Diversity of oncopharmacogenetic profile within Spanish population.

Consensus guidelines for genotype-guided fluoropyrimidine dosing based on variation in the dihydropyrimidine dehydrogenase (DPYD) gene before treatment have been firmly established. The prior pharmacogenetic report avoids the serious toxicity that inevitably occurred in a non-negligible percentage of the treated patients. The precise description of the allelic distribution of the variants of interest in our reference populations is information of great interest for the management of the prescription of these antineoplastic drugs. We characterized the allelic distribution of the UGT1A1*28 variant (rs3064744), as well as the DPYD*2A (rs3918290) variant, c.1679T>G (rs55886062), c.2846A>T (rs67376798) and c.1129-5923C>G (rs75017182; HapB3) in series of 5251 patients who are going to receive treatment with irinotecan and fluoropyrimidines, representative of Valencian, Aragonese and Western Andalusian populations.

Spinocerebellar ataxia type 15 caused by missense variants in the ITPR1 gene.

BACKGROUND AND PURPOSE: Spinocerebellar ataxia type 15 (SCA15) is a degenerative, adult onset autosomal dominant cerebellar ataxia, caused almost exclusively by deletions in the inositol 1,4,5 triphosphate receptor type 1 (ITPR1) gene (ITPR1). ITPR1 mediates calcium release from the endoplasmic reticulum, and particularly abounds in Purkinje cells. It plays a pivotal role in excitatory and inhibitory actions on Purkinje cells, and alterations in their balance cause cerebellar dysfunction in ITPR1 knockout mice. To date, only two single missense mutations have been reported to cause SCA15. They were considered pathogenic because cosegregation occurred with disease, and haploinsufficiency was hypothesized as their pathogenic mechanism. METHODS: In this study, three Caucasian kindreds with different heterozygous missense variants in ITPR1 are reported. The main clinical manifestation was a slowly progressive gait ataxia with onset after 40 years of age, with chorea in two patients and hand tremor in another one, concordant with manifestations found in SCA15. RESULTS: The three missense variants identified in ITPR1 were c.1594G>A; p.(Ala532Thr) in Kindred A, c.56C>T; p.(Ala19Val) in Kindred B, and c.256G>A; p.(Ala86Thr) in Kindred C. Every variant was labelled as of unknown significance; however, each one cosegregated with disease and was predicted to be pathogenic by in silico tests. CONCLUSIONS: The three ITPR1 missense variants found in this study exhibited cosegregation with disease, a result that sustains their pathogenicity. Further studies are needed to confirm the role of missense mutations in SCA15.

CSF1R-related Adult-onset Leukoencephalopathy: The First Reported Case in Spain.

Colony-stimulating factor 1 receptor-related adult-onset leukoencephalopathy is a primary microgliopathy characterized by a complex phenotype, which can be easily misdiagnosed with other leukoencephalopathy and neurodegenerative diseases such as frontotemporal dementia. It is estimated to be the most common adult-onset leukodystrophy. Here, we report the case of a 67-year-old man with a history of progressive impairment of behavioral and cognitive functions, including apathy, inhibition, tendency to mutism, and deficits in complex planning skills. Neurological examination revealed pyramidalism in the lower limbs. Brain imaging showed symmetrical confluent frontal leukoencephalopathy, bilateral frontal calcifications, and thinning of the corpus callosum. The diagnosis was confirmed by the identification of a heterozygous pathogenic variant in the colony-stimulating factor 1 receptor. As far as we know, this is the first documented case in Spain. In this paper, we aim to expand on clinical features and underline the importance of brain imaging for the diagnosis of an entity that we consider to be underdiagnosed.

Incidence of Huntington disease in a northeastern Spanish region: a 13-year retrospective study at tertiary care centre.

