A case of 49,XXXYY followed-up from infancy to adulthood with review of literature.

49,XXXYY is an extremely rare sex chromosomal aneuploidy (SCA), with only seven cases reported worldwide to date. Among these cases, only three have been documented into adulthood. Moreover, no cases of 49,XXXYY have been reported in Japan. This SCA has been identified in two scenarios: in vitro fertilization and abortion. Similar to 47,XXY, this aneuploidy is a type of Klinefelter syndrome. Aneuploidy of the X chromosome can lead to various progressive complications due to excess X chromosomes. Herein, we present the case of a Japanese man with 49,XXXYY. He exhibited developmental delays and external genitalia abnormalities since early infancy but was not closely monitored for these symptoms until the age of 3 years old. At that time, a chromosome test revealed his karyotype to be 49,XXXYY. Subsequent examinations were conducted due to various symptoms, including delayed motor development, intellectual disability, facial dysmorphisms, forearm deformities, hip dysplasia, cryptorchidism, micropenis, primary hypogonadism, and essential tremor. Since reaching puberty, he has undergone testosterone replacement therapy for primary hypogonadism, experiencing no complications related to androgen deficiency to date. He has maintained normal lipid and glucose metabolism, as well as bone density, for a prolonged period. There are no other reports on the long-term effects of testosterone treatment for the SCA. Appropriate testosterone replacement therapy is recommended for individuals with 49,XXXYY to prevent complications. This report will contribute to an enhanced understanding of the 49,XXXYY phenotype, aiding in the diagnosis, treatment, and genetic counseling of future cases.

Prevalence of germline GATA2 and SAMD9/9L variants in paediatric haematological disorders with monosomy 7.

Monosomy 7 (-7) occurs in various types of paediatric myeloid disorders and has a poor prognosis. Recent studies have demonstrated that patients with germline gain-of-function SAMD9/9L variants and loss-of-function GATA2 variants are prone to developing myelodysplastic syndrome (MDS) associated with -7. However, the prevalence of the genetic variants among paediatric haematologic disorders with -7 is unknown. The present study screened germline variants of GATA2 and SAMD9/9L in 25 patients with various types of paediatric haematological disorders associated with -7. The diagnoses of the 25 patients included MDS (n = 10), acute myeloid leukaemia (AML) and myeloid sarcomas (n = 9), juvenile myelomonocytic leukaemia (n = 3) and other disorders (n = 3). Seven patients with a germline pathogenic GATA2 variant were found. For SAMD9/9L screening, next-generation sequencing was used to detect low-abundance variants and found four novel germline variants. Functional analysis revealed that three out of the four variants showed growth-restricting capacity in vitro and thus, were judged to be pathogenic. Cases with GATA2 mutation tended to be older, compared to those with SAMD9/9L mutations. In conclusion, GATA2 and SAMD9/9L were sequenced in 25 patients with paediatric haematologic disorders associated with -7, and 40% of them were found to have some pathogenic germline variants in the three genes.

Unbalanced Y;7 Translocation between Two Low-Similarity Sequences Leading to SRY-Positive 45,X Testicular Disorders of Sex Development.

Unbalanced translocations of Y-chromosomal fragments harboring the sex-determining region Y gene (SRY) to the X chromosome or an autosome result in 46,XX and 45,X testicular disorders of sex development (DSD), respectively. Of these, Y;autosome translocation is an extremely rare condition. Here, we identified a 20-year-old man with a 45,X,t(Y;7)(q11.21;q35) karyotype, who exhibited unilateral cryptorchidism, small testis, intellectual disability, and various congenital anomalies. The fusion junction of the translocation was blunt, and the breakpoint-flanking regions shared only 50% similarity. These results indicate that Y;autosome translocations can occur between 2 low-similarity sequences, probably via nonhomologous end joining. Furthermore, translocations of a Ypterq11.21 fragment to 7q35 likely result in normal or only mildly impaired male-type sexual development, along with various clinical features of 7q deletion syndrome, although their effects on adult testicular function remain to be studied.

