, lamellar ichthyosis (IL) and nonbullous congenital ichthyosiformerythroderma (NCIE) were distinguished in this group. Currently, harlequin ichthyosis, self-healing collodion baby, andswimsuit ichthyosis are also included.Genetic study of ichthyosis is requested from the laboratory.

METHODS

A genetic study of the TGM-1 gene was carried out by means of DNA extraction and quantitative and qualitativeassessment of the DNA sample obtained, amplification of the coding region and flanking intronic areas of the TGM1gene and subsequent sequencing of the amplified fragments. The sequence obtained was analyzed and the variantsof interest were identified with respect to the reference genome (hg19).

RESULTS

The heterozygous presence of two pathogenic variants detected in the TGM1 gene that orient ARCI were identified:- Variant c.788G> A (p.Trp263Ter) is a nonsense type change.- Variant c.919C> G (p.Arg307Gly) is a missense type change.Given the results obtained, it was recommended to carry out the study in the parents which confirmed that each ofthem has one of the variants found in the child and allows to offer adequate genetic counseling to the family.

CONCLUSIONS

The importance of the genetic study lies, on the one hand, in confirming or ruling out clinical suspicion and, on theother, in offering genetic counseling.Once the trans configuration for parents is confirmed, the estimated risk of recurrence of having another affected child,in the event of future pregnancies, is 25% in each pregnancy, and may affect both sexes. 50% of their children will beunaffected carriers and the remaining 25% will be unaffected noncarriers.


Genetic diseases

T072

SHPRINTZEN-GOLDBERG CASE REPORT

A. Bravo Gomez 1, E. Llorente Martin 1, A.E. Zamora Trillo 1, C. Rodriguez Hernandez 1, M.A. Orera Clemente 1, M. Gonzalez

Estecha 11GREGORIO MARAÑON HOSPITAL (MADRID/SPAIN)

BACKGROUND-AIM

We present the case of a 44yeras old man previously diagnosed with Hodgking lymphoma, secondary hypothyroidism,> 100 basal cell carcinomas, iliofemoral thrombosis, aortic valve disease, mitral regurgitation, Wolff Parkinson Whitesyndrome, cryptorchidism and myotonia. No familial relevant pathologies.Height 191 cm with a decreased upper lower segment ratio. Microcephaly, dolichocephaly, hearing impairment, sunkeneyes, shallow orbits, pectus excavatum, arachnodactyly and venous dilation in both lateral regions of the abdomen.

METHODS

We performed NGS based exome connectivepathies panel (58 genes) including ABCC6, ACTA2, ACVR1, ADAMTS10,ADAMTS2, ADAMTSL4, COL11A1, COL11A2, COL1A1, COL1A2, COL3A1, COL4A1, COL5A1, COL5A2, COL9A1, COL9A2,COL9A3, ELN, FBLN5, FBN1, FBN2, FGFR3, FKBP14, FLNA, FMR1, KCNJ2, LTBP2, RIN2, SKI, SMAD3, SMAD4, TGFB1, TGFB2,TGFBR1, TGFBR2, TNXB.

RESULTS

We identify a heterozygous variant in the SKI gene: c.1735G> A, p.Ala579Thr, compatible with Sprintzen-Goldbergsyndrome (SGS).

CONCLUSIONS

Shprintzen-Goldberg syndrome (OMIM �182212) is an extremely rare (<1/1.000.000) connective tissue disordercharacterized by craniosynostosis, marfanoid body habitus, distinctive craniofacial features, skeletal abnormalitiesand aortic dilatation. Recently, mutations in exon 1 of the SKI gene were found in approximately 90% of reportedindividuals diagnosed with this syndrome. This gene is a known regulator of TGF-β signaling. On the other hand, it is aproto-oncogene that is associated with skin tumors and breast cancer. Most mutation are de novo.The variant identified in the patient sample is a missense variant that no has been previously reported, hence it wascategorized as a variant of uncertain significance. Additionally, 5 of the 8 predictors used as well as the predictive valueof CADD suggest that it may be deleterious.SGS is a very rare syndrome, so there are few cases reported in the literature and data on related genetic alterationsare scarce.Our patient´s phenotype fulfills diagnostic criteria of SGS, so we propose the reclassification of this variant as likelypathological, until more carriers are described and/or functional studies are performed.


Genetic diseases

T073

DETERMINATION OF HAEMOCHROMATOSIS GENOTYPE BY THE LAMP TECHNIQUE FROM LACAR MDX

H. Broutier 1, W. Masri 1, L. Detemmerman 2, T. Wtulich 1, L. Kalamar 1, C. Sautereau 1, S. Bonacci 1, H.A. Pichon - Cung1, E. Plouvier 11biochemistry department, Grand Hôpital de l'Est Francilien - site de Meaux (EST-Ile de France) – France2LaCAR MDX, Liège Science PARK, Liège, Belgique

BACKGROUND-AIM

Hereditary hemochromatosis (HH) is a common autosomal disorder of iron metabolism mainly affectingCaucasian populations. Two recurrent disease-associated mutations C282Y and H63D have been identified in thehemochromatosis gene (HFE). The aim of this study was to evaluate the LAMP technique to assess the genotype ofnucleotide-polymorphisms (SNPs) associated with HH (C282Y, H63D) in our laboratory.

METHODS

The LAMP Human Hemochromatosis KIT (LaCAR MDx) allows the qualitative detection of homozygous or heterozygousC282Y and H63D polymorphism by Loop-mediated isothermal amplification (LAMP) of DNA in a whole blood samplefrom patients in a very short time. The GenieIIITM automate is used. The positive control, containing both C282Yand H63D heterozygotes, allows the verification of the reagent quality. The negative control is essential to detectcontaminated reagents. The validation parameters such repeatability, intermediary precision, accuracy, inter-operatorvariability were evaluated using known genotype samples and external quality controls (EEQ: Instand/Orgentec). Theresults obtained by our method and those by «HFE CE TESTS» on LightCyclerII–Roche, our previous technique, werecompared on 50 patient samples.

