Characterization of Associated Nonclassical Phenotypes in Patients with Deletion in the WAGR Region Identified by Chromosomal Microarray: New Insights and Literature Review.

WAGR syndrome (Wilms' tumor, aniridia, genitourinary changes, and intellectual disability) is a contiguous gene deletion syndrome characterized by the joint deletion of PAX6 and WT1 genes, located in the short arm of chromosome 11. However, most deletions include other genes, leading to multiple associated phenotypes. Therefore, understanding how genes deleted together can contribute to other clinical phenotypes is still considered a challenge. In order to establish genotype-phenotype correlation in patients with interstitial deletions of the short arm of chromosome 11, we selected 17 patients with deletions identified by chromosomal microarray analysis: 4 new subjects and 13 subjects previously described in the literature with detailed clinical data. Through the analysis of deleted regions and the phenotypic changes, it was possible to suggest the contribution of specific genes to several nonclassical phenotypes, contributing to the accuracy of clinical characterization of the syndrome and emphasizing the broad phenotypic spectrum found in the patients. This study reports the first patient with a PAX6 partial deletion who does not present any eye anomaly thus opening a new set of questions about the functional activity of PAX6.

Syndromic hearing loss molecular diagnosis: Application of massive parallel sequencing.

Syndromic hearing loss accounts for approximately 30% of all cases of hearing loss due to genetic causes. Mutation screening in known genes is important because it potentially sheds light on the genetic etiology of hearing loss and helps in genetic counseling of families. In this study, we describe a customized Ion AmpliSeq Panel, specifically designed for the investigation of syndromic hearing loss. The Ion AmpliSeq Panel was customized to cover the coding sequences of 52 genes. Twenty-four patients were recruited: 17 patients with a clinical diagnosis of a known syndrome, and seven whose clinical signs did not allow identification of a syndrome. Of 24 patients sequenced, potentially causative mutations were found in nine, all of which belonged to the group with a previous clinical diagnostic and none in the group not clinically diagnosed. We were able to provide conclusive molecular diagnosis to six patients, constituting a diagnostic rate of 25% (6/24). In the group of patients with a suspected clinical diagnosis, the diagnostic rate was 35% (6/17). Of the nine different mutations identified, three are novel, and were found in patients with Waardenburg, Treacher Collins and CHARGE syndromes. Since all patients with a conclusive molecular diagnosis through this panel had a previous suspected clinical diagnosis, our results suggest that this panel was more effective in diagnosing this group of patients. Therefore, the panel demonstrated effectiveness in molecular diagnosis when compared to others in the literature, especially for patients with a defined clinical diagnosis.

Partial 1q Duplications and Associated Phenotype.

Duplications of the long arm of chromosome 1 are rare. Distal duplications are the most common and have been reported as either pure trisomy or unbalanced translocations. The paucity of cases with pure distal 1q duplications has made it difficult to delineate a partial distal trisomy 1q syndrome. Here, we report 2 patients with overlapping 1q duplications detected by G-banding. Array CGH and FISH were performed to characterize the duplicated segments, exclude the involvement of other chromosomes and determine the orientation of the duplication. Patient 1 presents with a mild phenotype and carries a 22.5-Mb 1q41q43 duplication. Patient 2 presents with a pure 1q42.13qter inverted duplication of 21.5 Mb, one of the smallest distal 1q duplications ever described and one of the few cases characterized by array CGH, thus contributing to a better characterization of distal 1q duplication syndrome.

Combined use of multiplex ligation-dependent probe amplification and automatic sequencing for identification of KAL1 defects in patients with Kallmann syndrome.

OBJECTIVE: To investigate the role of KAL1 abnormalities in Brazilian patients with Kallmann syndrome. DESIGN: In vitro experiments. SETTING: Academic medical center. PATIENT(S): One hundred fifteen Brazilian patients (98 men) with Kallmann syndrome. INTERVENTION(S): Peripheral blood leukocytes were used to obtain DNA. MAIN OUTCOME MEASURE(S): Direct sequencing and multiplex ligation-dependent probe amplification were used to identify KAL1 abnormalities. RESULT(S): We identified four KAL1 mutations (p.Met1?, p.Ala33Glyfs, p.Arg257*, and p.Trp462*) and two multiple exon deletions (exons 1-2 and 3-14) in six new male patients. Overall, 17 KAL1 defects (14.8%) were identified in the entire cohort of patients with Kallmann syndrome, including previously studied cases. KAL1-mutated patients presented with a more severe reproductive and nonreproductive phenotype (synkinesia, renal malformations, cryptorchidism, and anatomic olfactory abnormalities) in comparison with patients without KAL1 mutations. Intragenic deletions were one of the most often encountered defects (29.4%). These deletions can be missed by polymerase chain reaction (PCR) due to Yq11.2 KAL1 pseudogene (KALP) spurious amplification. CONCLUSION(S): These results indicate that intragenic multiexon deletions are one of the most frequent KAL1 abnormalities, which can be more accurately detected by multiplex ligation-dependent probe amplification. In addition, KAL1 sequencing results should be interpreted with caution, and stringency conditions of the PCR reaction should be adjusted to avoid pseudogene amplification.

Quantitative magnetic resonance imaging evaluation of the olfactory system in Kallmann syndrome: correlation with a clinical smell test.