DNA Methylation Status of SHOX-Flanking CpG Islands in Healthy Individuals and Short Stature Patients with Pseudoautosomal Copy Number Variations.

SHOX resides in the short arm pseudoautosomal region (PAR1) of the sex chromosomes and escapes X inactivation. SHOX haploinsufficiency underlies idiopathic short stature (ISS) and Leri-Weill dyschondrosteosis (LWD). A substantial percentage of cases with SHOX haploinsufficiency arise from pseudoautosomal copy number variations (CNVs) involving putative enhancer regions of SHOX. Our previous study using peripheral blood samples showed that some CpG dinucleotides adjacent to SHOX exon 1 were hypomethylated in a healthy woman and methylated in a woman with gross X chromosomal rearrangements. However, it remains unknown whether submicroscopic pseudoautosomal CNVs cause aberrant DNA methylation of SHOX-flanking CpG islands. In this study, we examined the DNA methylation status of SHOX-flanking CpG islands in 50 healthy individuals and 10 ISS/LWD patients with pseudoautosomal CNVs. In silico analysis detected 3 CpG islands within the 20-kb region from the translation start site of SHOX. Pyrosequencing and bisulfite sequencing of genomic DNA samples revealed that these CpG islands were barely methylated in peripheral blood cells and cultured chondrocytes of healthy individuals, as well as in peripheral blood cells of ISS/LWD patients with pseudoautosomal CNVs. These results, in conjunction with our previous findings, indicate that the DNA methylation status of SHOX-flanking CpG islands can be affected by gross X-chromosomal abnormalities, but not by submicroscopic CNVs in PAR1. Such CNVs likely disturb SHOX expression through DNA methylation-independent mechanisms, which need to be determined in future studies.

SHOX far-downstream copy-number variations involving cis-regulatory nucleotide variants in two sisters with Leri-Weill dyschondrosteosis.

SHOX haploinsufficiency leading to Leri-Weill dyschondrosteosis (LWD) and idiopathic short stature typically results from intragenic mutations or copy-number variations (CNVs) involving SHOX and/or its putative enhancer regions that are distributed in the genomic interval between 400 kb and 840 kb from Xpter/Ypter. Here, we report two sisters with LWD, who carried a deletion in the far-downstream region of SHOX. The 0.62 Mb deletion contained 50 single nucleotide polymorphisms (SNPs) and short insertions and deletions (indels), whose genotypes were linked to SHOX expression levels in the Genotype-Tissue Expression portal. Notably, most of these SNPs/indels accumulated within a ~20 kb interval that was positioned ~900 kb away from Xpter/Ypter. These SNPs/indels showed similar minor allele frequencies, indicating that they reside within a haplotype block. The ~20 kb interval was not evolutionarily conserved; however, it was associated with the previously determined peak of chromosome conformation capture profiling (4C)-seq. Importantly, the deletion in the present cases partially overlapped with CNVs of three previous cases with skeletal deformity and/or short stature. The results indicate that far-downstream CNVs constitute rare genetic causes of SHOX haploinsufficiency. These CNVs possibly impair SHOX expression through copy-number changes of a human-specific cis-regulatory haplotype block. This notion awaits further validation.

Two patients with MIRAGE syndrome lacking haematological features: role of somatic second-site reversion SAMD9 mutations.