RESULTS

Repeatability: study within a single series of 6 replicates from the same sample, respectively homozygous wild-type (normal, N), heterozygous (h) and homozygous mutated (H) for each mutation: no discrepancies. Intermediaryprecision: 5 series of analyses of the same N, h and H samples for each mutation: no discrepancies. This also validatedthe study of inter-operator variability. No discrepancies were observed.Accuracy: 18 EEQ were passed during the inter-method comparison study. On 50 patient samples, the genotypes arefor C282Y: 25N, 16h, 9H and for H63D: 27N, 18h, 5H. No discrepancies were observed. Performance monitoring viacomparison with other methods will be carried out through participation in the EEQ.

CONCLUSIONS

The LAMP Human Hemochromatosis KIT (LaCAR MDx) meets the requirements of the laboratory. The method has manyadvantages: very short time, on whole blood, easy to use, robust and low cost. The melting curve analysis is easy.Positive and negative internal quality controls validate the run and check for reagent quality and contamination.


Genetic diseases

T074

PREVALENCE AND PHENOTYPE, OF THE MOST FREQUENT BRCA1/BRCA2 MUTATIONS, RELATED TO THE HEREDITARYBREAST AND OVARIAN CANCER SYNDROME, IN FAMILIES FROM MURCIA (SOUTH-EAST OF SPAIN)

L. Rosado Jimenez 3, Y. Terkemani Mestre 3, R. Garcia Hernandez 2, M. Zafra Poves 1, A. Garcia Aliaga 3, M. Exposito

Garcia 3, M.D. Sarabia Meseguer 3, J.L. Alonso Romero 2, F. Ayala De La Peña 1, J.A. Noguera Velasco 3, F. Ruiz Espejo 31Genetic Counselling Unit, Department of Oncology, Morales Meseguer University General Hospital, Murcia, Spain2Genetic Counselling Unit, Department of Oncology, Virgen de la Arrixaca University Clinical Hospital, Murcia, Spain3Genomics Laboratory, Department of Clinical Analysis, Virgen de la Arrixaca University Clinical Hospital, Murcia, Spain

BACKGROUND-AIM

Hereditary Breast and Ovarian Cancer (HBOC) syndrome, which shows an autosomal dominant inheritance pattern, ismainly related to germline BRCA1/BRCA2 mutations. While in some regions, a wide variety of different mutations inboth genes is pictured, in others, specific mutations in BRCA1/BRCA2 have been reported with high frequency. Thisstudy describes which are the most recurrent BRCA1/BRCA2 mutations in the Murcian population (Spain) and theassociated phenotype.

METHODS

From April 2007 to June 2021, a genetic study was carried out in 2577 index cases (ICs) of families from Murcia (south-eastern Spain) with HBOC. The ICs were selected based on the clinical criteria recommended by the SEOM (SpanishMedical Oncology Society). BRCA1/BRCA2 variants detected by Sanger or Next Generation Sequencing technologieswere clinically classified according to databases (ClinVar and HGMD) and consulted bibliography. The ICs, whichpresented the most common BRCA1/BRCA2 mutations, were selected from the total of carriers, and, after that, agenotype/phenotype study was carried out.

RESULTS

Clinical diagnosis was confirmed in 10% of studied patients (252/2577), and 87 of them presented a recurrent BRCA1/BRCA2 mutation (35%).The most frequent mutations in BRCA1, with 39% of the total BRCA1 carriers, were: the c.68_69del, the c.212+1G>A,and the c.5123C>A. Three mutations (exon 2 deletion, c.3264dupT and c.9117G>A) were also identified, with 30% ofthe BRCA2 carriers. These recurrent mutations have been previously described in the Spanish population. In particular,the exon 2 deletion has only been found in Murcian and Catalan families.The most frequently observed phenotype in the 87 ICs was breast cancer (54%). Ductal carcinoma (85%) and triple-negative (67%) had the highest percentage in BRCA1 carriers, while ductal carcinoma (89%) and luminal A (61%) inBRCA2 ones, including in both genes a total of 16 bilateral BC cases (20%). Ovarian cancer had a percentage of 24%.

CONCLUSIONS

The high frequency of two mutations, c.68_69del and c.5123C>A, suggests the presence of Sephardic and AshkenaziJewish ancestors among the Murcian population.This study did not find evidence for phenotypic differences between patients, with a recurrent mutation, and BRCA1/BRCA2 carriers.


Genetic diseases

T075

PREIMPLANTATION, PRENATAL AND POSTNATAL DIAGNOSIS OF CONFINED PLACENTA MOSAICISM: A CASE REPORT

C. Laffitte 2, E. Mansilla 1, F. Garcia-Santiago 1, S. Arjona 2, A. Saez-Benito 21INGEMM, Hospital Universitario La Paz, Madrid2UG Laboratorios, Hospital Universitario Puerta del Mar, Cádiz

BACKGROUND-AIM

Mosaicism is defined as the existence of two or more cell lines with different chromosomal endowment in the sameindividual. In prenatal diagnosis, Confined Placenta Mosaicism (CPM) is considered when this mosaicism is found onlyin the placenta but not in the foetus. It happens when there is an abnormal chromosome division in cells that aredestined for the placenta, during embryo division. It can also occur after chromosomal rescue of an initial trisomy,generating two cell lines: one trisomic and one euploid.

