Defining The RCCX Phenotype

The RCCX gene cluster on chromosome 6 is one of the most complex regions of the human genome, and one that is likely a major player in chronic disease susceptibility. The RCCX gene region features a series of highly complex and anomalous activities, while the constituent genes in the region confer powerful effects throughout the physiology. This article is an attempt to define the RCCX phenotype from a clinical perspective, as a means to understanding which patients in the population likely suffer from complex illness, due to this highly unstable and complex genomic region. This article is largely theoretical because of the fact that RCCX genotyping has not been applied for widespread use. Credit is hereby given to Sharon Meglathery, MD for her pioneering clinical observations, which has led to the elucidation of the suspected RCCX phenotype from a clinical standpoint.

RCCX is a copy number variation (CNV) gene cluster comprised of 4 genes. Briefly these genes are:

• TNXB (Tenascin-X) – An extracellular matrix glycoprotein. Mutations of TNXB result in one form of Ehlers-Danlos Syndrome (EDS) that is associated with joint hypermobility and skin hyperelasticity. It is postulated here that genetic haploinsufficiency of TNXB is not required in order to produce a hypermobile phenotype, and that numerous adverse TNXB events will lead to hypermobility, and related complications. It is suspected that variations of the TNXB gene (or mutations within the CREB gene, or anomalous events within the chimeric TNXA pseudogene) alters how TNXB functions as a protein, and is likely a significant contributing factor to loss of normal extracellular matrix function. TNXB variations may contribute to inflammatory bowel diseases, organ prolapse in suspected RCCX phenotypes, and may play some role in neurological diseases, such as schizophrenia. Additionally, the TNXB protein regulates the function and availability of one of the most powerful cytokines and growth factors: TGFß (18). This fact has profound implications for the RCCX phenotype with respect to the function of the entire immune system, on neural growth, on stem cell differentiation, embryological development, and tissue remodeling. The transcriptional start points of the TNXB gene reside in the nearby CREB gene (28, 29). Therefore, mutations and deletions of CREB may influence how TNXB is expressed and functions.
• CYP21a2 (21-hydroxylase) – A cytochrome P450 enzyme involved in the formation of the stress hormone cortisol and the mineralcorticoid hormone aldosterone. The condition most associated with mutated CYP21a2 is CAH (congenital adrenal hyperplasia), which (in most cases) results in deficient cortisol and aldosterone. It is postulated here that due to the great variability of the CYP21a2 gene and it’s complex arrangements located within the RCCX cluster, there are more pathological processes that directly involve CYP21a2, which have yet to be discovered through scientific research. It is hypothesized that CYP21a2 variations are etiological in a wide range of neurological and psychiatric conditions. The transcriptional regulation of CYP21a2 exists within the 35th intron of the C4b gene. Therefore, mutations located in or near the genomic region of C4b may significantly affect CYP21a2 RNA and enzymatic expression.
• C4 (Complement C4) – Primary constituent of the complement immune system, an important arm of the innate immune system. C4 is integral to the lectin pathway of the complement immune system, as well as the subsequent formation of C3, C5, C6, C7 and C8. Complement C4 has also been shown to function as a key modulator of synaptic and dendritic pruning in the brain and central nervous system. Numerous associations exist between C4 variations, mutations, null alleles and deletions with respect to autoimmune disease, neurological diseases such as schizophrenia, and autism.
• STK19/RP1 – The precise function of the 4th gene within the RCCX cluster, STK19 (formerly RP1) is not known. The existing evidence based upon a two-hybrid yeast model suggests that STK19 possesses serine/threonine kinase activities, and may be involved in DNA transcription or replication (30).

CNV gene sequences such as RCCX comprise roughly 4-9% of the human genome yet are prone towards genomic instability, and increase the susceptibility to numerous diseases. In many respects, when discussing the RCCX gene cluster, many of the standard rules of genetics are challenged, as linearity simply does not apply. Even when discussing the copy number genotype itself (monomodular, bimodular, trimodular, quadrimodular), linearity does not apply, because there are far more anomalies that can occur aside from the copy number. New models of research are required in order to develop quantification of anomalous RCCX events within individual genotypes.