BACKGROUND: Despite the progress in the knowledge of Huntington disease (HD) in recent years, the epidemiology continues uncertain, so the study of incidence becomes relevant. This is important since various factors (type of population, diagnostic criteria, disease-modifying factors, etc.) make these data highly variable. Therefore, the genetic diagnosis of these patients is important, since it unequivocally allows the detection of new cases. METHODS: Descriptive retrospective study with 179 individuals. Incidence of HD was calculated from the ratio of number of symptomatic cases newly diagnosed per 100,000 inhabitants per year during the period 2007-2019 in Aragon (Spain). RESULTS: 50 (27.9%) incident cases of HD (CAG repeat length ≥ 36) were identified from a total of 179 persons studied. The remaining 129/179 (72.1%) were HD negative (CAG repeat length < 36). 29 (58.0%) females and 21 (42.0%) males were confirmed as HD cases. The overall incidence was 0.648 per 100,000 patient-years. 11/50 positive HD cases (22.0%) were identified by performing a predictive test, without clinical symptoms. The minimum number of CAG repeats found was 9 and the most common CAG length among HD negative individuals was 16. CONCLUSIONS: Our incidence lied within the range reported for other Caucasian populations. Implementation of new techniques has allowed to determine the exact number of CAG repeats, which is especially important in patients with triplet expansions in an HD intermediate and/or incomplete penetrance allele, both in diagnostic, predictive and prenatal tests.

Rasopathies case report: concurrence of two pathogenic variations de novo in NF1 and KRAS genes in a patient.

BACKGROUND: Rasopathies are a group of genetic malformative syndromes including neurofibromatosis 1, Noonan, LEOPARD, Costello, cardio-facio-cutaneous, Legius, and capillary malformation-arteriovenous malformation syndromes. CASE PRESENTATION: We present a female newborn that consulted at the emergency department with refusal to eat and sleepiness. A shortened femur, thickened nucal fold and suspect for agenesis of the corpus callosum were observed in prenatal ultrasound. Her phenotype included hypertelorism, antimongoloid obliquity of the palpebral fissure, prominent forehead, long filtrum, thickened nucal fold, separated nipples, widespread thickened skinfolds and café-au-lait spots. She had a systolic murmur due to pulmonary valve stenosis. The NF1 gene testing found the pathogenic variant p.E2586X (c.7756G > T) in exon 53, not described in any international database or scientific publications yet. Also, a mutation in the Kras gene was detected (p.Val14lle), which is associated with mild Noonan phenotype. Both variations were de novo. CONCLUSIONS: Not all genes and mutations have already been discovered, so it's important to document new findings, like our patient's, to enrich and update the international database and broaden all possible knowledge about rasopathies. This is the first case to be described presenting simultaneously two mutations in Kras and NF1 genes, whose possible synergic effect regarding its pathogenicity is unknown, but could be interesting towards therapeutic alternatives.

No increase in the CTG repeat size during transmission from parent with expanded allele: false suspicion of contraction phenomenon.

Objectives: Myotonic dystrophy type 1 (DM1), also known as Steinert's disease, is a chronic, progressive and disabling multisystemic disorder with a broad spectrum of severity that arises from an autosomal-dominant expansion of the Cytosine-Thymine-Guanine (CTG) triplet repeat in the 3' untranslated region of the DMPK gene (19q13.3). Case presentation: In this study, we report the case of a family with several intergenerational expansions of the CTG repeat, with an additional case of a false suspicion of contraction phenomenon due to TP-PCR limitations. Conclusions: The meiotic instability of the (CTG)n repeats leads to genetic anticipation where increased size of DM1 mutation and a more severe phenotype have been reported in affected individuals across generations. Even if extremely rare, a decrease in the CTG repeat size during transmission from parents to child can also occur, most frequently during paternal transmissions.

Association Between VDR and CYP24A1 Polymorphisms, Atopic Dermatitis, and Biochemical Lipid and Vitamin D Profiles in Spanish Population: Case-Control Study.