METHODS

We present the case of a pregnant woman by homologous IVF through transfer of an embryo diagnosedpreimplantationally with mosaicism in chromosome 3 due to the lack of an alternative. In addition, after the combinedscreening of the first trimester she showed a high risk of chromosome 21 trisomy. A prenatal genetic study wastherefore performed by amniocentesis at 15 weeks' gestation with karyotyping, Quantitative Fluorescence PolymeraseChain Reaction (QF-PCR) and Comparative Genomic Hybridisation array (a-CGH).

RESULTS

The study of fluid amniotic dismissed both anomalies in the foetus. After birth, the same genetic study was carriedout on the placental tissue, confirming a placental confined mosaicism of chromosome 3 of 10-15%.Preimplantation analysis of the embryo showed mosaicism on chromosome 3, in cells that would later give rise to theplacenta. Prenatal analysis of amniotic fluid ruled out its presence in the foetus, and postnatal analysis of placentaltissue confirmed CPM, resulting in the birth of an unaffected baby.

CONCLUSIONS

The presence of MPC can have clinical repercussions in pregnancy, due to compromised placental growth and thusfoetal development. Furthermore, in the case of chromosomal rescue, Uniparental Disomy (UPD) may occur in thefoetus. Therefore, after the transfer of an embryo with suspected mosaicism, it should always be studied and even ifthe study in fluid amniotic is normal, close monitoring of the pregnancy should be carried out in case of the possibilityof CPM.


Genetic diseases

T076

PREVALENCE OF MUTATIONS OF GILBERT SYNDROME AMONG GEORGIANS AND OTHER TRANSCAUCASIANS

N. Abramishvili 2, M. Barnovi 2, A. Chania 2, T. Dzagania 2, N. Jashiashvili 2, N. Kankia 2, G. Kvantaliani 2, N. Machavariani2, D. Metreveli 2, T. Phirtskhalaishvili 2, T. Ramishvili 2, I. Rtskhiladze 2, T. Sarishvili 2, E. Shatirishvili 2, A. Tavadze 2, L.

Zangurashvili 2, M. Post 1, D. Steinberger Prof. 11bio.logis Zentrum für Humangenetik, Frankfurt am Main, Germany2Medical Center Mrcheveli, Tbilisi, Georgia

BACKGROUND-AIM

Gilbert syndrome (GS) is a common autosomal dominant hereditary condition with incomplete penetrance andcharacterized by intermittent unconjugated hyperbilirubinemia in the absence of hepatocellular disease or hemolysis.It is associated with the reduced activity of uridine diphosphate glucuronosyltransferase (UGT-1A) in liver. The mostcommon genotype of Gilbert syndrome is the homozygous polymorphism A(TA)7TAA in the promoter of the gene forUDP-glucuronosyltransferase 1A1 (UGT1A1), which is a TA insertion into the promoter designated UGT1A1*28. Theprevalence of the mutant gene in European countries reaches 35–40 %, in certain ethnic groups in Africa more than 50%, in Asian countries it is slightly lower (16–33 %). The prevalence of GS in Georgia and other Transcaucasian countrieshas not been studied before.The Aim of our study was to evaluate the distribution of gene frequency in non-selected patientsof this ethnic group.

METHODS

We retrospectively analyzed data of PGS research from Medical Center Mrcheveli, in 99 patients from Georgiansand other Ethnical Caucasians. None from them were clinically GS suspected. The following data were collected:Gender, Age, Gene dispositions in each patient. Genetic study was performed in Bio.logis Zentrum für Humangenetik,within the test of PGS, using the Competitive Allele Specific PCR (KASP)-Technology by Laboratory of the GovernmentChemist (LGC) Genomics, Fragment analysis on the AB Prism sequencer. The methods applied detect simple bi-allelicSNPs (single nucleotide polymorphism), insertions, deletions, and duplications. Assignment of a genotype (referencesequence, alteration/variant or *-allele) is based on results at the positions investigated which are listed for eachanalysis.

RESULTS

We evaluated results of total analyzed 99 patients: 55 male, 44 female, mean Age 27 years (0-60 year). UGT-1Agene variant c.-40-13TA(6_7)TAA was found in the 80 patients (80.8). 51 patients (51,5%): 26 male, 25 female wereheterozygous, 19 patients (19,2%): 15 male, 4 female were homozygous. 29 of them were wildtype.

CONCLUSIONS

Based on these results, we can conclude that the incidence of GS mutations is very high in Georgians andTranscaucasians and its prevalence is higher compared to Europeans and Asians, more similar to certain ethnic Groupsin Africa.


Genetic diseases

T077

MOLECULAR CHARACTERIZATION OF PATIENTS WITH ALPHA-1-ANTITRYPSIN DEFICIENCY AND INCONSISTENTRESULTS BY ALLELE-SPECIFIC STUDY OF ALLELES S AND Z.

M.Á. Martínez Gallego 1, C. Prior De Castro 2, A. Jalvo Sánchez 2, R. De Sancho Martín 2, S. Rodríguez Novoa 2, R. Gómez

Rioja 1, R. Rodríguez 2, M. Fernández Elvira 2, R. Mena 2, C. Gómez-González 21Medicine Laboratory, Hospital Universitario La Paz, Madrid.2Molecular Genetic Laboratory, INGEMM, Hospital Universitario La Paz, Madrid.

BACKGROUND-AIM

Alpha-1-antitrypsin (AAT) deficiency (AATD) is a hereditary disorder caused by mutations in SERPINA1 gene, whichresults in the synthesis and secretion of defective AAT whose levels are genotype specific. AATD is associated withhepatic and pulmonary disease and exposure to some environmental factors, such as tobacco smoke and alcohol, canaffect its severity.Protease inhibitor (PI) M is the normal allele, while the two most frequent deficient alleles are PI*S and PI*Z. Otherrare variants have also been described.The aim of this study is to carry out the molecular analysis of SERPINA1 gene to verify whether discrepancies foundbetween decreased serum AAT levels and the obtained genotype by standardized molecular screening of S/Z allelesmay be due to rare variants.