Genetic variability of the RCCX cluster is influenced by many factors. Some of these include:

• Copy number: Copy numbers of genes are “hot spots” in the genome that are more susceptible to genetic instability, leading to transformational changes in how genes are expressed. An individual RCCX genotype is currently defined by the modular forms present:
  • RCCX monomodular genotype
  • RCCX bimodular genotype
  • RCCX trimodular genotype
  • RCCX quadrimodular genotype

• Unequal crossover: An RCCX genotype will present with gene duplications, deletions of pseudogenes and mutations of constituent genes. The degree of unequal crossover within RCCX genotypes can directly affect transcription and translation to respective RNA and protein/enzymatic products, because unequal crossing over can cause mutations and deletions of genes. Significantly, unequal crossing over will affect offspring, and this is why RCCX presents as a significant gene cluster from an evolutionary perspective. In some cases, the relationship between the pseudogenes and the active genes creates instability due to intergenic recombination.
• Chimeric Genes: The CYP21a2 gene has a pseudogene known as CYP21aP. The TNXB gene has a pseudogene known as TNXA. The TNXB/TNXA chimera and CYP21a2/CYP21aP chimeras may lead to complicating factors, leading towards certain diseases. So far the literature on the chimeric gene relationships is painfully lacking, as the predominant disease identified with respect to RCCX chimeric genes is CAH (congenital adrenal hyperplasia). A critical future area of RCCX research should identify how chimeric genes create genomic instabilities leading to other diseases.
• The length of the C4 gene – The C4 gene within the RCCX region will vary by copy number and by length. The length of the C4 gene is defined as “short” or “long” for each genotype. Evidence from the literature suggests that both the C4 copy number and the C4 length will affect the end C4 protein level.
• Endogenous retroviral insertion within the C4 gene and retrotransposon insertion within STK19/RP1. The presence of retroviruses within genes is known to affect gene expression, and may contribute to mutations to nearby genes. The same may be true for retrotransposons, which are retroviral-like structures. The C4 gene contains the HERV-K endogenous retrovirus within its 9th intron, and STK19/RP1 contains a retrotransposon within its 4th intron (44). Numerous diseases are known to be associated with HERV-K RNA expression, including autism, cancer and neurological diseases. It has been proposed in at least one study, that the HERV-K retrovirus may confer a protective effect in type 1 diabetes (15). The presence or absence of HERV-K is believed to affect C4 protein levels, implying a role for HERV-K in RNA transcription and/or C4 protein translation.
• Intergenic recombination – The constituent RCCX pseudogenes are believed to not directly transcribe, largely because of the high number of mutations contained within each pseudogene. However, in some cases, mutations within certain genomic regions of pseudogenes can affect the active gene, leading to instability. This occurrence is believed to be the central genomic etiology of CAH (congenital adrenal hyperplasia), with the CYP21a2 gene and its mutated pseudogene CYP21AP.
• Overlapping domains – RCCX genes overlap and share domains. Shared genomic regions, mutations of shared genomic regions, and complex events have been demonstrated to occur, including the recently termed CAH-X syndrome, whereby CAH (congenital adrenal hyperplasia) and hypermobile EDS have been shown to exist concurrently in certain individuals. This is due to anomalous events occurring between shared genomic regions located between CYP21a2 and the neighboring TNXB.
• Transcriptional introns located within neighboring genes – The fact that gene transcription can be controlled by neighboring genes is a phenomenon that raises many important questions. The 35th intron of the C4b gene contains the transcriptional regulation of neighboring CYP21a2 (27). Introns located within the nearby CREB gene (not considered part of RCCX) contain the transcriptional regulation start points for TNXB (28, 29).

RCCX & The Difficulty of Studying Complex Disease

One of the major problems with scientific research with respect to chronic disease is that it attempts to isolate and study single variables and link those to a complex disease. Chronic disease does not involve singular causes, linearity or singular variables, but rather a convergence of hundreds or thousands of variables simultaneously. Scientific papers that attempt to study multiple variables, and to affect those by using a combined therapeutic approach are often rejected for publication because they are labelled as too complex. Until more sophisticated models are employed, (which study the multi-variable nature of chronic disease), chronic disease research is prone to reductionism.

This central problem with scientific doctrine is a major challenge to RCCX research. While a considerable body of literature exists with respect to various components of the RCCX region, there has yet to be a publication that recognizes how this complex gene region is involved in chronic, complex, comorbid disease. An overwhelming amount of evidence implicates this genomic region as a central player, however new models of research are vital in order to prove the existence of an RCCX comorbid phenotype.