BACKGROUND: Atopic dermatitis (AD) is the most prevalent inflammatory skin disorder, characterized by impaired epidermal barrier function and an altered immune response, both of which are influenced by vitamin D deficiency. Single-nucleotide polymorphisms (SNPs) in VDR and CYP24A1 have been previously associated with AD. OBJECTIVE: We sought to characterize the associations between the VDR and CYP24A1 polymorphisms and the vitamin D and lipid biochemical profile in children diagnosed with AD. METHODS: A total of 246 participants (143 patients with AD and 103 healthy controls) were enrolled in this study. Genotyping for polymorphisms in VDR (rs2239185, rs1544410, rs7975232, rs2238136, rs3782905, rs2239179, rs1540339, rs2107301, rs2239182, and rs731236) and CYP24A1 (rs2248359 and rs2296241) was performed by allele-specific polymerase chain reaction using integrated fluidic circuit technology. Serum levels of calcium, phosphorus, and vitamin D were measured, and the biochemical lipid profile was determined. RESULTS: Among VDR SNPs, rs2239182 exerted a protective effect against the development of AD, whereas rs2238136 was identified as a risk factor for AD. The GCC haplotype (rs2239185-G, rs1540339-C, and rs2238136-C) appeared to protect against the development of AD. rs2239182-CC was associated with higher 25(OH)D concentrations, whereas rs2238136-TT, rs2239185-GA, and rs2248359-TT were present in a large proportion of patients with serum vitamin D deficiency. rs2239185-AA, rs2239182-CC, and rs1540339-CC were associated with higher serum total cholesterol; rs2239182-TT was associated with lower low-density lipoprotein cholesterol; and rs2239182-TC with lower high-density lipoprotein cholesterol. Both CYP24A1 SNPs (rs2296241-AA and rs2248359-TT) were associated with higher high-density lipoprotein cholesterol levels. CONCLUSIONS: The VDR SNP rs2238136 is a risk factor for AD and other SNPs in VDR and CYP24A1, which may lead to alterations in biochemical parameters that influence the risk of AD. Our findings highlight the complex genetic basis to AD and indicate that interrelationships between different genetic factors can lead to alterations in vitamin D metabolism or lipid profiles, which in turn may influence the development of AD.

First case of very late-onset FHL2 in Spain with two variants in the PRF1 gene.

Hemophagocytic lymphohistiocytosis (HLH) is a rare but fatal disorder characterized by the proliferation and infiltration of macrophages and hyperactivated T lymphocytes that escape from the physiological control pathways and favour the existence of an environment of excessive inflammation and tissue destruction. HLH has been classified into two types: a primary or familial autosomal recessive form, caused by mutations in genes encoding proteins involved in the granule-dependent cytotoxic pathway (familial hemophagocytic lymphohistiocytosis [FHL] types 1-5); and other secondary or acquired form, generally associated with infections, malignancy, autoimmune diseases, metabolic disorders or primary immunodeficiencies. Since the first familial hemophagocytic lymphohistiocytosis-2 (FHL2) causative mutation in the PRF1 gene was described in 1999, more than 200 mutations have been identified to date. Here, we report the first case of very late-onset FHL2 in a Spanish 72-year-old female with splenomegaly, hypertriglyceridemia, hypofibrinogenemia, pancytopenia and marrow hemophagocytosis harbouring in heterozygosity two PRF1 variants proposed as causative in this study. The heterozygous mutation c.445G>A (p.Gly149Ser) identified in the exon 2 results in a missense mutation previously described as a probable pathogenic variant associated with the development of FHL2. Affecting the same exon, c.272C>T (p.Ala91Val) is the most prevalent variant of this gene. Although it was initially classified as benign, recent studies support its potential pathogenic role, considering it a variant of uncertain significance associated with a risk of developing FHL2. The genetic confirmation of FHL made possible an adequate counselling to the patient and direct relatives and provided important information for her control and follow-up.

[AZF gene microdeletions in azoospermic-oligozoospermic males]. / Microdeleciones del gen AZF en varones azoospérmicos-oligozoospérmicos.

BACKGROUND AND OBJECTIVE: The presence of microdeletions in the Y-chromosome azoospermia factor (AZF) region (YCMs) is considered the most frequent genetic cause of male infertility along with Klinefelter syndrome. The objective of this study was to investigate the frequencies and type of YCMs in infertile men in Aragon and to analyze the relationship between sex hormones, sperm count and microdeletions in them. PATIENTS AND METHODS: Retrospective descriptive study of 644 men who during 2006-2019 were screened for YCMs using YChromStrip (Operón, Spain) by PCR+reverse hybridization, spermiogram, karyotype and quantification of sex hormones. RESULTS: The frequency of YCMs was 3.88% (25/644), not being detected in any patient with mild or normospermic oligozoospermia, that is, in sperm counts higher than 5×106/mL. The group of azoospermic patients was the one that presented a higher frequency of YCMs (14.58%, 14/96). Deletions in the AZFc region were the most frequent (68%). 20% (5/25) of patients with YCMs also presented some type of karyotype abnormality that included aneuploidies, deletions, duplications and/or translocations. Sperm count was significantly lower and FSH and LH concentrations significantly higher in the group of patients with YCMs. CONCLUSIONS: YCMs screening is a key test in the diagnostic approach to male infertility. Obtaining an adequate result allows choosing suitable assisted reproduction techniques, preventing unnecessary treatments and the transmission of genetic defects to offspring.