METHODS

A retrospective evaluation of 192 patients with AATD from a Spanish hospital was undertaken. They underwent anallele-specific study of S/Z alleles through TaqMan quantitative PCR.Discrepancies between serum AAT levels and the genotype determined by S/Z alleles molecular study were evaluatedaccording to both Gene Review and SEPAR AAT reference intervals.The five exons of the SERPINA1 gene were analyzed by Sanger sequencing in the patients with discrepant results.

RESULTS

Examination of 192 patients showed that 80.7% of them (n=155) are according to both Gene Review and SEPARreference intervals for each genotype, while 18.7% (n=36) are not and they are suspicious of having a rare variant.Of these 36 patients, 50% are heterozygous MS, 36.1% are homozygous MM and 13.8% are heterozygous MZ. In themolecular analysis, 50% showed rare variants: p.Phe76del (PI*MMalton/MPalermo) (38.9%), p.Asp280Val (PI*PLowell)(16.7%), p.Arg63Cys (PI*I) (16.7%), p.Pro393Leu (PI*MHeerlen) (11.1%), c.-5+2dupT (PI*Q0Madrid) (5.5%), p.Lys283Ter(PI*Q0Cairo) (5.5%) and p.Pro393Ser (PI*MWurzburg) (5.5%).

CONCLUSIONS

In the screening of S/Z alleles, 80.7% of patients had consistent genotype results.In discordant cases between AAT levels and S/Z genotype, it is suitable to expand the molecular study of the gene asrare variants don’t seem to be so rare and its identification can provide useful clinical information.The most frequent rare variant found was p.Phe76del.The clinical presentation of AATD depends on the genotype and environmental factors, therefore the accuratemolecular study of SERPINA1 gene would improve the diagnosis and management of AATD carriers, by the earlyrestriction of tabaco smoke and alcohol use, among others.


Genetic diseases

T078

FAMILIAL CASE OF PRIMARY BILATERAL MACRONODULAR ADRENAL HYPERPLASIA

R.M. Maria Jose 1, V.S. Gema M 1, R.R. Marta 11ANÁLISIS CLÍNICOS, HOSPITAL DE RIOTINTO, HUELVA, SPAIN

BACKGROUND-AIM

Primary macronodular adrenal hyperplasia (PMAH) is a rare cause of Cushing's syndrome (CS). Also known as ACTH-independent macronodular adrenal hyperplasia, it is characterized by the presence of functional adrenal macronodulesand increased cortisol production. It usually has a long and insidious prediagnostic course and diagnosis is usually laterwhen the patient presents CS. Although most of cases identified are sporadic, some familial cases have been described.Therefore, in presence of bilateral adrenal nodules, it is important to exclude a possible genetic origin, which couldresult an earlier identification and better management of this pathology.

METHODS

We present the case of patient with incidental finding of bilateral adrenal masses in abdominal computed tomography(CT) images. Clinical data were extracted from the Digital Clinical History of the Andalusian Health Service Diraya.Abdominal magnetic nuclear resonance with gadolinium showed multiple bilateral non-functioning adrenal adenomasand neuroradiological study showed no alterations in the pituitary region.It was determined the following Laboratory tests: basal serum cortisol, urinary free cortisol levels(Electrochemiluminescence, Cobas e-411, Roche Diagnostics) and plasma levels adrenocorticotropic hormone (ACTH)(Electrochemiluminescence, Roche Elecsys system). Physicians required Sanger sequencing analysis of ARMC5 gene.

RESULTS

A 45-years-old woman with no history of previous diseases or relevant family history with bilateral adrenal nodulesas radiological finding. There were no symptoms of hypercortisolism or typical cushingoid habitus in physicalexamination.Sanger sequencing analysis identified the c.2162T>C p.Leu721Pro germline variant in ARMC5 gene (16p11.2) inheterozygosis (NM_00121288767.1), described as variant of uncertain significance (VUS). The family segregation studyidentified the same variant in two paternal uncles (60 and 62 years old), a paternal cousin (44 years old) and thepatient's daughter (14 years old). Her only brother (46 years) does not have the variant and her father died at 61yearsold of myocardial infarction.Abdominal CT study in family members showed bilateral adrenal nodules in the cousin and paternal uncles, and the14-year-old daughter and the patient's brother showed no alterations.A progressive increase in nodule size is observed during follow-up and three years later, she presented autonomiccortisol secretion and symptoms of hypercortisolism (arterial hypertension, fatigue, headache, muscle weakness,weight gain and hyperglycemia). Laboratory data showed elevated basal serum cortisol (607 µg/dl [6.2-19.4]) and 24-hour urine free cortisol (1108 nmol/24h [100-379]), no circadian rhythm and no dexamethasone suppression (cortisolafter administration of dexamethasone 0.1 mg 20.9 µg/dl) and undetectable ACTH levels (<1.6 pg/ml [4.7-48.8]).Finally, a right adrenalectomy was performed and histopathological study confirmed diagnosis of macronodularhyperplasia. Three months after the intervention, cortisol and ACTH levels normalized.

CONCLUSIONS

In the present case, the result of the genetic study in the index case did not confirm the diagnosis of PMAH becausethe identified variant c.2162T>C p.(Leu721Pro) in ARMC5 gene has not been described as pathogenic. However, thefamilial segregation study shows the pathogenicity of the variant. Further studies would be necessary to determinethe genotype-phenotype correlation of the disease and the penetrance of the mutation in affected family members.