The fact that RCCX contains a remarkable parade of complex genomic events, the cluster can be likened to a house of cards or a set of dominos. The combined effect of these complex genomic interactions creates the impression that genomic instability is affecting a vastly under-reported percentage of people in the population. The best way to initially identify who in the population suffers from some adverse RCCX genotype is by:

1. Recognizing that the phenotype is going to express comorbid disease characteristics. That is, two or more overlapping diseases. These diseases, symptoms and conditions will be associated with a dysregulation of function attributable to the expression of RCCX genes. The suspected RCCX phenotype frequently suffers from a spectrum of conditions and symptoms which are in many ways traceable back to functions of RCCX genes, and the effects these genes exert physiologically.
2. Identifying the phenotype in families who share similar disease characteristics. Copy number variation gene sequences (CNV) and the associated unequal crossover are significant genomic events which affect evolution, and create variability within the human population. These facts implicate the RCCX region as a central genomic region with respect to familial inheritance, and associated disease susceptibilities.

The RCCX spectrum phenotype is best defined by group. Depending upon inter-individualistic RCCX genomic inheritance and interactions, the phenotype will not be linearly expressed in all individuals.

Identifying The Most Likely Candidates for RCCX Involvement in Comorbid Disease

Commercial or clinical genetic testing for RCCX is not currently available. In the instance where RCCX modular, CNV genotyping would be made available, it would not reveal the incredible complexities that occur within the region.

The following section is an attempt to identify the most probable candidates for advanced RCCX genotyping, based upon the logical associations between known RCCX genes, phenotypical characteristics and that of the published literature.

It is postulated here that various RCCX genotypes will experience a divergent and wide spectrum of symptoms and disease presentations, which tend to run through families. It is important to consider that disease susceptibility is not necessarily independently related to inherited RCCX genotype alone. Various epigenetic and environmental factors will converge leading to extremely complex interactions.

From a clinical perspective, it is important to identify these traits in the individual patient, as well as to identify the presence of these traits in family members. Investigating the presence of RCCX traits in families is a critical breaking point in the identification of RCCX genotypes.

Joint Hypermobility Syndromes (JHS) and/or Skin Hyperelasticity

It is not postulated here that all joint hypermobility conditions are attributable to RCCX. However, the presence of JHS and skin hyperelasticity are two starting points of clinical analysis because variations and mutations of the TNXB gene are associated with joint hypermobility and skin hyperelasticity. Furthermore, if an individual does not have joint hypermobility, this does not disqualify them for RCCX involvement either. The opposite situation has been considered and observed, namely that individuals with a lack of joint hypermobility may have an undesirable RCCX genotype. IF hypermobility or skin hyperelasticity is observed within a given individual (or within a relative), it is critical to consider the other major RCCX traits to see the overall pattern.

Joint hypermobility implies some degree of loss of regulation of the extracellular matrix (ECM). The ECM does far more than provides structure for cells, tissues and organs. The ECM is also integral in modulating the life cycle of the cell and in maintaining cell-to-cell communication. The ECM is critical for the regulation of a number of growth factors. RCCX phenotypes may have impaired growth factor regulation. This may be particularly true for TGFß-1 and VEGF, two growth factors that directly interact with TNXB domains (18).

Autoimmune Diseases Attributable to Complement C4 Deficiency, Complement C4 Involvement, or Specific C4 Copy Number

The number of C4 copies defines the RCCX modular genotypes: monomodular, bimodular, trimodular, quadrimodular. Variations of C4 genotype and C4 protein levels will have a direct effect on:

• The innate immune system, chiefly the pathways of complement lectin, classical and alternative
• Synaptic pruning mechanisms in the CNS

Variations of the complement C4 gene, and the C4 protein are associated with several autoimmune diseases. These include:

• Lupus – studies found lupus to be one of the autoimmune diseases most associated with C4 deficiency, with one study reporting a 75% frequency (20)
• Rheumatoid arthritis (RA) – associated with C4b deficiency (21)
• Type 1 diabetes (Juvenile Diabetes) – associated with lower C4 levels as well as low RCCX copy number (1)
• Celiac disease, associated with null alleles of C4a and C4b (22)
• Juvenile dermatomyositis – associated with C4a deficiency (23)
• Grave’s disease – associated with the C4a C4b genotype known as A2B2 (24)
• Behcet’s disease – associated with higher C4 levels, and increased copy numbers of C4a (25)
• Crohn’s disease – associated with lower C4 long variations, and higher C4 short variations. In Crohn’s, higher copy numbers are associated with higher C4 protein concentrations (26)
• Myasthenia Gravis – Associated with lower C4 and C3 levels (43)