[Allan-Herndon-Dudley syndrome: a diagnosis to rule out in any male infant with undiagnosed hypotonia]. / Síndrome de Allan-Herndon-Dudley: un diagnóstico a descartar ante todo lactante varón con hipotonía sin causa determinada.

Allan-Herndon-Dudley syndrome is a rare X-linked genetic disorder, caused by a deficiency of the monocarboxylate transporter 8 (MCT8), a specific transporter of thyroid hormones, with functions mainly at the brain level. The syndrome produces an early onset of severe neurological disorder, in which hypotonia predominates. OBJECTIVE: To present a rare case with an unexpected diagnosis, highlighting the usefulness of requesting a complete thyroid profile in every hypotonic male infant without a specific cause. CLINICAL CASE: A 10-month-old male infant with severe axial and peripheral hypotonia, global weakness with little spontaneous mobility, without head support or stable sitting. Complete metabolic and peripheral neurophysiological studies were performed. Genetic studies for spinal muscular atrophy, Prader Willi syndrome, and myotonic dystrophy were also performed. The trio exome analysis detected a probably pathogenic variant c.359C>T;p.(Ser120Phe), hemizygous in exon 1 of the SLC16A2 gene, inherited from the mother. Thyroid abnormalities as increased free triiodothyronine (T3) and thyroid-stimulating hormone (TSH), and delayed myelination were ob served. CONCLUSIONS: MCT8 deficiency should be considered in the case of the male infant with unex plained hypotonia and weakness without a determined cause. The diagnosis is guided by a thyroid profile including free T3 hormone, because it presents a characteristic thyroid profile with decreased free thyroxine (T4), increased free T3, and normal or slightly elevated TSH levels. In this case, the implementation of the trio exome analysis allows establishing an early certain diagnosis.

Initial Cerebellar Ataxia in Hereditary Adult-Onset Primary Lateral Sclerosis.

Cerebellar ataxia preceding the apparition of primary lateral sclerosis (PLS) is reported herein. Three individuals from 2 independent kindreds experienced ataxia before developing clinical signs of PLS. Disease onset was during the sixth decade or later, and an insidious onset, with progression exceeding 11 years, was observed. Pathochrony was homogenous, consisting of initial gait instability, followed by hand dysmetria 2 years later. During a 5-year follow-up, cerebellar ataxia remained the sole clinical manifestation, preceding the appearance of muscle stiffness, which progressed to a paraparesis, and then to a purely spastic quadriparesis, over 4 years; pseudobulbar dysarthria and dysphagia appeared later. At this disease stage, limb spasticity, hyperactive jaw and limb stretch reflexes, extensor plantar responses, and a spastic dysarthria were found on examination; limb dysmetria and an ataxo-spastic gait were also found. No muscle atrophy or fasciculation was observed. Among ancillary tests, electromyographic studies performed 6 years after disease onset revealed normal motor unit action potentials and absence of spontaneous activity, in 2 individuals. MRI revealed normal cerebellum and brainstem in 2 cases. Inheritance was dominant in both kindreds, and extensive genetic testing was negative. It is concluded that cerebellar ataxia preceded the appearance of a purely spastic spinobulbar syndrome (which fulfilled the clinical diagnostic criteria for PLS) during a 5-year period in 3 patients with a hereditary, adult-onset form of PLS; subsequent disease progression was equivalent to that of sporadic PLS. Further studies are needed to fully delineate the clinical and genetic spectra of adult-onset PLS.

Episodic Vestibulocerebellar Ataxia Associated with a CACNA1G Missense Variant.