Genetic diseases

T079

MOLECULAR CHARACTERIZATION OF HIGH-GRADE SEROUS OVARIAN CANCER USING MULTIGENE PANEL TESTING

Á. García Aliaga 3, L. Rosado Jiménez 3, Y. Terkemani Mestre 3, P. Sánchez Henarejos 2, M.D. Sarabia Meseguer 3, Á. Aliaga

Baño 3, M. Expósito García 3, M. Marin Vera 2, J.A. Macías Cerrolaza 1, J.A. Noguera Velasco 3, F. Ruiz Espejo 31Genetic Counselling Unit, Department of Oncology, Morales Meseguer University General Hospital, Murcia, Spain2Genetic Counselling Unit, Department of Oncology, Virgen de la Arrixaca University General Hospital, Murcia, Spain3Genomics Laboratory, Department of Clinical Analysis, Virgen de la Arrixaca University General Hospital, Murcia, Spain

BACKGROUND-AIM

Ovarian cancer is the gynaecological cancer with the highest mortality, with high-grade serous ovarian cancer (HGSOC)representing 75% of this pathology, characterized by having an aggressive course and being diagnosed in advancedstages. HGSOC is evidenced to be genetically unstable and approximately 50% of these tumors have some homologousrecombination deficiency (HRD), including BRCA1 and BRCA2 germline mutations. This study aims to determinethe diagnostic yield and the contribution of each gene in HGSOC using a multigene panel testing according to therecommendations of the National Comprehensive Cancer Network (NCCN) guide.

METHODS

From January 2018 to May 2021, a total of 70 patients selected in the Genetic Counselling Units from Murcia (Spain)according to the clinical criteria of selection of the Spanish Medical Oncology Society were studied.Genomic DNA was extracted from peripheral blood samples using the Maxwell Buffy Coat DNA kit (Promega). Moreover,a panel of 50 susceptibility genes in hereditary cancer, using the Hereditary OncoKitDx kit (Imegen), was used toperform the multigene panel testing. The DNA library was analysed by massive parallel sequencing in the MiSeqsequencer (Illumina). The bioinformatic study was carried out using Data Genomics software (Imegen), using a filter of20 genes related to hereditary breast and ovarian cancer (HBOC) according to the recommendations. All the pathogenicvariants (PV) were confirmed by Sanger sequencing.The study of the characterization of the variants was done with Human Gene Mutation Data base and ClinVar amongothers. Clinical histories were consulted on Agora Plus.

RESULTS

Of the 70 patients studied, 17 patients were carriers of a pathogenic variant related to HBOC, implying a diagnosticyield of 24.3%. Whereas 65% of the pathogenic variants were detected in BRCA1 (24%) and BRCA2 (41%), 35% of theremaining ones were detected in the genes: ATM, BRIP1, EPCAM, PALB2, PMS2, NF1 and STK11.Regarding the molecular characterization of the variants 53% were frameshift type, 23% missense, 12% synonymous,6% splicing, 6% gene rearrangement and no nonsense variants were detected.HGSOC was the most represented phenotype (82%), and it was detected in all PV carriers. Breast cancer (BC) + HGSOCphenotype (12%) was linked to BRCA2 and ATM mutations. Brugada syndrome (BrS) + HGSOC phenotype (6%) waslinked to an EPCAM mutation.

CONCLUSIONS

The genetic study of HGSOC using a panel of genes allows increasing the diagnostic yield more successfully than theanalysis of BRCA1/BRCA2 genes. It is also capable of detecting mutations in HRD involved genes. Furthermore, it candetect different cancer syndromes that share the same phenotype.


Genetic diseases

T080

IDENTIFICATION OF E92K MUTATION FOR THE FIRST TIME IN A MILDLY AFFECTED CYSTIC FIBROSIS TUNISIANPATIENT

S. Hadj Fredj 1, C. Sahli 1, Y. Amri 1, R. Othmani 1, R. Dabboubi 1, M. Othmani 1, F. Ouali 1, M. Khemiri 2, T. Messaoud 11Biochemistry and Molecular Biology Laboratory (LR00SP03), Children’s Hospital Bechir Hamza, Tunis, Tunisia2Pediatric department - Children’s Hospital Bechir Hamza, Tunis, Tunisia

BACKGROUND-AIM

Cystic fibrosis (CF) is the most common inherited recessive disorder in Caucasian populations but is uncommonin African populations. To date, more than 2000 sequence variants have been reported in the cystic fibrosistransmembrane conductance regulator (CFTR) gene but the spectrum and frequencies of these mutations changeaccording to the geographic and ethnic origin. In the present study, we report a rare c.274 G-->A (p.Glu92Lys, E92K)mutation in Cystic fibrosis Tunisian patient with a mild phenotype.

METHODS

Our study was carried out on a two month old female cystic fibrosis patient from a consanguineous marriage witha positive sweat test by pilocarpine iontophoresis (Exsudose technique). All 27 coding exons and their intron-exonjunctions of the CFTR gene were analyzed by direct sequencing.

RESULTS

Molecular study allowed us to identify for the first time in Tunisia the rare mutation c.274 G-->A (p.Glu92Lys, E92K)in a homozygous state in our patient touching the first transmembrane domain of the CFTR gene. This mutation wasfirstly described in a Spanish CF patient from Andalucia in heterozygous state presenting mild clinical manifestations.In the present study, the clinical features observed in our patient with a chronic cough without lung infections andpancreatic insufficiency was associated with a less severe missense mutation linked to residual CFTR function.

