Novel and recurrent variants in PAX6 in four patients with ocular phenotypes from Southeast Asia.

Aniridia is an autosomal dominant condition characterized by the complete or partial absence of the iris, often with additional presentations such as foveal hypoplasia, nystagmus, cataract, glaucoma and other ocular abnormalities. Most cases are caused by heterozygous mutations in the paired box 6 gene (PAX6), which codes for a transcription factor that regulates eye development. Four patients from our hospital who presented with ocular phenotypes were recruited for research sequencing with informed consent. Sanger sequencing of PAX6 coding exons or exome sequencing was performed on genomic DNA from venous blood samples. Variants in PAX6 were identified in the four patients. Two variants are recurrent single-nucleotide substitutions - one is a substitution found in a patient with bilateral aniridia, whereas the other is a splice variant in a patient with nystagmus and neuroblastoma. The other two variants are novel and found in two patients with isolated aniridia. Both are small duplications that are predicted to lead to premature termination. For the recurrent variants, the comparison of phenotypes for patients with identical variants would shed light on the mechanisms of pathogenesis, and the discovery of two novel variants expands the spectrum of PAX6 mutations.

Novel Variants and Clinical Characteristics of 16 Patients from Southeast Asia with Genetic Variants in Neurofibromin-1.

Neurofibromatosis type 1 (NF1) is one of the most common inherited disorders. It is caused by mutations in the neurofibromin-1 gene ( NF1 ) and affects the formation and growth of nerve tissues. More than 3,600 pathogenic variants in the NF1 gene have been identified from patients with most of the germline variants are from the Western populations. We found 16 patients (15 Chinese and 1 Asian Indian) who had heterozygous variants in NF1 through targeted next-generation sequencing. There were 15 different variants: 4 frameshift, 4 nonsense, 5 missense, and 2 splice variants. One nonsense variant and three frameshift variants had never been reported in any population or patient database. Twelve of the 16 patients met the NF1 diagnostic criteria, and each was found to have a pathogenic or likely pathogenic variant. Three different missense variants of unknown significance were discovered in the other four patients who did not meet NF1 diagnostic criteria. Our findings add four novel variants to the list of genetic mutations linked to NF1's various clinical manifestations.

Fibrous dysplasia in cardio-facio-cutaneous syndrome: A case report and review of literature.

Cardio-facio-cutaneous (CFC) syndrome (OMIM #:115150, 615278, 615279, 615280) is a rare genetic condition caused by variants in the RAS/mitogen-activated protein kinase (MAPK) signal transduction pathway. Up to 75% of cases are caused by mutations in the BRAF gene, whereas KRAS gene mutation has only been reported in <2% of cases. CFC syndrome is characterized by cardiac abnormalities, distinctive craniofacial dysmorphism, and various cutaneous abnormalities. Musculoskeletal and orthopedic manifestations are also prevalent in patients with CFC syndrome, among which the most common are skeletal deformities and joint laxities. Dysplastic bone disorders, on the other hand, have not been reported in CFC syndrome before. We report on a case of symmetrical polyostotic fibrous dysplasia (FD) in a patient with CFC syndrome with the KRAS(NM_004985.5):c.57G>C; p.Leu19Phe variant. The FDs were incidentally picked up, and patient was conservatively managed and remained asymptomatic on follow-up. The same variant was reported previously in a patient with Oculoectodermal Syndrome (OES), who developed polyostotic non-ossifying fibroma (NOF). This case explores FD as a possible new clinical feature of CFC syndrome, and when linked to the historical case of OES, explores whether the KRAS(NM_004985.5):c.57G>C; p.Leu19Phe mutation may potentially contribute to the development of dysplastic bone lesions in patients with this particular mutation.

A novel NSDHL variant in CHILD syndrome with gastrointestinal manifestations and localized skin involvement.