Episodic vestibulocerebellar ataxias are rare diseases, frequently linked to mutations in different ion channels. Our objective in this work was to describe a kindred with episodic vestibular dysfunction and ataxia, associated with a novel CACNA1G variant. Two individuals from successive generations developed episodes of transient dizziness, gait unsteadiness, a sensation of fall triggered by head movements, headache, and cheek numbness. These were suppressed by carbamazepine (CBZ) administration in the proband, although acetazolamide and topiramate worsened instability, and amitriptyline and flunarizine did not prevent headache spells. On examination, the horizontal head impulse test (HIT) yielded saccadic responses bilaterally and was accompanied by cerebellar signs. Two additional family members were asymptomatic, with normal neurological examinations. Reduced vestibulo-ocular reflex gain values, overt and covert saccades were shown by video-assisted HIT in affected subjects. Hearing acuity was normal. Whole-exome sequencing demonstrated the heterozygous CACNA1G missense variant c.6958G>T (p.Gly2320Cys) in symptomatic individuals. It was absent in 1 unaffected member (not tested in the other asymptomatic individual) and should be considered likely pathogenic. CACNA1G encodes for the pore-forming, α1G subunit of the T-type voltage-gated calcium channel (VGCC), in which currents are transient owing to fast inactivation, and tiny, due to small conductance. Mutations in CACNA1G cause generalized absence epilepsy and adult-onset, dominantly inherited, spinocerebellar ataxia type 42. In this kindred, the aforementioned CACNA1G variant segregated with disease, which was consistent with episodic vestibulocerebellar ataxia. CBZ proved successful in bout prevention and provided symptomatic benefit in the proband, probably as a result of interaction of this drug with VGCC. Further studies are needed to fully determine the vestibular and neurological manifestations of this form of episodic vestibulocerebellar ataxia. This novel disease variant could be designated episodic vestibulocerebellar ataxia type 10.

A study of crystalluria: effectiveness of including hygienic-dietary recommendations in laboratory reports.

Objectives: To assess the effectiveness of incorporating hygienic-dietary recommendations in laboratory reports in reducing the incidence of renal colic (RC). A study was performed to compare the incidence of RC in two groups of patients who had suffered at least a crystalluria event associated with the risk of urolithiasis. Recommendations were only incorporated in the laboratory reports of one group. Methods: A retrospective observational study. The study sample was composed of patients who had at least an episode of crystalluria associated with a higher risk of urolithiasis. The laboratory reports of patients in Group A (n=1,115), treated in 2017, did not include any hygienic-dietary recommendations, whereas patients in Group B (n=1,692), treated in 2018, received hygienic-dietary recommendations through their laboratory reports. χ2 and Mann-Whitney U test were used to assess differences based on sex, age, and type of urinary crystals. Results: The incidence of RC was 2.02 times higher in group A (2.24%) than in group B (1.12%). No significant differences were observed in the incidence of RC based on the type of urinary crystal. The incidence of RC was substantially higher in patients who suffered at least an event of crystalluria associated with a higher risk for urolithiasis as compared to the general population during the same period (0.46%, consistently with the incidence rates reported in the literature). Conclusions: The incorporation of messages alerting on the risk of urolithiasis and the inclusion of hygienic-dietary recommendations in laboratory reports may be useful for reducing the incidence of RC.

A novel variant in the COL4A3 gene: etiology of Alport syndrome type 2 in a 38-year-old male with suspected hereditary kidney disease.

Objectives: Patients with Alport syndrome develop progressive kidney function deterioration, sensorineural hearing loss, and ocular abnormalities. This condition is caused by mutations in COL4A5 (X-linked inheritance), COL4A3 and COL4A4 (autosomal dominant or recessive inheritance), and encoding type IV collagen α3, α4, and α5, respectively. If left untreated, clinical symptoms progress from microscopic hematuria to proteinuria, progressive kidney failure, and end-stage kidney disease. At present, kidney transplantation is the only effective approach. Next-generation sequencing is the method of choice for the diagnosis of this condition. Case presentation: We report the case of a young man with chronic kidney disease who eventually underwent transplantation. Molecular testing made it possible to determine the etiology of his clinical symptoms and autosomal recessive Alport syndrome type 2. The patient was found to be a compound heterozygote for two missense variants (trans configuration) in the COL4A3 gene: A likely pathogenic variant c.4981C>T (p.Arg1661Cys) in exon 52 inherited from the mother (described elsewhere), and another variant of uncertain significance, c.943G>A (p.Gly315Ser), in exon 17 inherited from the father that has not been previously reported in the literature or found in relevant databases. Conclusions: Following genetic confirmation, genetic counseling was provided to the patient and his direct relatives.