BACKGROUND: Myelodysplasia, infection, restriction of growth, adrenal hypoplasia, genital phenotypes and enteropathy (MIRAGE) syndrome is a recently described congenital disorder caused by heterozygous SAMD9 mutations. The phenotypic spectrum of the syndrome remains to be elucidated. METHODS AND RESULTS: We describe two unrelated patients who showed manifestations compatible with MIRAGE syndrome, with the exception of haematological features. Leucocyte genomic DNA samples were analysed with next-generation sequencing and Sanger sequencing, revealing the patients to have two de novoSAMD9 mutations on the same allele (patient 1 p.[Gln695*; Ala722Glu] and patient 2 p.[Gln39*; Asp769Gly]). In patient 1, p.Gln695* was absent in genomic DNA extracted from hair follicles, implying that the non-sense mutation was acquired somatically. In patient 2, with the 46,XX karyotype, skewed X chromosome inactivation pattern was found in leucocyte DNA, suggesting monoclonality of cells in the haematopoietic system. In vitro expression experiments confirmed the growth-restricting capacity of the two missense mutant SAMD9 proteins that is a characteristic of MIRAGE-associated SAMD9 mutations. CONCLUSIONS: Acquisition of a somatic nonsense SAMD9 mutation in the cells of the haematopoietic system might revert the cellular growth repression caused by the germline SAMD9 mutations (ie, second-site reversion mutations). Unexpected lack of haematological features in the two patients would be explained by the reversion mutations.

A novel SAMD9 mutation causing MIRAGE syndrome: An expansion and review of phenotype, dysmorphology, and natural history.

Germline gain-of-function variants in SAMD9 have been associated with a high risk of mortality and a newly recognized constellation of symptoms described by the acronym MIRAGE: Myelodysplasia, Infection, Restriction of growth, Adrenal insufficiency, Genital phenotypes, and Enteropathy. Here, we describe two additional patients currently living with the syndrome, including one patient with a novel de novo variant for which we provide functional data supporting its pathogenicity. We discuss features of dysmorphology, contrasting with previously described patients as well as drawing attention to additional clinical features, dysautonomia and hearing loss that have not previously been reported. We detail both patients' courses following diagnosis, with attention to treatment plans and recommended specialist care. Our patients are the oldest known with arginine-substituting amino acid variants, and we conclude that early diagnosis and multidisciplinary management may positively impact outcomes for this vulnerable group of patients.

Xp22.31 Microdeletion due to Microhomology-Mediated Break-Induced Replication in a Boy with Contiguous Gene Deletion Syndrome.

The Xp22.31 region is characterized by a low frequency of interspersed repeats and a low GC content. Submicroscopic deletions at Xp22.31 involving STS and ANOS1 (alias KAL1) underlie X-linked ichthyosis and Kallmann syndrome, respectively. Of the known microdeletions at Xp22.31, a common approximately 1.5-Mb deletion encompassing STS was ascribed to nonallelic homologous recombination, while 2 ANOS1-containing deletions were attributed to nonhomologous end-joining. However, the genomic bases of other microdeletions within the Xp22.31 region remain to be elucidated. Here, we identified a 2,735,696-bp deletion encompassing STS and ANOS1 in a boy with X-linked ichthyosis and Kallmann syndrome. The breakpoints of the deletion were located within Alu repeats and shared 2-bp microhomology. The fusion junction was not associated with nucleotide stretches, and the breakpoint-flanking regions harbored no palindromes or noncanonical DNA motifs. These results indicate that microhomology-mediated break-induced replication (MMBIR) can cause deletions at Xp22.31, resulting in contiguous gene deletion syndrome. It appears that interspersed repeats without other known rearrangement-inducing DNA features or high GC contents are sufficient to stimulate MMBIR at Xp22.31.

SOX2 nonsense mutation in a patient clinically diagnosed with non-syndromic hypogonadotropic hypogonadism.