BACKGROUND: CHILD syndrome is an X-linked dominant disorder associated with pathogenic mutations in the NSDHL gene. The condition is predominantly found in females as it is lethal in males. Most cases present at birth with extensive unilateral ichthyosiform erythroderma involving the trunk and limbs. Milder and less extensive presentations have been reported, leading to misdiagnosis especially during early childhood. METHODS AND RESULTS: We report an adult female of Malay ancestry who presented with minimal skin and limb involvement. She was only diagnosed in adulthood when she presented with gastrointestinal symptoms and worsening of skin manifestations. The clinical diagnosis was suspected after a combination of clinical, pathological and immunohistochemistry correlation, and molecularly confirmed with the discovery of a frameshift variant in NSDHL. The novel variant was inherited from her mother who had some linear hypopigmented patches over the medial aspects of both her arms and right forearm. CONCLUSION: We uncovered a novel frameshift variant associated with presentations that cast a new light on the clinical features of CHILD syndrome.

Epidermolytic ichthyosis in a child and systematized epidermolytic nevi in the mosaic parent associated with a KRT1 variant.

Epidermolytic ichthyosis and epidermolytic nevi share the same histopathological features of epidermolytic hyperkeratosis, characterized by distinctive vacuolar degeneration and hypergranulosis of the superficial epidermis. Both are caused by pathogenic variants in either of two keratin genes KRT1or KRT10, with epidermolytic ichthyosis presenting as a generalized phenotype and epidermolytic nevi presenting as a mosaic phenotype. We report a boy who presented as epidermolytic ichthyosis, with diffuse erythema, superficial erosions and flaccid blisters at birth progressing to generalized ichthyosis. He was found to have inherited a novel KRT1 variant from his mother who presented with extensive epidermolytic nevi or mosaic form of epidermolytic ichthyosis, with extensive, linear and Blaschkoid verrucous, hyperkeratotic plaques over the trunk and limbs. This case highlights the importance of recognising post-zygotic mosaicism which might be transmitted to a child, and the different presentations for germline and mosaic carriers.

Biallelic loss-of-function variants in WDR11 are associated with microcephaly and intellectual disability.

Heterozygous missense variants in the WD repeat domain 11 (WDR11) gene are associated with hypogonadotropic hypogonadism in humans. In contrast, knockout of both alleles of Wdr11 in mice results in a more severe phenotype with growth and developmental delay, features of holoprosencephaly, heart defects and reproductive disorders. Similar developmental defects known to be associated with aberrant hedgehog signaling and ciliogenesis have been found in zebrafish after Wdr11 knockdown. We here report biallelic loss-of-function variants in the WDR11 gene in six patients from three independent families with intellectual disability, microcephaly and short stature. The findings suggest that biallelic WDR11 variants in humans result in an overlapping but milder phenotype compared to Wdr11-deficient animals. However, the observed human phenotype differs significantly from dominantly inherited variants leading to hypogonadotropic hypogonadism, suggesting that recessive WDR11 variants result in a clinically distinct entity.

Case Report: Mosaicism of a novel nonsense variant in the neurofibromin gene underlies a mosaic generalized NF1 phenotype.

Neurofibromatosis 1 (NF1) is a neurocutaneous syndrome characterized by multiple café-au-lait macules, cutaneous neurofibromas or plexiform neurofibromas, iris Lisch nodules, axillary and inguinal freckling. Mosaicism in NF1 can either present as a generalized disease, or in a localized (segmental) manner. Mosaic generalized NF1 may have presentations that are similar to generalized NF1 or have a milder phenotype and hence may be under-recognised in clinical practice. We report a nonsense mutation in the NF1 gene in a 55-year old Chinese male with the mosaic generalized phenotype. He reported noticing increasing numbers of skin-colored papules over his face, neck, back and abdomen when he was about 40 years old. From both next-generation and Sanger sequencing data, the variant appeared to be mosaic and present at about 24%. It is in exon 39 and has not been reported in any database or published literature.

Epidermolysis bullosa with pyloric atresia associated with compound heterozygous ITGB4 pathogenic variants: Minimal skin involvement but severe mucocutaneous disease.