Discussion:

• The C4 protein is integral in the complement immune system
• As a participant in complement cascades, C4 is integral in the subsequent formation of Complements: C3, C3b, C5, C6, C7, C8, C9 (31)
• Deficiency of C4 will impair cell lysis and membrane attack mechanisms of the innate, complement systems and consequently increase vulnerability to pathogenic and toxic insults
• Peptides derived from C4b have shown capable of inhibiting TH1 cytokines, and it has been proposed that C4 functions to clear immune complexes and remove apoptotic cells (17).
• Two studies now have identified that complement C4 significantly influences T-regulatory cells (TREGs) as well as TGFß concentrations (16, 17). T-regulatory cells are a critical part of the immune system, which down-regulate hyperinflammatory states, leading to self tolerance. TGFß is critical for the differentiation of TREGs, and participates as an important anti-inflammatory cytokine.

Familial History of Autism or Autism Spectrum – Complement C4 & Synaptic Pruning

The association between family history of autism and autoimmune disease is established. One significant link is the C4b gene. Two separate studies found similar relationships between C4b null alleles and autism: the Egyptian study identified a 37% frequency, and the Utah/Oregon study identified a 42% frequency (3,4). Among Egyptian autistic patients who showed null alleles of the C4b gene, 40% of patients had family members who were found to have autoimmune disease, compared to 10% of controls (3). Therefore, individuals who have family members with autism diagnosis should be considered candidates for RCCX genotyping, and clinically should be considered as possible RCCX phenotypes.

Discussion:

• The C4 protein consists of two isoforms, C4a, and C4b, respectively.
• C4 has been found to be the most prevalent protein in the human brain that supports the connection between neurons (2).
• Aberrant pruning mechanisms are known to occur in autism, and autism features excess brain synapses (5).
• Null alleles of C4b have been observed in strong association with autism, with two separate studies identifying carriers between 37-42% of autistic children (3, 4). In the 2005 study, more than 50% of autistic children with C4b null alleles featured duplications of the C4a gene (4).
• The C4B*Q0 genotype, defined as having one or no copies of the C4b gene is associated with hyperreactive HPA axis function (9). This is believed to be due to the intrinsic relationship between C4 and CYP21a2.

Familial History of Schizophrenia – C4, TGFß, Synaptic Pruning & TNXB

Excessive pruning in the brain is a hallmark feature of schizophrenia, which links to C4-related mechanisms. A significant 2016 study identified the strongest genetic associations of schizophrenia linked to the HLA region, arising in part from allelic variations of the C4 gene located within the RCCX region. These tend to produce greater expression of C4a in the brain (6).

TNXB (tenascin-X), an abundantly present glycoprotein in the ECM, and part of the RCCX region has been found to be associated with schizophrenia in a 2017 study (7). A 2004 study found TNXB alleles to be associated with schizophrenia, including rs1009382, yet the mechanism of involvement has not been elucidated (8). One probable link between TNXB and schizophrenia is likely related to the fact that TNXB regulates the activation and function of TGFß. TGFß mediates neuronal refinement and mechanisms of microglia-mediated synaptic pruning, through modulation of complement proteins C3 and C1q (19). In a TNXB mutation or “negative event”, TGFß activation would be impaired. This directly impairs the function of neuronal refinement and pruning via complements C1q and C3.

One future area worth exploring with respect to schizophrenia and TNXB is the neighboring CREB gene located near TNXB. CREB is a cAMP responsive transcription factor, that controls the transcription of numerous genes including: BDNF, tyrosine hydroxylase, biological clock genes and neuropeptides such as somatostatin and CRH (32). Deficient CREB is associated with Alzheimer’s disease. Significantly, the CREB gene contains the transcriptional regulation of the TNXB gene. A single study investigating the association between TNXB and schizophrenia demonstrated a weak relationship between CREBL1 and schizophrenia. While the initial findings were weak, the researchers noted that disruption of cAMP in the brain leads to neurodegeneration (8).