Hypogonadotropic hypogonadism (HH) is a genetically heterogeneous condition that occurs either as an isolated disorder or as a component of congenital malformation syndromes. SOX2 is a causative gene of syndromic HH characterized by anophthalmia, microphthalmia, or coloboma and other neurological defects such as epilepsy. To date, the causal relationship between SOX2 abnormalities and non-syndromic HH remains speculative. Here, we identified a nonsense mutation of SOX2 in a male patient clinically diagnosed with non-syndromic HH. The patient had epilepsy but no additional clinical features. Ophthalmological examination revealed no abnormalities except for decreased thickness of the retinal nerve fiber layer. Audiometry showed mild sensorineural hearing impairment of both ears. Hormonal evaluation suggested isolated gonadotropin deficiency. Next-generation sequencing-based mutation screening of 13 major causative genes for HH identified a p.Lys35∗ mutation in SOX2 and excluded pathogenic mutations in other tested genes. The p.Lys35∗ mutation appeared to encode a non-functioning SOX2 protein that lacks 283 of 317 amino acids. The SOX2 mutation was absent in the maternal DNA sample, while a paternal sample was unavailable for sequence analysis. These results expand the clinical consequences of SOX2 haploinsufficiency to include non-syndromic HH. Systematic mutation screening using a next-generation sequencer and detailed evaluation of nonspecific ocular/neurological features may help identify SOX2 mutation-positive individuals among HH patients.

Complex X-Chromosomal Rearrangements in Two Women with Ovarian Dysfunction: Implications of Chromothripsis/Chromoanasynthesis-Dependent and -Independent Origins of Complex Genomic Alterations.

Our current understanding of the phenotypic consequences and the molecular basis of germline complex chromosomal rearrangements remains fragmentary. Here, we report the clinical and molecular characteristics of 2 women with germline complex X-chromosomal rearrangements. Patient 1 presented with nonsyndromic ovarian dysfunction and hyperthyroidism; patient 2 exhibited various Turner syndrome- associated symptoms including ovarian dysfunction, short stature, and autoimmune hypothyroidism. The genomic abnormalities of the patients were characterized by array-based comparative genomic hybridization, high-resolution karyotyping, microsatellite genotyping, X-inactivation analysis, and bisulfite sequencing. Patient 1 carried a rearrangement of unknown parental origin with a 46,X,der(X)(pter→ p22.1::p11.23→q24::q21.3→q24::p11.4→pter) karyotype, indicative of a catastrophic chromosomal reconstruction due to chromothripsis/chromoanasynthesis. Patient 2 had a paternally derived isochromosome with a 46,X,der(X)(pter→ p22.31::q22.1→q10::q10→q22.1::p22.31→pter) karyotype, which likely resulted from 2 independent, sequential events. Both patients showed completely skewed X inactivation. CpG sites at Xp22.3 were hypermethylated in patient 2. The results indicate that germline complex X-chromosomal rearrangements underlie nonsyndromic ovarian dysfunction and Turner syndrome. Disease-causative mechanisms of these rearrangements likely include aberrant DNA methylation, in addition to X-chromosomal mispairing and haploinsufficiency of genes escaping X inactivation. Notably, our data imply that germline complex X-chromosomal rearrangements are created through both chromothripsis/chromoanasynthesis-dependent and -independent processes.

Systematic molecular analyses of SHOX in Japanese patients with idiopathic short stature and Leri-Weill dyschondrosteosis.

The etiology of idiopathic short stature (ISS) and Leri-Weill dyschondrosteosis (LWD) in European patients is known to include SHOX mutations and copy-number variations (CNVs) involving SHOX and/or the highly evolutionarily conserved non-coding DNA elements (CNEs) flanking the gene. However, the frequency and types of SHOX abnormalities in non-European patients and the clinical importance of mutations in the CNEs remains to be clarified. Here, we performed systematic molecular analyses of SHOX for 328 Japanese patients with ISS or LWD. SHOX abnormalities accounted for 3.8% of ISS and 50% of LWD cases. CNVs around SHOX were identified in 16 cases, although the ~47 kb deletion frequently reported in European patients was absent in our cases. Probably damaging mutations and benign/silent substitutions were detected in four cases, respectively. Although CNE-linked substitutions were detected in 15 cases, most of them affected poorly conserved nucleotides and were shared by unaffected individuals. These results suggest that the frequency and mutation spectrum of SHOX abnormalities are comparable between Asian and European patients, with the exception of a European-specific downstream deletion. Furthermore, this study highlights the clinical importance and genetic heterogeneity of the SHOX-flanking CNVs, and indicates a limited clinical significance of point mutations in the CNEs.