We report a case of junctional epidermolysis bullosa with pyloric atresia (JEB-PA) with minimal skin involvement but severe protein-losing enteropathy and airway involvement. Genetic analysis revealed heterozygous mutations in the ITGB4 gene encoding integrin ß4 protein. Parental testing confirmed inheritance of frameshift variant (c.794dupC) as maternal and splice site variant (c.1608C>T/p.Cys536Cys) as paternal. Immunofluorescence mapping of her skin revealed a subepidermal blister with decreased and frayed integrin ß4 at both the floor and the roof of the blister, while the intestinal mucosa showed complete absence of integrin ß4. We review the literature and discuss the differential expression of integrins in the skin and gastrointestinal tract, as well as the role of chronic inflammation in the pathogenesis of EB.

Innate Immune and Neuronal Genetic Markers Are Highly Predictive of Postoperative Pain and Morphine Patient-Controlled Analgesia Requirements in Indian but Not Chinese or Malay Hysterectomy Patients.

OBJECTIVE: Pain severity and opioid requirements in the postoperative period show substantial and clinically significant inter-patient variation due mainly to factors such as age, surgery type, and duration. Genetic factors have not been adequately assessed except for the neuronal OPRM1 rs1799971 and COMT rs4680, whereas the contribution of innate immune signaling pathway genetics has seldom been investigated. SETTING: Hospital surgical ward. SUBJECTS: Women (107 Indian, 184 Malay, and 750 Han Chinese) undergoing total hysterectomy surgery. METHODS: Morphine consumption, preoperative pain, and postoperative pain were evaluated in relation to genetic variability comprising 19 single-nucleotide polymorphisms (SNPs) in 14 genes involved in glial activation, inflammatory signaling, and neuronal regulation, plus OPRM1 (1 SNP) and COMT (3 SNPs). RESULTS: Pre- and postoperative pain and age were associated with increased and decreased morphine consumption, respectively. In Chinese patients, only 8% of the variability in consumption could be explained by these nongenetic and genetic (BDNF, IL1B, IL6R, CRP, OPRM1, COMT, MYD88) factors. However, in Indian patients, 41% of morphine consumption variability could be explained by age (explaining <3%) and variants in OPRM1 rs1799971, CRP rs2794521, TLR4 rs4986790, IL2 rs2069762, COMT rs4818, TGFB1 rs1800469, and IL6R rs8192284 without controlling for postoperative pain. CONCLUSIONS: This is the highest known value reported for genetic contributions (38%) to morphine use in the acute postoperative pain setting. Our findings highlight the need to incorporate both genetic and nongenetic factors and consider ethnicity-dependent and nonadditive genotypic models in the assessment of factors that contribute to variability in opioid use.

Self-improving dystrophic epidermolysis bullosa: First report of clinical, molecular, and genetic characterization of five patients from Southeast Asia.

Self-improving dystrophic epidermolysis bullosa is a rare subtype of dystrophic epidermolysis bullosa (DEB) characterized by significant improvement in skin fragility within the first few years of life. Genetic inheritance has previously been reported as autosomal dominant or recessive with both forms harboring mutations in COL7A1. To date, there have been no reports of this rare clinical entity from various Southeast Asian ethnicities. Here, we describe the clinical and molecular features of five patients from the Southeast Asia region who presented with predominantly acral-distributed blisters and erosions in the first few days of life. Blistering resolved over several months, without appearance of new blisters. By immunofluorescence, intraepidermal retention of Type VII collagen was observed in all patient skin biopsies when investigated with antibody staining. Genetic analysis of four patients revealed pathogenic variants in COL7A1 which have not been previously reported. The clinical diagnosis in these rare patients is confirmed with molecular histology and genetic characterization.

Singapore Undiagnosed Disease Program: Genomic Analysis aids Diagnosis and Clinical Management.