Management of postanalytical processes in the clinical laboratory according to ISO 15189:2012 Standard requirements: considerations on the review, reporting and release of results.

The objective of this paper is to share some considerations about the management of postanalytical processes in relation to the review, reporting and release of test results in accordance with UNE-EN ISO 15189:2013 Standard requirements. The scope of this paper includes postanalytical activities and the personnel involved (laboratory management and staff). We describe the criteria and information required to review and validate analytical results and ensure that clear reports are sent to requesters. These criteria also guarantee that results are transcribed in a reliable way and that all necessary information is provided for the correct interpretation of results. Likewise, the requirements for the correct release of laboratory results are described, with special emphasis on the release of alarming or critical results. In some European countries, clinical laboratories are required to hold partial or full ISO 15189 accreditation, which is a global trend. Therefore, understanding ISO 15189 requirements is imperative for a progressive and more effective implementation of the Standard.

Management of post-analytical processes in the clinical laboratory according to ISO 15189:2012. Considerations about the management of clinical samples, ensuring quality of post-analytical processes, and laboratory information management.

ISO 15189:2012 establishes the requirements for clinical sample management, ensuring quality of process and laboratory information management. The accreditation authority, ENAC in Spain, established the requirements for the authorized use of the label in reports issued by accredited laboratories. These recommendations are applicable to the postanalytical processes and the professionals involved. The Standard requires laboratories to define and document the duration and conditions of sample retention. Laboratories are also required to design an internal quality control scheme to verify whether postanalytical activities attain the expected standards. Information management requirements are also established and laboratories are required to design a contingency plan to ensure the communication of laboratory results. Instructions are finally provided about the correct use of the accreditation label in laboratory reports. A range of nations and scientific societies support that clinical laboratories should be required to obtain accreditation. With ISO 15189 being the most specific standard for demonstrating technical performance, a clear understanding of its requirements is essential for proper implementation.

Effect of AGG Interruptions on FMR1 Maternal Transmissions.

There are four classes of CGG repeat alleles in the FMR1 gene: normal alleles have up to 44 repeats; patients with Fragile X Syndrome have more than 200 repeats; those between 55 and 200 CGGs are considered FMR1 premutation alleles, because they are associated with maternal expansions of the number of CGGs in the next generation and finally, alleles between 45 and 54 CGGs are called intermediate or gray zone alleles. In these last categories, the stability depends on the presence of AGG interruptions, which usually occurs between 9 and 10 CGGs. In this context, we have studied retrospectively 66 women with CGG repeats between 45 and 65, and their offspring. In total 87 transmissions were analyzed with triplet repeat primed PCR using AmplideX® FMR1 PCR (Asuragen, Austin, TX, USA) and we found that alleles with CGG repeats between 45 and 58 do not expand in the next generation except two cases with 56 repeats and 0 AGG interruptions. Furthermore, we have found four females with alleles with more than 59 CGG repeats and 2 AGG interruptions that do not expand either. Alleles from 56 CGG repeats without AGGs expand in all cases. In light of these results and those of the literature, we consider that the risk of unstable transmissions should be based on the presence or absence of AGG interruptions and not on the classical cutoffs which define each category of FMR1 alleles. The application of these results in the genetic and reproductive counseling is essential and AGG interruptions should always be studied.

A variant of the gene HARS detected in the clinical exome: etiology of a peripheral neuropathy undiagnosed for 20 years.

Objectives: Describe a case with axonal Charcot-Marie-Tooth (CMT) type 2W, a neurological disease characterized by peripheral neuropathy typically involving the lower limbs and causing gait alterations and distal sensory-motor impairment. Case presentation: We report this case, where the application of massive genetic sequencing (NGS) with clinical exome in a molecular genetics laboratory enabled to detect the presence of candidate variants of the clinic of the patient. Conclusions: The variant detected in HARS gene suggests that this variant could be causative of the symptoms of the patient, who went undiagnosed for 20 years and experienced an exacerbation of symptoms over time.