OBJECTIVES: To measure olfactory bulbs and sulci using dedicated magnetic resonance imaging (MRI) sequences and specific measurement tools in Kallmann syndrome (KS) patients with a well-established genotype and phenotype, as well as correlate MRI findings with a clinical smell test. METHODS: MRI was performed in 21 patients with KS and 16 healthy volunteers; olfactory dysfunction was assessed using the Smell Identification Test (UPSIT), a qualitative suprathreshold olfaction test. Coronal turbo spin echo T2-weighted and volumetric T1-weighted gradient echo sequences were acquired in a 1.5T system. ImageJ software was used to obtain olfactory bulb volumes and olfactory sulcus depths and lengths. Data were analyzed with SPSS 15.0 and the Kappa index was used to evaluate the agreement between the UPSIT and MRI. RESULTS: The UPSIT showed 14 patients with anosmia and 6 with moderate hyposmia. Eighteen patients (85%) presented altered rhinencephalon structures in the MRI. Sixteen patients (76%) presented olfactory bulb aplasia (14/16 bilaterally), and these patients presented a total of 16 aplastic sulci. There was moderate agreement between the MRI quantitative evaluation and the UPSIT (overall Kappa = 0.55), but when considering the presence of aplastic bulbs and anosmia, we found almost perfect agreement (Kappa = 0.87). Three patients had normal rhinencephalon structures, including one with a KAL1 gene mutation. CONCLUSION: Olfactory bulb and sulcus aplasia were the most common findings in KS patients. We objectively demonstrated agreement between MRI findings and the smell test, especially the presence of bulb aplasia and anosmia. Therefore, our findings help ascertain MRI accuracy in the diagnosis of KS, differentiating patients with hypogonadotropic hypogonadism with an apparently normal or difficult to evaluate sense of smell.

Kallmann syndrome and mirror movements: White matter quantitative evaluation with magnetic resonance imaging.

Kallmann syndrome (KS), characterized by the association of hypogonadotropic hypogonadism and anosmia, may present many other phenotypic abnormalities, including neurologic features as involuntary movements, called mirror movements (MM). MM etiology probably involves a complex mechanism comprising corticospinal tract abnormal development associated with deficient contralateral motor cortex inhibitory system. In this study, in order to address previous hypotheses concerning MM etiology, we identified and quantified white matter (WM) alterations in 21 KS patients, comparing subjects with and without MM and 16 control subjects, using magnetization transfer ratio (MTR) and T2 relaxometry (R2). Magnetization transfer and T2 double-echo images were acquired in a 1.5 T system. MTR and R2 were calculated pixel by pixel to initially create individual maps, and then, group average maps, co-registered with MNI305 stereotaxic coordinate system. After analysis of selected regions of interest, we demonstrated areas with higher T2 relaxation time and lower MTR values in KS patients, with and without MM, differently involving corticospinal tract projection, frontal lobes and corpus callosum. Higher MTR was observed only in pyramidal decussation when compared in both groups of patients with controls. In conclusion, we demonstrated that patients with KS have altered WM areas, presenting in a different manner in patients with and without MM. These data suggest axonal loss or disorganization involving abnormal pyramidal tracts and other associative/connective areas, relating to the presence or absence of MM. We also found a different pattern of alteration in pyramidal decussation, which can represent the primary area of neuronal disarrangement.

Clinical assessment and molecular analysis of GnRHR and KAL1 genes in males with idiopathic hypogonadotrophic hypogonadism.

OBJECTIVE: The pathogenesis of idiopathic hypogonadotrophic hypogonadism (IHH) is mostly unclear. We characterized the clinical findings and molecular analysis of GnRHR and KAL1 genes in 26 Brazilian males with IHH with and without hyposmia/anosmia. Design Clinical assessment was performed for endocrine status, olfactory structure and function, renal lesion, and mirror movement. The diagnosis of Kallmann syndrome (KS) included HH and the clinical complaint of hyposmia/anosmia or decreased olfactory acuity obtained by the Smell Identification Test (SIT). We analysed GnRHR and KAL1 genes using the polymerase chain reaction (PCR) direct sequencing method. RESULTS: A variable degree of HH was observed, including various clinical abnormalities, such as cryptorchidism, hearing loss, strabismus, cleft lip/palate, high-arched palate, dental agenesis, psychiatric disorders, learning dysfunction, and bimanual synkinesia. Twenty-two out of 26 patients with IHH (85%) were classified as KS. Abnormalities of olfactory bulbs/sulci were observed in 79% of KS patients. One-third of KS patients had renal defects and 45.5% had a positive family history. GnRHR gene sequence analysis showed no mutations. KAL1 sequence analysis identified two novel missense mutations: c.1061A to G in exon 7 (N304S) and c.1583C to A in exon 10 (S478X). We also observed a 14-bp deletion within exon 11 that caused a premature termination. According to the National Center for Biotechnology Information (NCBI)-Single Nucleotide Polymorphism (SNP) database, two previously described polymorphisms (rs808119 and rs809446) were detected. CONCLUSION: KAL1 mutations accounted for 12% of KS patients. This low prevalence of KAL1 mutations indicates that other genes, such as the fibroblast growth factor receptor 1 (FGFR1) gene or other as yet undiscovered genes, epigenetic events and/or environmental factors might be involved in the aetiology and phenotypic variability of KS.