OBJECTIVE: Use next-generation sequencing (NGS) technology to improve our diagnostic yield in patients with suspected genetic disorders in the Asian setting. DESIGN: A diagnostic study conducted between 2014 and 2019 (and ongoing) under the Singapore Undiagnosed Disease Program. Date of last analysis was 1 July 2019. SETTING: Inpatient and outpatient genetics service at two large academic centres in Singapore. PATIENTS: Inclusion criteria: patients suspected of genetic disorders, based on abnormal antenatal ultrasound, multiple congenital anomalies and developmental delay. EXCLUSION CRITERIA: patients with known genetic disorders, either after clinical assessment or investigations (such as karyotype or chromosomal microarray). INTERVENTIONS: Use of NGS technology-whole exome sequencing (WES) or whole genome sequencing (WGS). MAIN OUTCOME MEASURES: (1) Diagnostic yield by sequencing type, (2) diagnostic yield by phenotypical categories, (3) reduction in time to diagnosis and (4) change in clinical outcomes and management. RESULTS: We demonstrate a 37.8% diagnostic yield for WES (n=172) and a 33.3% yield for WGS (n=24). The yield was higher when sequencing was conducted on trios (40.2%), as well as for certain phenotypes (neuromuscular, 54%, and skeletal dysplasia, 50%). In addition to aiding genetic counselling in 100% of the families, a positive result led to a change in treatment in 27% of patients. CONCLUSION: Genomic sequencing is an effective method for diagnosing rare disease or previous 'undiagnosed' disease. The clinical utility of WES/WGS is seen in the shortened time to diagnosis and the discovery of novel variants. Additionally, reaching a diagnosis significantly impacts families and leads to alteration in management of these patients.

Identification of KMT2D and KDM6A variants by targeted sequencing from patients with Kabuki syndrome and other congenital disorders.

Kabuki syndrome (KS) is a rare congenital disorder characterized by distinctive facies, postnatal growth deficiency, cardiac defects and skeletal anomalies. Studies have determined that pathogenic variants of the lysine-specific methyltransferase 2D (KMT2D) and lysine-specific demethylase 6A (KDM6A) genes are the major causes of KS. The two genes encode different histone-modifying enzymes that are found in the same protein complex that is critical for cell differentiation during development. Here we report the results from next-generation sequencing of genomic DNA from 13 patients who had a clinical diagnosis of KS based on facial dysmorphism and other KS-specific cardinal phenotypes. Nine of the 13 patients were confirmed to be carrying heterozygous pathogenic KMT2D variants, seven of which were truncating and two were missense substitutions. Overall, we uncovered 11 novel variants - nine in KMT2D and two in KDM6A. Seven of the novel variants (all KMT2D) were likely causative of the KS phenotype. Our study expands the number of naturally occurring KMT2D and KDM6A variants. The discovery of novel pathogenic variants will add to the knowledge on disease-causing variants and the relevance of missense variants in KS.

Palmoplantar keratoderma, oral involvement, and homozygous CTSC mutation in two brothers from Cambodia.

Haim-Munk syndrome (HMS) and Papillon-Lefevre syndrome (PLS) are phenotypic variants of palmoplantar keratoderma (PPK) with progressive early-onset periodontitis and dental caries. HMS and PLS have been associated with homozygous or compound heterozygous mutations in the lysosomal protease gene Cathepsin C (CTSC). There have been only a few documented cases of CTSC mutations in patients from South-East Asia. We report the clinical findings of two Cambodian brothers who presented with diffuse, demarcated PPK with transgrediens extending to the elbows and knees, as well as pachyonychia and dental caries. Arachnodactyly and periodontitis were also found in the older brother. Next-generation sequencing unveiled a homozygous missense variant in CTSC (NM_001814.5: c.1337AC: p.(Asp446Ala)) in both brothers. Both parents were heterozygous for the variant, while an unaffected older brother was homozygous for the wild-type allele. Our study adds to the spectrum of mutations and associated clinical presentations for this rare genodermatosis.

The spectrum of genetic variants and phenotypic features of Southeast Asian patients with Noonan syndrome.