CYP21a2 Haplogroups: PCOS, CAH, Hyperandrogenemia, Sodium Wasting, Brain & Limbic Volume Variations

The CYP21a2 gene generates 21-hydroxylase, central to the formation of the adrenal stress hormone cortisol and the mineralcorticoid aldosterone. Moderate to severe deficiencies of CYP21a2 leads to insufficient cortisol and aldosterone production. Classically this is represented in the disease CAH, congenital adrenal hyperplasia. In most cases of CAH, the CYP21a2 bottleneck leads to a shunt of progesterone towards the androgen pathway (there have been reports of rare forms of CAH featuring elevated cortisol and HPA axis activities). Deficient CYP21a2 is most commonly associated with:

• CAH (congenital adrenal hyperplasia)
• Hirsutism
• Hyperandrogenemia
• PCOS and ovarian cyst formation
• Sodium deficiency and salt wasting

Discussion:

Insufficient 21-hydroxylase leads to a shunt of progesterone towards the formation of androstenedione, which can then convert into other androgenic hormones, including testosterone. In females, it is postulated here that RCCX phenotypes are prone towards PCOS, ovarian cysts, hirsutism and hyperandrogenemia. These individuals often express joint hypermobility and/or skin hyperelasticity, characteristic of the involvement with the neighboring TNXB gene. Blood pressure may tend to be low, due in part to electrolyte insufficiency, and salt wasting caused by low aldosterone levels.

CYP21a2: Decreased Amygdala Volume, Emotional Processing Disturbances & Various “Psychiatric” Presentations

It has been identified in the literature that children with congenital adrenal hyperplasia (CAH) have decreased amygdala volume (10). Adolescents with CAH showed greater amygdala activation during an exercise involving the presentation of negative faces to study subjects (11). The decreased amygdala volume and associated increased amygdala activation suggests unique limbic brain involvement due to insufficient glucocorticoids during development, among those with CAH.

It is postulated here that other CYP21a2 haplotypes (apart from those with CAH) will experience similar emotional processing disturbances. A characteristic trait of certain RCCX phenotypes is having a highly empathic nature. This may be due in part to unique phenotype presentations in the limbic organs such as the amygdala, as well as variability of function to the related vagus nerve.

The relationship between psychiatric disorders among patients with CAH (congenital adrenal hyperplasia) has been described for decades (12). What is dramatically understudied are the relationships between mild 21-hydroxylase deficiencies and psychiatric illnesses. Common “psychiatric” presentations among suspected RCCX phenotypes include:

• Anxiety disorders
• Sensory and emotional processing disturbances
• Enhanced susceptibility to PTSD

The implications for the involvement of CYP21a2 in a wide spectrum of psychiatric and neurochemical-related illnesses is based on the following:

• CYP21a2 RNA has been detected in the brain and central nervous system (13). It’s role here is not clearly defined, but likely relates to neurosteroid pathways, such as those associated with pregnanes (14).
• Cortisol has a direct effect on brain development and behavior. Aberrant cortisol due to variations of CYP21a2 will influence: behavior, responses to stress, increase the susceptibility of trauma-induced psychiatric illness, and inhibit the normal functioning mechanisms of the HPA axis.

TGFß, TNXB & VEGF: Tissue Remodeling, Immune, Synaptic Pruning, Embryological, Stem Cell Differentiation, Bowel Disorders, Vascular & Endothelial Functions

TGFß & TNXB

The fibrinogen-like domains of TNXB regulate the bioavailability of TGFß (18). Any possible adverse genetic activities which affect TNXB may impair TGFß bioavailability. This may include: Mutated TNXB, “dominant negative events” involving TNXB, mutations in or near the TNXB fibrinogen-like domain, chimeric TNXB/TNXA activities, mutations or deletions of CREB (which contain the transcriptional start points of TNXB), unfolded TNXB or other anomalous, undefined TNXB events. The implications of dysregulated TGFß due to TNXB has profound implications on:

• Immunological Homeostasis – TGFß is ubiquitous in the immune system. It exists as a cytokine with both pro and anti-inflammatory effects. It acts as one of the most significant immunosuppressant cytokines.
  • Dysregulated TGFß signaling may be etiological in autoimmune diseases, among suspected RCCX phenotypes
• Embryological Development – TGFß is centrally involved in embryological development. This includes brain development. Mice bred to be deficient in type 2 TGFß receptors developed enlarged midbrain (33).
  • Joint hypermobility syndromes are known to feature increases in amygdala volume (34). For a subset of suspected RCCX phenotypes this may be due to loss of TGFß signaling.
• Synaptic Pruning – TGFß is centrally involved in synaptic pruning mechanisms. TGFß mediates neuronal refinement and mechanisms of microglia-mediated synaptic pruning, through modulation of complement proteins C3 and C1q (19). Aberrant pruning mechanisms are etiological in neurological diseases such as: schizophrenia, autism, ALS, Huntington’s Parkinson’s.
• Inflammatory Bowel Diseases – Inflammatory bowel diseases such as ulcerative colitis involve aberrant TGFß signaling (37), and singular nucleotide polymorphisms (SNP’s) of TNXB have shown to be associated with ulcerative colitis (38). TGFß is central in the gut, and has been proposed as a master regulatory cytokine of the gut microbiota (39).
• Pathogenesis of Endometriosis – There is a high rate of frequency of endometriosis in EDS (35). TGFß signaling is centrally involved in the pathogenesis of endometriosis (36).