Functional variants in a TTTG microsatellite on 15q26.1 cause familial nonautoimmune thyroid abnormalities.

Insufficient thyroid hormone production in newborns is referred to as congenital hypothyroidism. Multinodular goiter (MNG), characterized by an enlarged thyroid gland with multiple nodules, is usually seen in adults and is recognized as a separate disorder from congenital hypothyroidism. Here we performed a linkage analysis of a family with both nongoitrous congenital hypothyroidism and MNG and identified a signal at 15q26.1. Follow-up analyses with whole-genome sequencing and genetic screening in congenital hypothyroidism and MNG cohorts showed that changes in a noncoding TTTG microsatellite on 15q26.1 were frequently observed in congenital hypothyroidism (137 in 989) and MNG (3 in 33) compared with controls (3 in 38,722). Characterization of the noncoding variants with epigenomic data and in vitro experiments suggested that the microsatellite is located in a thyroid-specific transcriptional repressor, and its activity is disrupted by the variants. Collectively, we presented genetic evidence linking nongoitrous congenital hypothyroidism and MNG, providing unique insights into thyroid abnormalities.

(Epi)genetic and clinical characteristics in 84 patients with pseudohypoparathyroidism type 1B.

OBJECTIVE: Pseudohypoparathyroidism type 1B (PHP1B) caused by methylation defects of differentially methylated regions (DMRs) on the GNAS locus can be categorized into groups according to etiologies and methylation defect patterns of the DMRs. The aim of this study was to clarify the clinical characteristics of each group. DESIGN: Comprehensive molecular analyses consisting of methylation, copy number, and microsatellite analyses. METHODS: Eighty-four patients with PHP1B were included in this study. We classified them into 5 groups, namely, autosomal dominant inheritance-PHP1B (Group 1, G1), sporadic-PHP1B (G2), and atypical-PHP1B (G3-G5), based on the methylation defect patterns in 4 DMRs on the GNAS locus and etiologies and evaluated the clinical findings in each group and compared them among the groups. RESULTS: G2 had the youngest age and the highest serum intact parathyroid hormone levels among the 5 groups at the time of diagnosis. The most common symptoms at the time of diagnosis were tetany in G1, and seizures or loss of consciousness in G2. Albright's hereditary osteodystrophy and PHP-suggestive features were most frequently observed in the G2 proband. Nine patients had neurodevelopmental disorders (NDs) consisting of mild to borderline intellectual disability and/or developmental delay. There were no significant correlations between the average methylation ratios of 7 CpG sites in the GNAS-A/B:TSS-DMR and hormonal and biochemical findings. CONCLUSION: This study revealed the differences in some clinical characteristics, particularly clinical features, and ages at the time of diagnosis between G2 and other groups and detailed NDs observed in some patients with PHP1B.

SOX10 Mutation Screening for 117 Patients with Kallmann Syndrome.