BACKGROUND: Noonan syndrome (NS) is an autosomal dominant disorder that belongs to a group of developmental disorders called RASopathies with overlapping features and multiple causative genes. The aim of the study was to identify mutations underlying this disorder in patients from Southeast Asia and characterize their clinical presentations. METHODS: Patients were identified from the hospital's Genetics clinics after assessment by attending clinical geneticists. A targeted gene panel was used for next-generation sequencing on genomic DNA extracted from the blood samples of 17 patients. RESULTS: Heterozygous missense variants were identified in 13 patients: eight were in PTPN11, three in SOS1, and one each in RIT1 and KRAS. All are known variants that have been reported in patients with NS. Of the 13 patients with identified variants, 10 had short stature, the most common feature for NS. Four of the eight patients with PTPN11 variants had atrial septal defect. Only two had pulmonary stenosis which is reported to be common for PTPN11 mutation carriers. Another two had hypertrophic cardiomyopathy, a feature which is negatively associated with PTPN11 mutations. CONCLUSIONS: Our study provides the mutation and phenotypic spectrum of NS from a new population group. The molecular testing yield of 76% is similar to other studies and shows that the targeted panel approach is useful for identifying genetic mutations in NS which has multiple causative genes. The molecular basis for the phenotypes of the remaining patients remains unknown and would need to be uncovered via sequencing of additional genes or other investigative methods.

Investigation of variants in estrogen receptor genes and perinatal depression.

OBJECTIVES: Depressive symptoms are common during pregnancy and after childbirth. Estrogen levels fluctuate greatly during the course of pregnancy and may contribute to mood instability. The first aim of this case-control study was to investigate whether variants in the two estrogen receptor genes might contribute to the genetic susceptibility to pregnancy-related depression using controls that were screened for postnatal depression. The second aim was to uncover new variants in the two estrogen receptor genes. PATIENTS AND METHODS: Our study sample comprised 554 control subjects who had Edinburgh Postnatal Depression Scale (EPDS) scores below 7 at postnatal screening, and 159 patients with clinically diagnosed pregnancy-related depression. They were genotyped for four single-nucleotide polymorphisms (SNPs) and a dinucleotide repeat in the two genes: estrogen receptor α (ESR1) and estrogen receptor ß (ESR2). Fifty-six cases with personal and/or family history of depression of psychiatric disorders were selected for resequencing of the two genes. RESULTS: There was no statistically significant association with perinatal depression for all five variants. However, there was a trend toward higher frequencies of the genotypes associated with higher risk of depression for rs2077647 and rs4986938 in the case group. From resequencing, two novel ESR1 variants were identified from two different patients. CONCLUSION: Our study that used screened controls with low EPDS scores and cases with clinically diagnosed pregnancy-related depression could not replicate the association with depression for any of the SNPs for both genotype and allele frequencies. Two novel SNPs were identified and could be further investigated in a larger sample set.

Novel mutations in the ciliopathy-associated gene CPLANE1 (C5orf42) cause OFD syndrome type VI rather than Joubert syndrome.

Mutations in CPLANE1 (previously known as C5orf42) cause Oral-Facial-Digital Syndrome type VI (OFD6) as well as milder Joubert syndrome (JS) phenotypes. Seven new cases from five unrelated families diagnosed with pure OFD6 were systematically examined. Based on the clinical manifestations of these patients and those described in the literature, we revised the diagnostic features of OFD6 and include the seven most common characteristics: 1) molar tooth sign, 2) tongue hamartoma and/or lobulated tongue, 3) additional frenula, 4) mesoaxial polydactyly of hands, 5) preaxial polydactyly of feet, 6) syndactyly and/or bifid toe, and 7) hypothalamic hamartoma. By whole or targeted exome sequencing, we identified seven novel germline recessive mutations in CPLANE1, including missense, nonsense, frameshift and canonical splice site variants, all causing OFD6 in these patients. Since CPLANE1 is also mutated in JS patients, we examined whether a genotype-phenotype correlation could be established. We gathered and compared 46 biallelic CPLANE1 mutations reported in 32 JS and 26 OFD6 patients. Since no clear correlation between paired genotypes and clinical outcomes could be determined, we concluded that patient's genetic background and gene modifiers may modify the penetrance and expressivity of CPLANE1 causal alleles. To conclude, our study provides a comprehensive view of the phenotypic range, the genetic basis and genotype-phenotype association in OFD6 and JS. The updated phenotype scoring system together with the identification of new CPLANE1 mutations will help clinicians and geneticists reach a more accurate diagnosis for JS-related disorders.