CONCLUSIONS

A wide spectrum of molecular defects occurs in the CFTR gene may be associated with a severe or mild phenotypedepending on the type of mutations and its localization. Determination of the common CFTR mutations in a specificpopulation allows confirming the clinical diagnosis and facilitates the development of a rapid and accurate assayfor prenatal diagnosis. Functional studies and bioinformatics analysis of this mutation help to better understandphenotype/genotype correlation.


Genetic diseases

T081

THE PREVALENCE, AUTOIMMUNE AND GENETIC MARKERS OF LATENT AUTOIMMUNE DIABETES OF ADULTS IN THECOASTAL AREA /SYRIA

A. Alfarwi 11Al Andalus University for Medical Sciences

BACKGROUND-AIM

The aims of our study are to assess the frequency, clinical characteristics, islet β-cell autoimmune markers ( ICA, GADA,IA-2, and IAA) , and HLA-DQB1 association of latent autoimmune diabetes of adults (LADA) in the diabetic center ofLatakia/ Syria.

METHODS

Based on glutamic acid decarboxylase autoantibodies positivity , a population of 254 type 2 diabetics, males andfemales aged 35 to 75 years, were subdivided and studied in terms of the clinical and laboratory characteristics. Also, asubgroup of patients (n=70) with c-peptide ≤1.2 ng/ml were studied in terms of the frequency of islet β-cell autoimmunemarkers ( ICA, GADA, IA-2, and IAA) . Moreover, LADA (35-75 years) and type 1 diabetics (4-33 years) were evaluatedin terms of susceptible and protective DQB1 genotypes in comparison with normal control.

RESULTS

GADAs were positive (GADA+) in 45 diabetic patients versus 209 type 2 diabetics with GADA negative (GADA-). Inboth subgroups, GADA+ and GADA-, no significant differences were observed in terms of anthropometric and clinicalfeatures except for BMI which was significantly lower in GADA+ subgroup (27.6±4.8 vs. 29.8±5.9; P= 0.02). Significantpoor glycemic control was detected in terms of fasting blood sugar (FBS) (221.6±77.9 vs 182±66.7; P=0.001), glucosuria(60% vs. 41.6%; P=0.025) , and ketonuria (22.2% vs. 3.8%; P<0.0001) in LADA patients (GADA+) versus type 2 diabetics(GADA-). By subdividing the studied sample into tertiles of type 2 (GADA- <5 IU/ml) , GADA+ ≤50 IU/ml, and GADA+ >50IU/ml diabetics, the tertile with high GADA titers (>50 IU/ml) presented significantly low BMI (P=0.012) and c-peptidelevels (P<0.002) in comparison with type 2 and GADA≤ 50 IU/ml tertiles. Based on the positivity of at least one of theislet β-cell autoantibodies (ICA, GADA, IA-2, or IAA) LADA was identified. A subgroup of type 2 diabetic pateints (n=70)with c-peptide ≤1.2 ng/ml were studied and patients with positive IAA who were treated with insulin were excluded.The majority of patients (53 out of 70 patients) diagnosed as having autoimmune diabetes based on the positivityof ICA+ (sensitivity 94%; n=50), IAA+ (sensitivity 37%; n=20), GADA+ (sensitivity 22%; n =12) and/or IA2+ (sensitivity4%; n=2). Both GADA+ and IA2+ patients were ICA+ positive as well. In IAA+ positive patients (n= 20), 3 subjects werediagnosed as having LADA based on only IAA positivity. Therefore, the frequency of positivity of both ICA+ and IAA+ covered all the studied group of LADA. As far as the HLA-DQB1 genotypes association with LADA is concerned , ourresults showed a significant increase in HLA-DQB1 *0302/x (x= any other associated DQB1 allele) susceptible genotypein comparison with normal control group (56.25 vs. 7.1 %; p=0.007). In classic type 1 diabetics, the frequency of highrisk genotypes *0201/x (79.2 vs. 7.1%; p= < 0.0001), *0302/x (58.3 vs. 7.1%; p=0.002), and *0201/*0302 (45.8 vs. 0%;p=0.003) was significantly detected in comparison with the normal control group. The significance of *0601/x as aprotective genotype was negatively associated with classic type 1 subjects but not with LADA patients.

CONCLUSIONS

Overall the prevalence of LADA was 17.7% in the studied type 2 diabetics. LADA patients showed similar laboratory andclinical features as type 2 diabetics with the exception of low BMI levels and poor glycemic control. Diagnostically, bothICA and IAA almost have 100% diagnostic value in identifying LADA in type 2 diabetics with c-peptide ≤1.2 ng/ml. Thesignificance of HLA-DQB1 genotypes associated with *0201 and *0302 susceptible alleles and *0601 protective onewas confirmed in classic type 1 diabetes group and only *0302 susceptible allele was significantly increased in LADA.


Genetic diseases

T082

ANALYSIS OF ATXN2 CAG-REPEATS IN AMYOTROPHIC LATERAL SCLEROSIS (ALS) IN SPANISH PATIENTS

L. Martínez Figueras 1, M. Arriba Domènech 3, E. Llorente Martín 3, A. Bravo Gómez 3, I. Catalina Álvarez 4, R. Muñoz-

Pacheco Román 3, M.D. García González 3, J. Guillén Reyes 3, J.L. Muñoz Blanco 2, B. Ezquieta Zubicaray 11Instituto Investigación Sanitaria Gregorio Marañón and Laboratorio Diagnóstico Molecular, Servicio de Bioquímica.Hospital General Universitario Gregorio Marañón, Madrid2Instituto Investigación Sanitaria Gregorio Marañón and Unidad ELA-Neuromuscular, Servicio de Neurología. HospitalGeneral Universitario Gregorio Marañón, Madrid3Laboratorio Diagnóstico Molecular, Servicio de Bioquímica. Hospital General Universitario Gregorio Marañón, Madrid4Unidad ELA-Neuromuscular, Servicio de Neurología. Hospital General Universitario Gregorio Marañón, Madrid

BACKGROUND-AIM

ALS is a neurodegenerative disease of upper and lower motor neurons. About 5-10% of patients are familial cases(fALS). Over the years, some genes (such as SOD1 or C9orf72) have been related to ALS but its origin and risk factorsremain largely unknown.Ataxin-2 (ATXN2) presents polyglutamine (polyQ) expansions, encoded by CAG-repeats at ATXN2 exon 1. In the lastfew years, some studies have reported the presence of intermediate-length repeats (27-33) associated with increasedrisk of ALS.Aim: to determine the ATXN2-alleles distribution in our population and compare the intermediate-length repeatsalleles frequency (CAG repeats ≥27) in ALS patients vs control cases.