INTRODUCTION: Kallmann syndrome (KS) is a genetically heterogeneous condition characterized by hypogonadotropic hypogonadism (HH) and olfactory dysfunction. Although SOX10, a causative gene for Waardenburg syndrome (WS) and peripheral demyelinating neuropathy, central demyelination, WS, and Hirschsprung disease (PCWH) has previously been implicated in KS, the clinical significance of SOX10 variants as the cause of KS remains uncertain. PATIENTS AND METHODS: A total of 117 patients with KS underwent mutation screening of SOX10 and 14 other causative genes for KS/HH. Rare SOX10 variants were subjected to in silico and in vitro analyses. We also examined clinical data of the patients and their parents with SOX10 variants. RESULTS: Sequence analysis identified 2 heterozygous variants of SOX10 (c.1225G > T, p.Gly409* and c.475C > T, p.Arg159Trp) in patients 1-3, as well as in the parents of patients 1 and 3. The variants were assessed as pathogenic/likely pathogenic, according to the American College of Medical Genomics guidelines. Both variants lacked in vitro transactivating activity for the MITF promoter and exerted no dominant-negative effects. Patients 1-3 carried no pathogenic variants in other genes examined. The patients presented with typical KS, while such features were absent in the parents of patients 1 and 3. None of the 5 variant-positive individuals exhibited hypopigmentation, while 1 and 2 individuals exhibited complete and partial hearing loss, respectively. CONCLUSION: These results provide evidence that SOX10 haploinsufficiency accounts for a small percentage of KS cases. SOX10 haploinsufficiency is likely to be associated with a broad phenotypic spectrum, which includes KS without other clinical features of WS/PCWH.

WDR11 is another causative gene for coloboma, cardiac anomaly and growth retardation in 10q26 deletion syndrome.

10q26 deletion syndrome is caused by a rare chromosomal abnormality, and patients with this syndrome present with an extensive and heterogeneous phenotypic spectrum. Several genes, such as EMX2 and FGFR2, were identified as the cause genital anomalies and facial dysmorphism in 10q26 deletion syndrome. However, the critical region for 10q26 deletion syndrome is not determined and the precise relationships between the causative genes and the phenotypes are still controversial. WD repeat domain 11 (WDR11), located at 10q25-26, was recently identified as a causative gene in hypogonadotropic hypogonadism, but other clinical phenotypes caused by WDR11 variants have not been identified. In this study, we have identified a WDR11 missense mutation, NM_018117.11: c.2108G > A; p.(Arg703Gln); ClinVar accession SCV000852064, in a two-year-old boy with severe growth retardation, ventricular septal defect, and coloboma symptoms. The case suggests that WDR11 is partially responsible for the clinical features of 10q26 deletion syndrome and provides novel insights into the pathophysiology of this syndrome.

A case report and literature review of monoallelic mutation of GHR.

Background Monoallelic mutations of GHR have been described in idiopathic short stature (ISS), although the significance of these remain unclear. We report a case of ISS with novel monoallelic S219L mutation of GHR and discuss the possible significance of monoallelic GHR mutation in ISS. Case presentation The proband, a 13.9-year-old Japanese boy, had severe short stature (-3.8 standard deviation [SD]). Serum insulin-like growth factor (IGF)-I level and growth hormone (GH) secretion was normal. His parents were nonconsanguineous and had normal stature. Genetic analyses revealed a novel monoallelic missense variation in exon 7 of GHR (S219L). The proband's mother had the same variation. S219L might be the novel mutation judging from there being no registration of it as a single-nucleotide polymorphism (SNP) in any database, evolutional conservation of Ser219, in silico analyses, and computational molecular visualization analysis. Furthermore, a review of the literature showed that the median height of missense mutation carriers of GHR was relatively low. Conclusions We propose the possibility that monoallelic mutation of GHR increases the susceptibility to short stature.

MIRAGE syndrome with recurrent pneumonia probably associated with gastroesophageal reflux and achalasia: A case report.

Aspiration pneumonia is a common complication of myelodysplasia, infection, restriction of growth, adrenal hypoplasia, genital phenotypes, and enteropathy (MIRAGE) syndrome. However, the detailed clinical course of aspiration pneumonia in neonates and infants diagnosed with this disorder remains unclear. We report a case of a 2-yr-old girl diagnosed with MIRAGE syndrome during the early neonatal period. The patient developed 3 episodes of aspiration pneumonia until 4 mo of age, and this complication was attributed to esophageal hypoperistalsis secondary to achalasia and gastroesophageal reflux. Enteral feeding via a duodenal tube effectively prevented further episodes of aspiration pneumonia in this patient.