TNXB & VEGF-B (vascular endothelial growth factor-B)

The TNXB protein interacts with VEGF-B, as well as the VEGF-R1 receptor. (18). Unlike its counterpart VEGF-A, VEGF-B regulates the growth and survival of blood vessels during pathological states. VEGF-B is critical for the survival of newly formed blood vessels, as well as smooth muscle cells and vascular stem cells (42). How this plays out among RCCX phenotypes is not known. It is worth speculating that during inflammatory insults, such as autoimmune flares, lack of VEGF-B activation could lead to vascular complications and symptoms. This may involve hypoperfusion of blood supply to peripheral tissues, symptoms of neuropathy and burning, discoloration of fingers and toes, and symptoms of hypoxia or pseudohypoxia.

Future Testing For RCCX Genotypes & Additional Questions

This section is to propose a list of tests for suspected RCCX genotypes in order to establish involvement between RCCX and associated dysregulation of physiology.

From a research perspective, studying the relationship between biomarkers may prove more useful than studying the individual markers by themselves. Because of the overlapping nature of RCCX genes, biomarker relationships may be useful to establish the differences of RCCX phenotype groups.

• Total Complement C4 – Low blood levels of C4 are associated with certain autoimmune diseases. Low levels of C4 are associated with C4 long/short genotypes and with fewer copy numbers of the C4 gene.
• CD4+ CD25+ Regulatory T-cells (TREG’s) – Because C4 regulates TREG’s cell activities as well as TGFß, quantifying levels of TREGs could prove to be an essential tracking marker with respect to C4 activities and the activities of TGFß-1.
• TGFß-1 – The difficulty with interpreting TGFß-1 levels is that the suspected issue is more related to qualitative versus quantitative problems. Because TNXB regulates TGFß maturation and differentiation, the quantitative levels of TGFß-1 may not reflect the problem occurring. Instead, studying the 3-way relationship between TREG’s, TGFß-1 and C4 may prove to be more fruitful.
• Tenascin protein levels – The utility of tenascin protein levels is not well understood. However, establishing baseline levels of TNXB could prepare deeper investigations into relationships with TGFß-1, and the downstream effects of loss of TGFß signaling (such as autoimmunity and bowel disorders). Additionally, it is worth considering how a loss of TNXB function may be related to other tenascins, such as TNXC.
• VEGF-B – The TNXB protein is known to interact with VEGF-B. Investigating the relationship is useful especially as it relates to vascular and endothelial-related complications.
• C4b-Derived Peptides – Peptides derived from C4b have shown to have TH1 immunomodulatory functions. Exploring the derivatives of the C4 protein isoforms is something that mass spectrometry and metabolomics profiling may be useful for. C4b and C4a peptides (their presence or absence) may be an important future area of research for neurological and inflammatory diseases.
• Related Complement Proteins – How does low RCCX copy number or various C4 short/long genotypes affect related complement genes such as: Factor B, C3, C2? How does the genomic “hostspot” created by the RCCX CNV affect nearby genes? How does the HERV-K retrovirus (within C4) and retrotransposon (within STK19/RP1) affect nearby genes? These important questions may lead to answers related to how CNV’s affect multiple genes and pathways.
• Cortisol – In most cases of CYP21a2 mutations that cause CAH, cortisol levels are low. There have been few cases of CAH with elevated cortisol reported in literature (40). Future testing should look at cortisol in blood and urine in relationship to C4 protein levels, as well as C4 genotype (short/long and C4 copy numbers), because of the fact that intron 35 of the C4b gene controls CYP21a2 transcription. Over 150 CYP21a2 haplogroups have been identified (40, 41). This great diversity suggests a wide range of genomic influence on cortisol expression.