METHODS

DNA from 71 controls and 74 ALS cases was extracted from peripheral blood. The group of “controls” was composed byanonymized DNA samples from patients for who a TTR (transthyretin) gene sequencing was performed due to a PNP(polyneuropathy)-suspicion. We performed polymerase chain reaction (PCR) to determine the length of normal andexpanded alleles at ATXN2 (forward primer was marked with the 6FAM-fluorophore). PCR products were resolved in acapillary electrophoresis on an ABI 3730xl sequencer. Size analysis was determined using GeneMapper v4.0 software(Applied Biosystem).

RESULTS

Most subjects enrolled in this study (cases and controls) were homozygous for 22 CAG repeats (74.7% in controls,73.1% in ALS patients) or heterozygous for 22/23 repeats (15.5% in controls, and 20.9% in cases) at ATXN2. Four controlsamples (5.6%) harboured an intermediate-length polyQ expansion compared to three ALS patients (4.1%). 27 CAG-repeats alleles were found in five (3 controls, 2 ALS patients) and 28 and 30 CAG-repeats in one control and one patient,respectively.

CONCLUSIONS

As previously reported, 22/22 and 22/23 genotypes were the most frequent. However, no significant differences in thedistribution of ≥27CAG repeats between ALS patients and our control group were observed.The percentage of “control cases” carrying pathological alleles was higher than that reported previously (1.8%). Thesesubjects were not diagnosed with ALS but presented with other neurological symptoms, which might be related to theresults obtained.Further studies are required to define how intermediate length polyQ expansions contribute to ALS pathogenesis.


Genetic diseases

T083

MOLECULAR CHARACTERIZATION OF A NOVEL VARIANT IN THE MITOCHONDRIAL COMPLEX I NDUFB11 GENEASSOCIATED WITH NEONATAL LETHAL CARDIOMYOPATHY.

G. Amate-García 1, M.J. Ballesta-Martínez 2, P. Serrano-Lorenzo 1, R. Garrido 1, A. González-Quintana 1, A. Blázquez 1, C.

Ugalde 1, J.C. Rubio 1, J. Arena 1, I. García-Consuegra 1, M. Morán 1, E. Guillén-Navarro 2, M.Á. Martín 11Mitochondrial Diseases Laboratory, Biochemistry deparment. Hospital Universitario ‘12 de Octubre’. Instituto deInvestigación Hospital Universitario ‘12 de Octubre’ (imas12), Madrid, Spain. Center for Biomedical Network Research onRare Diseases (CIBERER)2Section of Medical Genetics, Paediatrics department. Hospital Clínico Universitario Virgen de la Arrixaca, IMIB, UMU,Murcia, Spain. CIBERER-ISCIII

BACKGROUND-AIM

The X-linked NDUFB11 gene encodes a complex I (CI) structural subunit of the mitochondrial OXPHOS system. Wereport a neonatal patient exitus at 48 hours diagnosed with hypertrophic cardiomyopathy (HCM), lactic acidosis andisolated CI muscle deficiency, and the molecular and functional characterization of a new variant in NDUFB11.

METHODS

Genetic analysis was performed with a specific Next-Generation Sequencing (NGS) panel- OXPHOS panel- and Xchromosome inactivation (XCI) was analysed by HUMARA test. Molecular analysis were carried out in skeletal muscleand myocardium by qRT-PCR, Western-blot (WB), Blue Native-Polyacrylamide Gel Electrophoresis (BN-PAGE) and CI in-gel activity (CI-IGA).

RESULTS

The family history revealed fatal heart disease and an adult maternal aunt with mild HCM. We prioritized c.338G>Avariant in hemizygosis state in the NDUFB11 gene, predicting the amino acid substitution p.(Arg113Lys), located in thelast nuclei base of exon 2 of the canonical transcript, and classified as probably pathogenic according to ACMG/AMPcriteria. In the asymptomatic mother and maternal aunt with MHC, XCI revealed a biased pattern towards the mutatedallele. cDNA analysis showed increased levels of alternative transcript (492 bp) and absence of the canonical transcript(462 bp). Tissue analysis of OXPHOS proteins by WB showed a lack of NDUFB11 protein and other CI structural subunitsin myocardium, and a decrease of these in skeletal muscle. The study of supercomplexes (SCs) by BN-PAGE showed adecrease in SC I+III2+IV1-2 (respirasome), isolated CI, increase in SC III2+IV, and accumulation of complex IV dimer.Likewise, a decrease in CI activity was observed by CI-IGA in myocardium.

CONCLUSIONS

The cDNA study showed a splicing alteration which allowed us to redefine the predicted molecular effect of thec.338G>A-NDUFB11 variant. OXPHOS protein analysis demonstrated a deleterious effect of this variant in OXPHOSassembly/stability.


Genetic diseases

T084

EVALUATION OF THE PERFORMANCE OF THE PROMEGA MSI-LMR KIT IN THE DETECTION OF MICROSATELLITEINSTABILITY IN LYNCH SYNDROME

I. Hidalgo Mayoral 3, A. Almeida Santiago 2, B. Hidalgo Calero 3, J.M. Sánchez-Zapardiel 3, I. Rey Prieto 3, V. Carrero

Blázquez 3, I. González Martínez 3, I. Salces 1, J. Díaz-Tasende 1, L. Robles 41Consulta de alto riesgo de Digestivo, Servicio de Digestivo, Hospital 12 de octubre2Fundación para la Investigación Biomédica del Hospital Universitario 12 de Octubre3Laboratorio de Cáncer Hereditario, Servicio de Bioquímica Clínica, Hospital 12 de Octubre4Unidad de Cáncer Familiar, Servicio de Oncología Médica, Hospital 12 de Octubre

BACKGROUND-AIM

Lynch syndrome is characterized by alterations in genes involved in the MMR system (Mismatch Repair). Theinactivation of this system leads to a characteristic molecular phenotype: microsatellite instability (MSI).One of the most common approaches for the detection of MSI is the fluorescent PCR analysis system based on theuse of quasi-monomorphic markers. The objective of the study is to evaluate the performance of a panel composedof four classic MSI markers with four additional mononucleotide markers with greater repeat length (BAT-52, BAT-56,BAT-59 and BAT-60).

METHODS

We selected a cohort of 31 individuals with suspected Lynch Syndrome. They all showed abnormality of the MMR proteinexpression on immunohistochemistry but a previous study of stable microsatellites (MSI Analysis System, v1.2).Healthy tissue and tumour tissue samples (colorectal and/or extracolonic) were processed in parallel by fluorescent PCRusing the Promega MSI-LMR Kit. Fragment analysis was performed by capillary electrophoresis (Applied Biosystems®3110xL Genetic Analyzer). Microsatellite instability was established on the number of altered markers: ≥2 MSI-H (high),1 MSI-L (low), and 0 MSS (stable microsatellites).

RESULTS

23% of the cases were reclassified: 4/26 MSS as MSI by the new kit (two as MSI-L and two as MSI-H) and 4/4 MSI-Las MSI-H.5.7% of the cases could not be evaluated because there was no amplification of the largest markers. These onescorresponded to the oldest samples.

CONCLUSIONS

The LMR kit shows higher sensitivity than the MSI Analysis System, v1.2 in the detection of MSI. It may be usefulin patients with abnormalities in MMR protein expression on immunohistochemistry that do not manifests MSI withthe previous panel. Although additional studies are necessary, this technique is more dependent on the quality of thesample and it has potential for other extracolonic approaches.


Genetic diseases

T085

STUDY OF THE GENOTYPE-PHENOTYPE CORRELATION IN A COHORT OF 1204 PATIENTS WITH FAMILIALADENOMATOUS POLYPOSIS IN A THIRD LEVEL HOSPITAL

A. Almeida Santiago 2, I. Hidalgo Mayoral 8, J.M. Sánchez-Zapardiel 8, M. De Miguel Reyes 8, D. Rueda Fernández 8, S.

Tapial 2, F. Sainz De Baranda 8, D. Marrupe 5, M. Soria 4, J.d.D. García Díaz 6, C. Lacambra 7, S. Hernando 3, L. Robles 9, J.

Díaz-Tasende 1, I. Salces 1, I. González Martínez 8, B. Hidalgo Calero 81Consulta de alto riesgo de Digestivo, Servicio de Digestivo, Hospital 12 de octubre2Fundación para la Investigación Biomédica del Hospital Universitario 12 de Octubre3Hospital Universitario de Alcorcón4Hospital Universitario de Getafe5Hospital Universitario de Móstoles6Hospital Universitario Príncipe de Asturias7Hospital Universitario Severo Ochoa8Laboratorio de Cáncer Hereditario, Servicio de Bioquímica Clínica, Hospital 12 de Octubre9Unidad de Cáncer Familiar, Servicio de Oncología Médica, Hospital 12 de Octubre

BACKGROUND-AIM

Familial adenomatous polyposis is a genetic condition characterized by multiple adenomatous polyps distributedthroughout the entire colon that increase the risk of developing colorectal cancer. Clinical presentation is variable,distinguishing between: familial adenomatous polyposis (FAP-APC gene) and polymerase proofreading-associatedpolyposis (PPAP- POLE/POLD1 genes) with an autosomal dominant inheritance pattern, MUTYH-associated polyposis(MAP) and NTHL1-associated polyposis (NAP) with an autosomal recessive inheritance pattern.The present work has been conducted with the aim of reviewing the mutational spectrum and the genotype-phenotypecorrelation of the set of genes previously proposed.

METHODS

We selected a cohort of 1204 patients diagnosed with adenomatous polyposis syndrome referred from 2008 to thepresent. The methodological approach was based on the combination of the techniques of high resolution melting(HRM), NGS and/or Sanger sequencing and MLPA.

RESULTS

Deleterious variants were detected in 168 patients (13.9%): 63 cases in APC (5.2%), 100 cases in MUTYH (8.3%, of which55% had biallelic mutations), 4 cases in POLE (0.3%) and 1 case in POLD1 (0.1%).The results obtained correlate the gene with the number of adenomas and the age of debut. There is a predominanceof missense variants in the MUTYH gene compared to a predominance of truncating variants in the APC gene. APCgene presents more severe phenotypes in variants with large rearrangements or variants located in the MCR region(mutation cluster region).

CONCLUSIONS

Our results are consistent with the genotype-phenotype correlations described in the literature. Knowledge ofmutational frequencies and genotype-phenotype correlations allow the implementation of management guidelinesadjusted to our population.


clin chem lab med 2021; 59, special suppl, pp s94 s835

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